Preparation Method and Application of a Dynamically Adjustable Curvature Composite Hydrogel Substrate

Abstract

The present disclosure provides a method for preparing a dynamically adjustable curvature composite hydrogel substrate and its application in cell stimulation. This method uses a step-by-step polymerization method to prepare the composite hydrogel substrate, including: using a cylindrical lens template for reverse molding to prepare a polydimethylsiloxane negative etching mold with a semi-circular stripe structure; using this polydimethylsiloxane negative etching mold to prepare the first layer of polyacrylamide hydrogel layered substrate; using a polymerization solution of N-isopropylacrylamide doped with nano-gold to polymerize on the surface of the first layer of polyacrylamide hydrogel layered substrate to form the second layer of poly-N-isopropylacrylamide temperature-sensitive hydrogel, obtaining a PNIPAM-NPG / PA composite hydrogel substrate. Under 808 nm infrared laser irradiation, the curvature of this composite hydrogel can be dynamically and reversibly regulated. This preparation method is simple, the production process is mild, and the economic cost is low, providing a new idea for the research on the mechanical regulation mechanism of cell behaviors such as cell migration and cell polarization.

Description

Technical Field

[0001] The present disclosure relates to the technical field of hydrogels and biomaterials, and particularly to a method for preparing a dynamically adjustable curvature composite hydrogel substrate and an application of the composite hydrogel substrate in cell stimulation. Background Art

[0002] Mechanical signals such as cell-matrix interaction, cell-cell contact, and various physical fields (mechanical force, heat, electric field, etc.) in the microenvironment around cells are key factors affecting cell function and fate. The cell microenvironment is a complex collection composed of multiple factors and variable in space and time. The physical structure, mechanical properties, etc. of the extracellular matrix in the microenvironment also exhibit very complex spatio-temporal dynamic change characteristics. In order to better simulate the influence of the mechanical signal mechanism in the extracellular matrix (ECM) on cell behavior (such as cell adhesion, migration, polarization, and differentiation, etc.) in vitro, researchers have designed and developed various stimulus-responsive materials affected by temperature, pH, light, etc., and used them to simulate the dynamically changing microenvironment in vivo to further explore the influence mechanism of mechanical signals on cell function.

[0003] However, the changes of current stimulus-responsive materials are mostly irreversible or uniform deformation. Therefore, it is necessary to design and develop stimulus-responsive materials with anisotropy and dynamic deformation in vitro to more accurately simulate the dynamically changing cell microenvironment in vivo.

[0004] Hydrogels have excellent biocompatibility and can obtain stimulus responsiveness to pH, electrochemistry, magnetic field, light, temperature, etc. by introducing some groups. Poly-N-isopropylacrylamide (PNIPAM) is a typical temperature-sensitive polymer material. Because of its lower critical solution temperature (32 - 38 °C) and functions such as shape memory and self-regulation similar to cells and some intelligent biological systems, it is an ideal gel material for reproducing the dynamic microenvironment. Summary of the Invention

[0005] (I) Technical Problems to be Solved

[0006] In view of this, the present disclosure mainly provides a method for preparing a dynamically adjustable curvature composite hydrogel substrate and an application of the composite hydrogel substrate in cell stimulation, so as to realize the preparation of a PNIPAM-NPG / PA composite hydrogel substrate and realize reversible dynamic curvature mechanical stimulation of cells.

[0007] (II) Technical Solutions

[0008] To achieve the above object, the present disclosure provides a method for preparing a dynamically adjustable curvature composite hydrogel substrate. This method uses a stepwise polymerization method to prepare the composite hydrogel substrate, specifically including:

[0009] Step S1: Use a cylindrical lens template to prepare a negative PDMS mold with a semi-circular stripe structure by reverse molding;

[0010] Step S2: Use the PDMS negative mold to prepare the first layer of polyacrylamide (PA) hydrogel layered substrate;

[0011] Step S3: Use a polymerization solution of N-isopropylacrylamide (NIPAM) doped with nano-gold (NPG) to polymerize on the surface of the first layer of polyacrylamide hydrogel layered substrate to form the second layer of poly-N-isopropylacrylamide (PNIPAM) thermosensitive hydrogel, obtaining a PNIPAM-NPG / PA composite hydrogel substrate.

[0012] In the above solution, the use of a cylindrical lens template to prepare a negative PDMS mold with a semi-circular stripe structure in Step S1 includes:

[0013] Mix prepolymer A liquid and curing agent B liquid in a volume ratio of 10:1 to prepare a PDMS prepolymer solution. After mixing with a rubber mixer, drop it between a plasma-treated cover glass and a cylindrical lens template, and evacuate to remove air bubbles;

[0014] Cover the surface of the cover glass with a weight of 200 - 400 g, put it into an oven for heating and curing, then immerse it in anhydrous ethanol to peel off the PDMS from the cylindrical lens template, obtaining a PDMS negative mold with a semi-circular stripe structure.

[0015] In the above solution, the cylindrical lens template has a bottom periodic cylindrical array structure with wavelengths of 170 μm and 340 μm, the heating temperature of the oven is 65 - 85 °C, and the heating time is 2 - 10 hours.

