A method for manufacturing a near-infrared light responsive polyaniline hydrogel film intelligent cell culture container

Abstract

The application provides a preparation method of a near-infrared light responsive polyaniline hydrogel film intelligent cell culture container, and the existing near-infrared light responsive cell culture container has the problems of complex preparation process, long cycle, high cost, low efficiency, poor biocompatibility and poor stability. The hydrophilic treatment of the cell culture container (substrate) can increase the number of hydroxyl groups on the surface, thereby increasing the binding force between the conjugated polymer hydrogel film and the cell culture container, and making the cell culture container more stable. First, a spin coater is used to spin coat a mixed solution uniformly stirred on the cell culture container to obtain a film, and then a one-step ultraviolet polymerization method is used to generate a substrate hydrogel film on the surface of the cell culture container. Then, aniline hydrochloride solution and ammonium persulfate prepared in advance are added dropwise on the substrate hydrogel film, and a polyaniline composite hydrogel film is obtained through aniline radical polymerization.

Description

Technical Field

[0001] The present invention belongs to the technical field of responsive cell culture containers, and in particular relates to a method for manufacturing a near-infrared light responsive polyaniline hydrogel film intelligent cell culture container. Background Art

[0002] Detaching adherent cells from the surfaces of cell culture plates, flasks, or dishes is one of the most fundamental and frequently performed experimental procedures in cell and tissue engineering. Currently, the most commonly used method for cell detachment is trypsin digestion. Trypsin is a type of protease that acts as a digestive enzyme in vertebrates. It hydrolyzes extracellular matrix (ECM) proteins between cells and between cells and the surface of the culture vessel, thereby separating cells. Therefore, in animal cell culture, trypsin can be used to disperse cells within tissues and detach adherent cells. However, in addition to digesting intercellular matrix proteins, prolonged exposure to trypsin can also degrade cell membrane proteins, potentially damaging cells. Therefore, when using trypsin to digest cells, strict control of parameters such as trypsin concentration, duration of exposure, temperature, and number of rinses is crucial. Trypsin solutions are most effective when used in a weakly alkaline solution at 37°C. However, even the slightest negligence can damage the integrity of cell membrane structure and function, leading to decreased cell activity and even cell death.

[0003] In response to the above problems, a variety of methods have been developed in the past few decades to regulate cell behavior, mainly including mechanical scraping, temperature control, pH control, ion control, magnetization control and light control. The light control method stands out among many separation technologies due to its outstanding advantages such as strong versatility, fast separation speed and good controllability. Among the light control methods, near-infrared light is more popular because it causes minimal damage to cells. The core of the near-infrared light control method is the design and construction of the photothermal surface, which refers to a surface embedded or coated with a photothermal agent such as precious metal nanomaterials, semiconductor nanomaterials, carbon-based nanomaterials and conjugated polymers. The surface can absorb light of appropriate wavelengths (usually in the near-infrared region) and convert light energy into thermal energy, allowing cell sheets to detach.

[0004] Since conjugated polymers absorb in the near-infrared region, when irradiated with a near-infrared laser, the electrons in the conjugated polymer will jump to a singlet excited state, and when the electrons fall back from the singlet excited state to the ground state, the energy will be released in the form of light and heat. At present, the main method for modifying conjugated polymers on the surface of cell culture plates is solution casting polymerization. The solution casting polymerization method mainly uses surface binding forces to adhere the conjugated polymer to the surface of the cell culture plate (see Xiao C, Liang W, Hasi QM, et al. Ag / polypyrrole co-modified poly (ionic liquid) shydrogels as efficient solar generators for desalination [J]. Materials Today Energy, 2020, 16: 100417.).

[0005] Although the above-mentioned methods can achieve controlled cell detachment, the existing methods still have the following shortcomings: the solution casting polymerization method also has strict requirements on the surface properties of the substrate. Generally, the substrate surface needs to be treated accordingly to generate some groups on the substrate surface to increase its surface binding force. Then, a thin film is directly polymerized on the surface. The film and the substrate are connected by hydrogen bonds, van der Waals forces, etc., so that the film and the substrate surface are tightly adhered together; however, the binding force is weak, and the modified cell culture plate has poor stability in use. These problems will not only increase the production cost of near-infrared light-responsive cell culture plates, but also greatly reduce their production efficiency.

[0006] Therefore, developing and designing a simple, inexpensive, and efficient method to produce near-infrared light-responsive cell culture containers with good stability is of great significance for promoting the market development of near-infrared light-responsive cell culture containers. Summary of the Invention

[0007] The purpose of the present invention is to solve the problems of complex manufacturing process, long cycle, high cost, low efficiency, poor biocompatibility and poor stability of existing near-infrared light-responsive cell culture containers, and to provide a method for manufacturing a near-infrared light-responsive polyaniline hydrogel film intelligent cell culture container.