[0016] In the above solution, the use of the PDMS negative mold to prepare the first layer of polyacrylamide (PA) hydrogel layered substrate in Step S2 includes:

[0017] Use ultrasound to clean the glass substrate in a methanol solution and dry it in a fume hood; then prepare a 4% (v / v) 3-aminopropyltriethoxysilane (APES) acetone solution and drop it on the surface of the glass substrate. After drying in a fume hood, rinse it with water, and fix it with a glutaraldehyde solution and then dry it to obtain a glass substrate for use;

[0018] Add acrylamide monomer (AM) and methylene bisacrylamide (Bis-Acryamide) crosslinker to deionized water to prepare a polymer solution containing acrylamide and methylene bisacrylamide crosslinker. After vacuum degassing for 0.5 - 2 minutes, add ammonium persulfate (APS) and tetramethylethylenediamine (TEMED), and quickly drip the gel mixture solution onto the surface of the glass substrate to be used after mixing;

[0019] Cover the negative PDMS mold from one side of the gel solution to the other side. After 20 - 40 minutes, the gel mixture polymerizes. Peel the negative PDMS mold from the surface of the hydrogel to form the first layer of PA hydrogel on the substrate surface, and obtain the first layer of polyacrylamide (PA) hydrogel layered substrate.

[0020] In the above scheme, in the step of using glutaraldehyde solution for fixation and then air drying to obtain the glass substrate to be used, the concentration of the glutaraldehyde solution is 0.05 - 2.0% (v / v), and the curing time is 15 - 40 minutes; in the polymer solution containing acrylamide and methylene bisacrylamide crosslinker, the concentration of acrylamide is 3 - 10% (v / v), and the concentration of methylene bisacrylamide is 0.02 - 0.4% (v / v); in the step of adding ammonium persulfate (APS) and tetramethylethylenediamine (TEMED), the concentration of APS is 1 - 3% (v / v), and the concentration of TEMED is 0.1 - 0.5% (v / v).

[0021] In the above scheme, in step S3, the use of a polymerization solution of N-isopropylacrylamide (NIPAM) doped with nano-gold (NPG) to polymerize on the surface of the first layer of polyacrylamide hydrogel layered substrate to form the second layer of poly-N-isopropylacrylamide (PNIPAM) thermosensitive hydrogel, and obtain the PNIPAM-NPG / PA composite hydrogel substrate, including:

[0022] Soak the cleaned glass cover slip in a 5% (v / v) toluene solution of dichlorodimethylsilane (DCDMS) for 1 - 3 minutes, and then rinse with ethanol and water in turn to remove the excess DCDMS to obtain a hydrophobic cover slip treated with DCDMS;

[0023] Add N-isopropylacrylamide, methylene bisacrylamide (Bis-Acryamide) crosslinker and doped nano-gold (NPG) to deionized water to prepare a polymer solution containing N-isopropylacrylamide, methylene bisacrylamide (Bis-Acryamide) crosslinker and doped nano-gold (NPG). After vacuum degassing for 0.5 - 2 minutes, add APS and TEMED, and quickly drip the gel mixture solution onto the surface of the first layer of polyacrylamide (PA) hydrogel layered substrate after mixing;

[0024] Cover the DCDMS-treated hydrophobic cover glass from the side where the gel mixture solution is dropped to the other side. After 20 - 40 minutes, the gel mixture polymerizes, and then peel the hydrophobic cover glass from the hydrogel surface to obtain the PNIPAM-NPG / PA composite hydrogel substrate.

[0025] In the above solution, in the polymer solution containing N-isopropylacrylamide, methylenebisacrylamide (Bis-Acryamide) cross-linking agent, and doped nanogold (NPG), the concentration of N-isopropylacrylamide is 5 - 15% (v / v), the concentration of methylenebisacrylamide (Bis-Acryamide) cross-linking agent is 0.02 - 1.0% (v / v), the doped nanogold (NPG) is in powder form or aqueous nanogold solution, and its concentration is 0.2 - 2%; in the step of adding APS and TEMED, the concentration of APS is 1 - 3% (v / v), and the concentration of TEMED is 0.1 - 0.5% (v / v).

[0026] In the above solution, after obtaining the PNIPAM-NPG / PA composite hydrogel substrate in step S3, it further includes: soaking the PNIPAM-NPG / PA composite hydrogel substrate in phosphate buffer solution (PBS, pH 7.4) for standby.

[0027] The present disclosure also provides the application of the dynamically adjustable curvature composite hydrogel substrate prepared by the above method in cell stimulation, including:

[0028] Step 1: Couple proteins on the hydrogel surface;

[0029] Wash the PNIPAM-NPG / PA composite hydrogel 2 - 5 times with PBS, drop 200 μL of sulfo-SANPAH reagent, and irradiate it under ultraviolet light for 20 - 40 minutes. Then wash it 2 - 5 times with 50 mM HEPES buffer solution at pH 8.5, incubate with fibronectin or rat tail type I collagen, and place it in a 4°C refrigerator overnight;

[0030] Step 2: Inoculate and stimulate cells;

[0031] Aspirate the protein solution incubated in step 1, wash it 2 - 5 times with PBS, sterilize it under ultraviolet light for 20 - 40 minutes, then inoculate cells, incubate them in a cell culture incubator for 1 - 3 hours, and then place them under an infrared laser for stimulation, and further observe the cell morphology.

[0032] In the above solution, the cell types inoculated in step 2 are A549, MDCK, NIH / 3T3, MCF-7, or IEC-6, and the cell seeding density is 10 4 cells / cm2 ; In Step 2, the infrared laser has a wavelength of 808 nm, a power of 0 - 5 W, a frequency of 0.1 - 10 Hz, and a spot size of 0.5 - 20 mm.

[0033] (III) Beneficial Effects

[0034] The preparation method of the dynamically tunable curvature composite hydrogel substrate provided by the present disclosure and the application of this composite hydrogel substrate in cell stimulation have the following advantages compared with the existing technologies:

[0035] 1. The preparation method of the dynamically tunable curvature composite hydrogel substrate provided by the present disclosure uses the step-by-step polymerization method to polymerize PNIPAM and PA into a composite hydrogel, and adds gold nanoparticles to the PNIPAM layer, which can convert the absorbed infrared light into heat energy, increase the temperature in the local area, realize the surface curvature structure, and achieve the preparation of the dynamically tunable curvature composite hydrogel substrate.

[0036] 2. The preparation method of the dynamically tunable curvature composite hydrogel substrate provided by the present disclosure can enable the PNIPAM-NPG / PA composite hydrogel to overcome the limitation of irreversible changes of ordinary gel materials under stimulation response by adjusting the frequency of the infrared laser, and conveniently realizes the reversible dynamic curvature mechanical stimulation of cells.

[0037] 3. The preparation method of the dynamically tunable curvature composite hydrogel substrate provided by the present disclosure and its application in cell stimulation use a lens mold and gradually polymerize NIPAM and AM monomers to economically and conveniently prepare the PNIPAM-NPG / PA composite hydrogel, and successfully realize the dynamic regulation of the substrate curvature with an 808 nm infrared laser.

[0038] 4. The preparation method of the dynamically tunable curvature composite hydrogel substrate provided by the present disclosure and its application in cell stimulation. Under the irradiation of an 808 nm infrared laser, the composite hydrogel can realize the dynamic reversible regulation of curvature, and then cells can be inoculated on the surface of the composite hydrogel to explore the mechanical mechanism of cell regulation by dynamic curvature.

[0039] 5. The preparation method of the dynamically tunable curvature composite hydrogel substrate provided by the present disclosure has a simple preparation method, a mild production process, and a low economic cost, providing a new idea for the research on the mechanical regulation mechanism of cell behaviors such as cell migration and cell polarization. Description of the Drawings

[0040] To more fully understand the present disclosure and its advantages, the specific description of the drawings is given below, where:

[0041] Figure 1 is a flowchart of the method for preparing a dynamically tunable curvature composite hydrogel substrate according to an embodiment of the present disclosure.

[0042] Figure 2 It is a process flow chart for preparing a dynamically tunable curvature composite hydrogel substrate according to an embodiment of the present disclosure.

[0043] Figures 3A to 3C It is a three-dimensional contour scan of PNIPAM-NPG / PA in a dynamically tunable curvature composite hydrogel substrate prepared according to an embodiment of the present disclosure. Among them, Figure 3A is the two-dimensional topography scanning area of the composite hydrogel surface, Figure 3B is the three-dimensional topography contour map of the composite hydrogel, Figure 3C is to quantitatively characterize the curvature height of the three-dimensional surface topography of the composite hydrogel.

[0044] Figure 4 It is the result of dynamically stimulating cells with PNIPAM-NPG / PA in a dynamically tunable curvature composite hydrogel substrate prepared according to Example 1 of the present disclosure. Detailed implementation manners

[0045] Hereinafter, embodiments of the present disclosure will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are merely exemplary and are not intended to limit the scope of the present disclosure. In the following detailed description, for the sake of explanation, many specific details are set forth to provide a comprehensive understanding of the embodiments of the present disclosure. However, obviously, one or more embodiments can also be implemented without these specific details. In addition, in the following description, descriptions of well-known structures and technologies are omitted to avoid unnecessarily confusing the concepts of the present disclosure.

[0046] The method for preparing a dynamically tunable curvature composite hydrogel substrate provided by the present disclosure adopts a step-by-step polymerization method. Specifically, PNIPAM and PA are polymerized into a composite hydrogel, and nano-gold particles are added to the PNIPAM layer, which can convert the absorbed infrared light into heat energy, increase the temperature in the local area, and realize the surface curvature structure. In addition, by adjusting the frequency of the infrared laser, the PNIPAM-NPG / PA composite hydrogel can overcome the limitation of irreversible changes of ordinary gel materials under stimulus response, and conveniently realize reversible dynamic curvature mechanical stimulation of cells.

[0047] As Figure 1 and Figure 2 shown, Figure 1 is a method flow chart for preparing a dynamically tunable curvature composite hydrogel substrate according to an embodiment of the present disclosure, Figure 2 is a process flow chart for preparing a dynamically tunable curvature composite hydrogel substrate according to an embodiment of the present disclosure. This method uses a step-by-step polymerization method to prepare a composite hydrogel substrate, specifically including:

[0048] Step S1: using a cylindrical lens template to prepare a polydimethylsiloxane (PDMS) negative-etching mold with a semicircular stripe structure, specifically comprising:

[0049] Prepolymer A liquid and curing agent B liquid are mixed at a volume ratio of 10:1 to prepare a polydimethylsiloxane (PDMS) prepolymer liquid, and the prepolymer liquid is mixed by a glue mixer and then dropped between the plasma-treated cover glass and the cylindrical lens template, and the air bubbles are removed by vacuuming; and

[0050] The surface of the cover glass was covered with a weight of 200-400 g, placed in a drying oven for heating and curing, and then immersed in anhydrous ethanol to peel off the PDMS and the cylindrical lens template to obtain a polydimethylsiloxane (PDMS) negative engraving mold with a semicircular stripe structure.

[0051] In this step, the cylindrical lens template is a bottom periodic cylindrical array structure containing wavelengths of 170 μm and 340 μm, the heating temperature of the drying box is 65-85° C., and the heating time is 2-10 hours.