[0008] To achieve the above objectives, the technical solutions provided by the present invention are:

[0009] The method for preparing a near-infrared light-responsive polyaniline hydrogel film is special in that it comprises the following steps:

[0010] 1) Dissolve sodium alginate in water and stir evenly at room temperature to obtain solution I;

[0011] Add acrylamide, acrylic acid, N,N-methylenebisacrylamide and ammonium persulfate to solution I and mix well to obtain solution II;

[0012] 2) pouring the solution II obtained in step 1) onto the substrate with a hydrophilic surface treatment and performing spin coating, and then irradiating the substrate with an ultraviolet lamp to obtain a composite hydrogel film (i.e., a base hydrogel film), wherein the composite hydrogel film has a uniform porous structure;

[0013] 3) Add aniline hydrochloride to water to obtain solution III;

[0014] Add ammonium persulfate to water to obtain solution VI;

[0015] In order to better control the amount of reaction materials used, solution III and solution VI are alternately added dropwise to the upper surface of the composite hydrogel film obtained in step 2) until the product polyaniline grows evenly on the composite hydrogel film (i.e., after sufficient amounts of solution III and solution VI are added to immerse the composite hydrogel film, the product polyaniline grows in situ in the pores of the composite hydrogel film and also forms a layer on its upper surface). After soaking in water to remove impurities, a polyaniline hydrogel film is obtained on the substrate.

[0016] Furthermore, in step 1), the mass volume ratio of sodium alginate to water is 1:20-40 g / mL, and stirring is carried out at room temperature for 30-60 min;

[0017] The mass ratio of sodium alginate, acrylamide, acrylic acid, N,N-methylenebisacrylamide and ammonium persulfate is 25-60:1000-3000:200-500:1-10:1-10.

[0018] Furthermore, step 2) is specifically as follows:

[0019] The solution II obtained in step 1) is poured onto the substrate with hydrophilic surface treatment, and is spin-coated at a speed of 500 to 3000 r / min for 1 to 5 minutes using a spin coater, and then the substrate is irradiated with an ultraviolet lamp for 5 to 20 minutes to obtain a composite hydrogel film.

[0020] Furthermore, in step 3), the mass volume ratio of the aniline hydrochloride to water is 0.1 to 0.5 g / ml;

[0021] The mass volume ratio of ammonium persulfate to water is 0.2-1 g / ml.

[0022] At the same time, the present invention also provides a near-infrared light-responsive polyaniline hydrogel film, which is special in that it is prepared by the above method.

[0023] Also, the application of the near-infrared light-responsive polyaniline hydrogel film prepared by the above method as a photothermal surface in the preparation of a near-infrared light-responsive intelligent cell culture container.

[0024] In addition, the present invention provides a method for preparing a near-infrared light-responsive intelligent cell culture container, the special feature of which is that a near-infrared light-responsive polyaniline hydrogel film is prepared according to the above method on a cell culture container with a hydrophilic surface treatment to obtain a near-infrared light-responsive intelligent cell culture container; that is, the cell culture container is directly used as a substrate, and a near-infrared light-responsive polyaniline hydrogel film is modified thereon and used as a photothermal surface, which is conducive to cell detachment and transfer.

[0025] Furthermore, the cell culture container is a cell culture plate, a cell culture flask or a cell culture dish. The material of the cell culture container can be any material, for example, commercial cell culture containers made of existing glass, polystyrene, polypropylene, polyvinyl chloride, polyethylene and the like are applicable.

[0026] Also, a near-infrared light-responsive intelligent cell culture container obtained by the above preparation method, and its application in cell sheet desorption and separation.

[0027] Principle of the present invention:

[0028] The present invention performs hydrophilic treatment on a cell culture container (substrate), which increases the hydroxyl groups on the surface, thereby increasing the binding force between the conjugated polymer hydrogel film and the cell culture container and making it more stable. First, a spin coater is used to spin-coat the stirred mixed solution on the cell culture container to obtain a film, and then a one-step ultraviolet polymerization method is used to generate a base hydrogel film on the surface of the cell culture container; thereafter, a configured aniline hydrochloride solution and ammonium persulfate are successively added dropwise to the base hydrogel film, and a polyaniline composite hydrogel film is obtained by aniline free radical polymerization. Although pure PANI hydrogel has a good near-infrared response effect, its rigid structure is fragile and has poor stability, while the synthesized composite hydrogel has excellent mechanical properties, which enhances the stability of the material. In addition, the uniform porous structure of the base gel allows polyaniline to be uniformly filled therein, ensuring that the composite gel can uniformly absorb near-infrared light. Cell sheets are cultured on the polyaniline composite hydrogel film, which can increase the temperature of the cell sheet by absorbing near-infrared light. When a certain temperature is reached, the cell sheet detaches from the composite hydrogel film, achieving the effect of cell sheet separation.

[0029] Advantages of the present invention:

[0030] 1. The present invention utilizes a one-step UV polymerization method and an in-situ polymerization method to prepare a polyaniline, sodium alginate, and acrylamide composite conductive hydrogel. Under near-infrared light irradiation, the composite conductive hydrogel rapidly reaches equilibrium temperature. Its excellent anti-swelling properties and near-100% photothermal conversion efficiency demonstrate significant potential in near-infrared light response. This demonstrates the high photothermal conversion efficiency and rapid thermal equilibrium achieved by the polyaniline film prepared by the present invention. This is primarily due to the uniform pore structure of the base hydrogel film synthesized using the present method. The polyaniline synthesized using the in-situ polymerization method uniformly fills and adheres to the pores, enabling uniform absorption of near-infrared light.