[0052] Step S2: using the polydimethylsiloxane negative-engraving mold to prepare a first layer of polyacrylamide (PA) hydrogel layered substrate, specifically comprising:

[0053] The glass substrate was cleaned in a methanol solution using ultrasound and dried in a fume hood; then a 4% (v / v) 3-aminopropyltriethoxysilane (APES) acetone solution was prepared and added dropwise to the surface of the glass substrate, which was placed in a fume hood to dry and then rinsed with water, fixed with a glutaraldehyde solution and dried to obtain a glass substrate to be used;

[0054] Adding acrylamide monomer (AM) and bis-acrylamide (Bis-Acryamide) crosslinking agent to deionized water to prepare a polymer solution containing acrylamide and bis-acrylamide crosslinking agent, vacuum degassing for 0.5-2 minutes, optionally 1 minute, adding ammonium persulfate (APS) and tetramethylethylenediamine (TEMED), mixing well, and then quickly dropping the gel mixture solution onto the surface of the glass substrate to be used; and

[0055] The polydimethylsiloxane (PDMS) negative-engraving mold is covered from one side of the gel solution to the other side. After 20-40 minutes, optionally 30 minutes, the gel mixture is polymerized, and the polydimethylsiloxane (PDMS) negative-engraving mold is peeled off from the hydrogel surface to form a first layer of PA hydrogel on the substrate surface, thereby obtaining a first layer of polyacrylamide (PA) hydrogel layered substrate.

[0056] In this step, in the step of using a glutaraldehyde solution for fixation and then air-drying to obtain a glass substrate for use, the concentration of the glutaraldehyde solution is 0.05 - 2.0% (v / v), and the curing time is 15 - 40 minutes; in the polymer solution containing acrylamide and methylene bisacrylamide crosslinker, the concentration of acrylamide is 3 - 10% (v / v), and the concentration of methylene bisacrylamide is 0.02 - 0.4% (v / v); in the step of adding ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TEMED), the concentration of APS is 1 - 3% (v / v), and the concentration of TEMED is 0.1 - 0.5% (v / v).

[0057] Step S3: Polymerize a solution of N-isopropylacrylamide (NIPAM) doped with nano-gold (NPG) on the surface of the first-layer polyacrylamide hydrogel layered substrate to form a second-layer poly-N-isopropylacrylamide (PNIPAM) thermosensitive hydrogel, obtaining a PNIPAM-NPG / PA composite hydrogel substrate, which specifically includes:

[0058] Immerse the cleaned glass coverslip in a 5% (v / v) toluene solution of dichlorodimethylsilane (DCDMS) for 1 - 3 minutes, and then rinse with ethanol and water in sequence to remove the excess DCDMS, obtaining a hydrophobic coverslip treated with DCDMS;

[0059] Add N-isopropylacrylamide, methylene bisacrylamide (Bis-Acryamide) crosslinker, and doped nano-gold (NPG) to deionized water to prepare a polymer solution containing N-isopropylacrylamide, methylene bisacrylamide (Bis-Acryamide) crosslinker, and doped nano-gold (NPG). After vacuum degassing for 0.5 - 2 minutes, optionally 1 minute, add APS and TEMED. After mixing evenly, quickly drop the gel mixture solution onto the surface of the first-layer polyacrylamide (PA) hydrogel layered substrate; and

[0060] Cover the hydrophobic coverslip treated with DCDMS from the side where the gel mixture solution is dropped to the other side. After 20 - 40 minutes, optionally 30 minutes, the gel mixture polymerizes. Peel the hydrophobic coverslip from the surface of the hydrogel to obtain a PNIPAM-NPG / PA composite hydrogel substrate.

[0061] In this step, in the polymer solution containing N-isopropylacrylamide, methylenebisacrylamide (Bis-Acrylamide) crosslinker, and doped nano-gold (NPG), the concentration of N-isopropylacrylamide is 5-15% (v / v), the concentration of methylenebisacrylamide (Bis-Acrylamide) crosslinker is 0.02-1.0% (v / v), the doped nano-gold (NPG) is in powder form or aqueous nano-gold solution, and its concentration is 0.2-2%; in the step of adding APS and TEMED, the concentration of APS is 1-3% (v / v), and the concentration of TEMED is 0.1-0.5% (v / v).

[0062] Figures 3A to 3C Shows the three-dimensional contour scan of PNIPAM-NPG / PA in the dynamically tunable curvature composite hydrogel substrate prepared according to an embodiment of the present disclosure. Among them, Figure 3A Is the two-dimensional topography scanning area of the composite hydrogel surface, Figure 3B Is the three-dimensional topography contour map of the composite hydrogel, Figure 3C Is to quantitatively characterize the curvature height of the three-dimensional surface topography of the composite hydrogel.

[0063] In the method for preparing a dynamically tunable curvature composite hydrogel substrate according to an embodiment of the present disclosure, after obtaining the PNIPAM-NPG / PA composite hydrogel substrate in step S3, it further includes: soaking the PNIPAM-NPG / PA composite hydrogel substrate in phosphate buffer solution (PBS, pH 7.4) for standby.

[0064] Based on Figure 1 And Figure 2 The method for preparing a dynamically tunable curvature composite hydrogel substrate shown, the present disclosure also provides an application of the dynamically tunable curvature composite hydrogel substrate prepared by the method in cell stimulation, specifically including:

[0065] Step 1: Couple proteins on the hydrogel surface;

[0066] Wash the PNIPAM-NPG / PA composite hydrogel with PBS 2-5 times, optionally 3 times, add 200 μL of sulfo-SANPAH reagent, and irradiate under ultraviolet light for 20-40 minutes, optionally 30 minutes. Then wash with 50 mM HEPES buffer solution Ph8.5 2-5 times, optionally 3 times, incubate with fibronectin or rat tail type I collagen, and place it in a 4°C refrigerator overnight;

[0067] Step 2: Inoculate and stimulate cells;

[0068] Aspirate the protein solution incubated in Step 1, wash it 2 - 5 times with PBS, optionally 3 times, sterilize it with ultraviolet light for 20 - 40 minutes, optionally 30 minutes, inoculate cells, incubate in a cell culture incubator for 1 - 3 hours, optionally 2 hours, place it under an infrared laser for stimulation, and further observe the cell morphology.

[0069] In this step, the types of inoculated cells are A549, MDCK, NIH / 3T3, MCF - 7 or IEC - 6, and the inoculated cell density is 10 4 cells / cm 2 ; the wavelength of the infrared laser is 808 nm, the power is 0 - 5 W, the frequency is 0.1 - 10 Hz, and the spot size is 0.5 - 20 mm.

[0070] The present invention will be further illustrated by the following examples, but is not limited to the following examples.

[0071] Example 1

[0072] This Example 1 provides a preparation method of a dynamically adjustable curvature composite hydrogel substrate and its application in cell stimulation, which specifically includes the following steps:

[0073] Step (1): Prepare a PDMS negative etching template;

[0074] Mix the prepolymer A liquid and the curing agent B liquid in a volume ratio of 10:1 to prepare a polydimethylsiloxane (PDMS) prepolymer solution. After mixing with a mixer, drop it between the plasma - treated coverslip and the cylindrical lens mold, evacuate to remove air bubbles, and use a lens mold with a bottom layer periodic columnar array structure of 340 μm. Cover the coverslip surface with a weight of about 200 g, put it into an oven for heating and curing, the heating temperature is 65 °C, and the heating time is 5 hours. Immerse it in anhydrous ethanol and slowly peel off the PDMS and the lens mold to obtain the corresponding PDMS negative etching mold.

[0075] Step (2): Treat the glass substrate;

[0076] First, clean the glass substrate with ultrasound in a methanol solution and dry it in a fume hood. Then, prepare a 4% (v / v) 3 - aminopropyltriethoxysilane (APES) acetone solution and drop it on the surface of the glass substrate. After drying in a fume hood, rinse it with water. Then, fix it with an aqueous glutaraldehyde solution and dry it for standby.

[0077] Step (3): Prepare the first layer (bottom layer) of PA hydrogel;

[0078] Prepare a polymer solution containing AM (5%, v / v) and Bis-Acryamide (0.2%, v / v) crosslinker in deionized water. After vacuum degassing for 1 minute, add APS (2%, v / v) and TEMED (0.2%, v / v). After mixing, quickly drop the gel mixture solution onto the surface of the glass substrate prepared in step (2). Then cover the PDMS mold prepared in step (1) from one side of the gel solution to the other side. After 30 min, the gel mixture polymerizes, and slowly peel the mold from the hydrogel surface to form the first layer of PA hydrogel on the substrate surface.

[0079] Step (4): Prepare a hydrophobic coverslip;

[0080] Immerse the cleaned glass coverslip in a 5% (v / v) dichlorodimethylsilane (DCDMS) toluene solution for 1 - 3 min, and then rinse with ethanol and water in sequence to remove the excess DCDMS. Obtain a DCDMS-treated hydrophobic coverslip.

[0081] Step (5): Prepare the second layer (top layer) of PNIPAM-NPG hydrogel;

[0082] Prepare a polymer solution containing N-isopropylacrylamide (10%, v / v), Bis-Acryamide crosslinker (0.05%, v / v), and NPG (0.3%, v / v) in deionized water. After vacuum degassing for 1 minute, add APS (2%, v / v) and TEMED (0.2%, v / v). After mixing, quickly drop the gel mixture solution onto the surface of the PAAm hydrogel prepared in step (3). Then cover the hydrophobic coverslip prepared in step (4) from one side of the gel solution to the other side. After 30 min, the gel mixture polymerizes, and slowly peel the coverslip from the hydrogel surface. Finally, obtain the PNIPAM-NPG / PA composite hydrogel, and then immerse it in phosphate buffer solution (PBS, pH 7.4) for further use.

[0083] Wherein, Figures 3A to 3C shows the three-dimensional contour scan of PNIPAM-NPG / PA in the dynamically tunable curvature composite hydrogel substrate prepared according to the embodiments of the present disclosure. Among them, Figure 3A is the two-dimensional topography scan area of the composite hydrogel surface, Figure 3B is the three-dimensional topography contour map of the composite hydrogel, Figure 3C is to quantitatively characterize the curvature height of the three-dimensional surface topography of the composite hydrogel.

[0084] Based on the above steps (1) - step (5), the PNIPAM-NPG / PA composite hydrogel is obtained. In this Example 1, it is used for anisotropically stretching cells, specifically including the following steps:

[0085] Step (6): Coupling protein on the hydrogel surface;

[0086] Wash the PNIPAM-NPG / PA composite hydrogel 3 times with PBS, add 200 μL of sulfo-SANPAH reagent, irradiate under ultraviolet light for 30 min, then wash 3 times with 50 mM HEPES buffer at pH 8.5, incubate with rat tail type I collagen, and place it in a 4 °C refrigerator overnight.

[0087] Step (7): Inoculating and stimulating cells;

[0088] Aspirate the protein solution incubated in step (6), wash 3 times with PBS, sterilize under ultraviolet light for 30 min, and then inoculate A549 cells (10 4 cells / cm 2 ). Incubate in a cell incubator for 2 h, then place it under an infrared laser (400 mW, 0.1 Hz) for stimulation, and further observe the cell morphology.

[0089] Specifically, as Figure 4 shown, Figure 4 shows the results of dynamic curvature stimulation of cells by PNIPAM-NPG / PA in the dynamically tunable curvature composite hydrogel substrate prepared according to Example 1 of the present disclosure.

[0090] Example 2

[0091] This Example 2 provides a preparation method of a dynamically tunable curvature composite hydrogel substrate and its application in cell stimulation, which specifically includes the following steps:

[0092] Step (1): Preparing a PDMS negative etching template;

[0093] Mix the prepolymer A liquid and the curing agent B liquid in a volume ratio of 10:1 to prepare a polydimethylsiloxane (PDMS) prepolymer solution. After mixing with a mixer, drop it between a plasma-treated coverslip and a cylindrical lens mold, evacuate to remove air bubbles, and use a lens mold with a bottom layer periodic columnar array structure of 170 μm. Cover the coverslip surface with a weight of about 200 g, place it in an oven for heating and curing, the heating temperature is 65 °C, and the heating time is 5 h. Immerse it in anhydrous ethanol and slowly peel off the PDMS and the lens mold to obtain the corresponding PDMS negative etching mold.

[0094] Step (2): Treating the glass substrate;

[0095] First, clean the glass substrate in a methanol solution using ultrasound and dry it in a fume hood. Subsequently, prepare a 4% (v / v) solution of 3-aminopropyltriethoxysilane (APES) in acetone, drop it onto the surface of the glass substrate, let it dry in the fume hood, and then rinse it thoroughly with water. Then fix it with an aqueous glutaraldehyde solution and dry it for later use.

[0096] Step (3): Prepare the first layer (bottom layer) of PA hydrogel;

[0097] Prepare a polymer solution containing AM (8%, v / v) and Bis-Acryamide (0.4%, v / v) cross-linker in deionized water. After vacuum degassing for 1 minute, add APS (1%, v / v) and TEMED (0.1%, v / v), mix well, and quickly drop the gel mixture solution onto the surface of the glass substrate prepared in step (2). Then cover the PDMS mold prepared in step (1) from one side of the gel solution to the other side. After 30 minutes, the gel mixture polymerizes, and slowly peel the mold from the surface of the hydrogel to form the first layer of PA hydrogel on the substrate surface.

[0098] Step (4): Prepare a hydrophobic cover glass;

[0099] Soak the cleaned glass cover glass in a 5% (v / v) toluene solution of dichlorodimethylsilane (DCDMS) for 1 - 3 minutes, and then rinse the excess DCDMS with ethanol and water in sequence. Obtain a DCDMS-treated hydrophobic cover glass.

[0100] Step (5): Prepare the second layer (top layer) of PNIPAM-NPG hydrogel.

[0101] Prepare a polymer solution containing N-isopropylacrylamide (5%, v / v), Bis-Acryamide cross-linker (0.03%, v / v), and NPG (0.5%, v / v) in deionized water. After vacuum degassing for 1 minute, add APS (2%, v / v) and TEMED (0.2%, v / v), mix well, and quickly drop the gel mixture solution onto the surface of the PA hydrogel prepared in step (3). Then cover the hydrophobic cover glass prepared in step (4) from one side of the gel solution to the other side. After 30 minutes, the gel mixture polymerizes, and slowly peel the cover glass from the surface of the hydrogel. Finally, obtain the PNIPAM-NPG / PA composite hydrogel, and then soak it in phosphate buffer solution (PBS, pH 7.4) for further use.

[0102] Wherein, Figures 3A to 3C shows the three-dimensional contour scan of PNIPAM-NPG / PA in the dynamically tunable curvature composite hydrogel substrate prepared according to the embodiments of the present disclosure. Wherein, Figure 3Ais the scanning area of the two-dimensional topography of the composite hydrogel, Figure 3B is the three-dimensional topography contour map of the composite hydrogel, Figure 3C is to quantitatively characterize the curvature height of the three-dimensional surface topography of the composite hydrogel.

[0103] Based on the above steps (1)-(5), the PNIPAM-NPG / PA composite hydrogel was obtained. In Example 2 of the present invention, it was used for anisotropically stretching cells, which specifically included the following steps:

[0104] Step (6): Coupling proteins on the hydrogel surface;

[0105] Wash the PNIPAM-NPG / PAAm composite hydrogel 3 times with PBS, add 200 μL of sulfo-SANPAH reagent, and irradiate it under an ultraviolet lamp for 30 min. Then wash it 3 times with 50 mM HEPES buffer at pH 8.5, incubate with rat tail type I collagen, and place it in a 4°C refrigerator overnight.

[0106] Step (7): Inoculating and stimulating cells

[0107] Aspirate the protein solution incubated in step (6), wash it 3 times with PBS, sterilize it under ultraviolet light for 30 min, and then inoculate MDCK cells (1×10 4 cells / cm 2 ). After incubating in a cell incubator for 2 h, place it under an infrared laser (400 mW, 0.1 HZ) for stimulation, and further observe the cell morphology.

[0108] So far, the present disclosure has been described in detail with reference to the accompanying drawings. Based on the above description, those skilled in the art should have a clear understanding of the present disclosure.

[0109] It should be noted that in the accompanying drawings or the text of the specification, the implementation manners that are not depicted or described are all forms known to those of ordinary skill in the art in the technical field to which the present invention belongs, and no detailed description is given. In addition, the above definitions of each element are not limited to the various specific structures, shapes or manners mentioned in the embodiments, and those of ordinary skill in the art can make simple changes or substitutions to them.

[0110] Of course, according to actual needs, the present disclosure may also include other parts, which are not described herein in detail because they have nothing to do with the innovative points of the present disclosure.

[0111] Similarly, it should be understood that, for the purpose of streamlining the present disclosure and facilitating the understanding of one or more of the various disclosed aspects, in the foregoing description of the exemplary embodiments of the present disclosure, various features of the present disclosure are sometimes grouped together into a single embodiment, figure, or description thereof. However, the disclosed method should not be construed as reflecting an intention that the claimed present disclosure requires more features than those expressly recited in each claim. Rather, as reflected in the following claims, the disclosed aspects lie in less than all the features of the single foregoing disclosed embodiment. Thus, the claims following the detailed description are hereby expressly incorporated into the detailed description, with each claim standing on its own as a separate embodiment of the present disclosure.

[0112] In addition, in the drawings or the description of the specification, similar or identical parts are denoted by the same reference numerals. The technical features in the various embodiments exemplified in the specification may be freely combined to form a new solution on the premise of no conflict. Additionally, each claim may be regarded as a separate embodiment by itself, or the technical features in each claim may be combined to form a new embodiment. Further, although exemplary values of parameters may be provided herein, it should be understood that the parameters need not exactly equal the corresponding values, but may approximate the corresponding values within acceptable error tolerances or design constraints.

[0113] Unless there are technical obstacles or contradictions, the above various embodiments of the present disclosure may be freely combined to form additional embodiments, and these additional embodiments are all within the protection scope of the present disclosure.

[0114] Although the present disclosure has been described in conjunction with the accompanying drawings, the embodiments disclosed in the drawings are intended to exemplarily illustrate the preferred embodiments of the present disclosure and should not be construed as a limitation to the present disclosure. The dimensional ratios in the drawings are merely illustrative and should not be construed as a limitation to the present disclosure.

[0115] Although some embodiments of the general inventive concept of the present disclosure have been shown and described, those of ordinary skill in the art will understand that changes may be made to these embodiments without departing from the principles and spirit of the general inventive concept, and the scope of the present disclosure is defined by the claims and their equivalents.

[0116] The above-described specific embodiments have further elaborated on the purpose, technical solutions, and beneficial effects of the present disclosure. It should be understood that the above are only specific embodiments of the present disclosure and are not used to limit the present disclosure. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present disclosure should be included within the protection scope of the present disclosure.

Claims

1. A preparation method of a dynamically adjustable curvature composite hydrogel substrate, characterized in that, the method prepares the composite hydrogel substrate by a stepwise polymerization method, specifically including: Step S1: Use a cylindrical lens template to mold a negative engraving mold of polydimethylsiloxane (PDMS) with a semi-circular stripe structure; Step S2: Use the polydimethylsiloxane negative engraving mold to prepare the first layer of polyacrylamide (PA) hydrogel layered substrate; Step S3: Use a polymerization solution of N-isopropylacrylamide (NIPAM) doped with nano-gold (NPG) to polymerize on the surface of the first layer of polyacrylamide hydrogel layered substrate to form the second layer of poly-N-isopropylacrylamide (PNIPAM) thermosensitive hydrogel, obtaining a PNIPAM-NPG / PA composite hydrogel substrate.

2. The preparation method of the dynamically adjustable curvature composite hydrogel substrate according to claim 1, characterized in that, in Step S1, the use of a cylindrical lens template to mold a negative engraving mold of polydimethylsiloxane (PDMS) with a semi-circular stripe structure includes: Mix a prepolymer A liquid and a curing agent B liquid in a volume ratio of 10:1 to prepare a polydimethylsiloxane (PDMS) prepolymer solution, mix it evenly with a rubber mixer and then drop it between a plasma-treated cover glass and a cylindrical lens template, and evacuate to remove air bubbles; Cover the surface of the cover glass with a weight of 200-400 g, put it into an oven for heating and curing, and then immerse it in anhydrous ethanol to peel the PDMS from the cylindrical lens template to obtain a negative engraving mold of polydimethylsiloxane (PDMS) with a semi-circular stripe structure.

3. The preparation method of the dynamically adjustable curvature composite hydrogel substrate according to claim 2, characterized in that, the cylindrical lens template is a bottom-layer periodic cylindrical array structure containing wavelengths of 170 μm and 340 μm, the heating temperature of the oven is 65-85 °C, and the heating time is 2-10 hours.

4. The preparation method of the dynamically adjustable curvature composite hydrogel substrate according to claim 2, characterized in that, in Step S2, the use of the polydimethylsiloxane negative engraving mold to prepare the first layer of polyacrylamide (PA) hydrogel layered substrate includes: Use ultrasound to clean the glass substrate in a methanol solution and dry it in a fume hood; then prepare a 4% (v / v) 3-aminopropyltriethoxysilane (APES) acetone solution, drop it on the surface of the glass substrate, dry it in a fume hood, rinse it with water, and fix it with a glutaraldehyde solution and then dry it to obtain a glass substrate for use; Add acrylamide monomer (AM) and methylene bisacrylamide (Bis-Acryamide) crosslinking agent to deionized water to prepare a polymer solution containing acrylamide and methylene bisacrylamide crosslinking agent. After vacuum degassing for 0.5-2 minutes, add ammonium persulfate (APS) and tetramethylethylenediamine (TEMED), mix evenly and quickly drop the gel mixture solution on the surface of the glass substrate for use; The polydimethylsiloxane (PDMS) negative-engraving mold is covered from one side of the gel solution to the other side. After 20-40 minutes, the gel mixture is polymerized, and the polydimethylsiloxane (PDMS) negative-engraving mold is peeled off from the hydrogel surface to form a first layer of PA hydrogel on the substrate surface, thereby obtaining a first layer of polyacrylamide (PA) hydrogel layered substrate.

5. The method for preparing the dynamically adjustable curvature composite hydrogel substrate according to claim 4, It is characterized in that In the step of fixing with glutaraldehyde solution and then drying to obtain a glass substrate to be used, the concentration of the glutaraldehyde solution is 0.05-2.0% (v / v) and the curing time is 15-40 minutes; In the polymer solution containing acrylamide and methylene bisacrylamide crosslinking agents, the concentration of acrylamide is 3-10% (v / v), and the concentration of methylene bisacrylamide is 0.02-0.4% (v / v); In the step of adding ammonium persulfate (APS) and tetramethylethylenediamine (TEMED), the concentration of APS is 1-3% (v / v), and the concentration of TEMED is 0.1-0.5% (v / v).

6. The method for preparing the dynamically adjustable curvature composite hydrogel substrate according to claim 4, It is characterized in that In step S3, the N-isopropylacrylamide (NIPAM) polymer solution doped with nanogold (NPG) is polymerized on the surface of the first layer of polyacrylamide hydrogel layered substrate to form a second layer of poly-N-isopropylacrylamide (PNIPAM) thermosensitive hydrogel to obtain a PNIPAM-NPG / PA composite hydrogel substrate, comprising: The cleaned glass coverslip was immersed in a 5% (v / v) dichlorodimethylsilane (DCDMS) toluene solution for 1-3 minutes, and then the excess DCDMS was washed with ethanol and water in sequence to obtain a hydrophobic coverslip treated with DCDMS; N-isopropylacrylamide, bisacrylamide (Bis-Acryamide) crosslinker and doped nano-gold (NPG) were added to deionized water to prepare a polymer solution containing N-isopropylacrylamide, bisacrylamide (Bis-Acryamide) crosslinker and doped nano-gold (NPG), and after vacuum degassing for 0.5-2 minutes, APS and TEMED were added, and after mixing, the gel mixture solution was quickly dropped onto the surface of the first layer of polyacrylamide (PA) hydrogel layered substrate; The hydrophobic cover glass treated with DCDMS was covered from one side to which the gel mixture solution was dropped to the other side. After 20-40 minutes, the gel mixture polymerized, and the hydrophobic cover glass was peeled off from the hydrogel surface to obtain a PNIPAM-NPG / PA composite hydrogel substrate.

7. The method for preparing the dynamically adjustable curvature composite hydrogel substrate according to claim 6, It is characterized in that In the polymer solution containing N-isopropylacrylamide, methylenebisacrylamide (Bis-Acrylamide) crosslinker, and doped nanogold (NPG), the concentration of N-isopropylacrylamide is 5-15% (v / v), the concentration of methylenebisacrylamide (Bis-Acrylamide) crosslinker is 0.02-1.0% (v / v), and the doped nanogold (NPG) is in powder form or aqueous nanogold solution with a concentration of 0.2-2%; In the step of adding APS and TEMED, the concentration of APS is 1-3% (v / v), and the concentration of TEMED is 0.1-0.5% (v / v).

8. The method for preparing a dynamically tunable curvature composite hydrogel substrate according to claim 6, characterized in that, after obtaining the PNIPAM-NPG / PA composite hydrogel substrate in step S3, it further includes: Soaking the PNIPAM-NPG / PA composite hydrogel substrate in phosphate buffer PBS with a pH value of 7.4 for standby.

9. Application of the dynamically tunable curvature composite hydrogel substrate prepared by the method according to any one of claims 1 to 8 in cell stimulation.

10. The application of the dynamically tunable curvature composite hydrogel substrate according to claim 9 in cell stimulation, characterized in that, including: Step 1: Coupling proteins on the hydrogel surface; Washing the PNIPAM-NPG / PA composite hydrogel 2-5 times with PBS, dropping 200 μL of sulfo-SANPAH reagent, irradiating under an ultraviolet lamp for 20-40 minutes, then washing 2-5 times with 50 mM HEPES buffer at pH 8.5, incubating with fibronectin or rat tail type I collagen, and placing it in a 4°C refrigerator overnight; Step 2: Inoculating and stimulating cells; Absorbing the protein solution incubated in step 1, washing 2-5 times with PBS, sterilizing with ultraviolet light for 20-40 minutes, inoculating cells, incubating in a cell culture incubator for 1-3 hours, then placing it under an infrared laser for stimulation, and further observing the cell morphology.

11. The application of the dynamically tunable curvature composite hydrogel substrate according to claim 10 in cell stimulation, characterized in that, The cell types inoculated in Step 2 are A549, MDCK, NIH / 3T3, MCF-7 or IEC-6, and the cell seeding density is 10 4 cells / cm 2 ; in step 2, the infrared laser has a wavelength of 808 nm, a power of 0-5 W, a frequency of 0.1-10 Hz, and a spot size of 0.5-20 mm.

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