[0031] 2. The polyaniline hydrogel prepared by the present invention has good biocompatibility. Polyacrylamide and sodium alginate have excellent biocompatibility. By mixing polyacrylamide, sodium alginate and polyaniline together, the biocompatibility of the entire photothermal surface can be greatly improved.

[0032] 3. The present invention uses oxygen plasma treatment to perform hydrophilic treatment on the cell culture container (substrate), so that the base hydrogel film is bonded to the surface of the substrate through more hydrogen bonds (chemical bonds), which has good stability and is more solid.

[0033] 4. The method of the present invention is used to modify the near-infrared light-responsive polyaniline hydrogel film on the cell culture container. The process steps are simple, the cycle is short, the efficiency is high, the raw materials for preparation are widely available, and the cost is lower than that of the existing method. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is a scanning electron micrograph of the base hydrogel prepared in Example 1;

[0035] Figure 2 This is a scanning electron microscope image of the substrate hydrogel composited with polyaniline prepared in Example 1;

[0036] Figure 3 is an optical image of the polyaniline hydrogel film cell culture dish prepared in Example 1;

[0037] Figure 4 is an optical microscope image of human chronic myeloid leukemia cells cultured using a polyaniline hydrogel film cell culture dish in Example 1;

[0038] Figure 5 is the desorption efficiency of the polyaniline hydrogel film cell culture dish in Example 1 in response to the photothermal reaction of human chronic myeloid leukemia cells;

[0039] Figure 6 is the desorption efficiency of the polyaniline hydrogel film cell culture dish in Example 1 in response to the photothermal response of human chronic myeloid leukemia cells after the second reuse;

[0040] Figure 7 These are the results of cell activity testing after culturing cells for 24 hours and 72 hours using the polyaniline hydrogel film cell culture dish in Example 1. DETAILED DESCRIPTION

[0041] The present invention will be described in further detail below with reference to the accompanying drawings and specific embodiments:

[0042] Unless otherwise specified, the present invention has no special requirements on the sources of the raw materials used, and commercially available products known to those skilled in the art can be used.

[0043] The present invention provides a method for manufacturing a near-infrared light-responsive polyaniline hydrogel film-based intelligent cell culture container, comprising the following steps:

[0044] 1) Dissolve sodium alginate in deionized water and stir at room temperature for 30-60 minutes to obtain Solution I;

[0045] Adding acrylamide, acrylic acid, N,N-methylenebisacrylamide and ammonium persulfate to solution I and mixing them evenly to obtain solution II;

[0046] The mass ratio of sodium alginate, acrylamide, acrylic acid, N,N-methylenebisacrylamide and ammonium persulfate is 25-60:1000-3000:200-500:1-10:1-10;

[0047] 2) pouring the solution II obtained in step 1) into a cell culture container with a hydrophilic surface treatment, and spin-coating the container at a speed of 500 to 3000 rpm for 1 to 5 minutes using a spin coater, and then irradiating the container under ultraviolet light for 5 to 20 minutes to obtain a composite hydrogel film;

[0048] 3) adding aniline hydrochloride to deionized water to obtain solution III; the mass volume ratio of aniline hydrochloride to deionized water is 0.1 to 0.5 g / ml;

[0049] Ammonium persulfate is added to deionized water to obtain solution VI; the mass volume ratio of ammonium persulfate to deionized water is 0.2 to 1 g / ml;

[0050] Solution III and solution VI are alternately added dropwise onto the upper surface of the composite hydrogel film obtained in step 2) until polyaniline is evenly coated on the surface. Finally, the container is immersed in deionized water to remove impurities, thereby obtaining a polyaniline hydrogel film-based cell culture container.

[0051] The above process is used to make a near-infrared light-responsive intelligent cell culture container. The process parameters of the specific example are as follows:

[0052] Example 1

[0053] 1) Dissolve 2 g of sodium alginate in 80 mL of deionized water and stir at room temperature for 45 min to obtain Solution I.

[0054] Take 10 mL of solution I, add 1.0 g acrylamide, 0.2 g acrylic acid, 0.0072 g N,N-methylenebisacrylamide, and 0.0060 g ammonium persulfate, and mix well to obtain solution II;

[0055] 2) Pour solution II obtained in step 1) into a cell culture container with a hydrophilic surface treatment, and spin coat it at a speed of 1000 r / min for 3 minutes using a spin coater, and then irradiate it under ultraviolet light for 10 minutes to obtain a composite hydrogel film, such as Figure 1 The uniform porous structure shown;

[0056] 3) Add 3 g of aniline hydrochloride to 10 mL of deionized water to obtain solution III;

[0057] Add 5 g of ammonium persulfate to 10 mL of deionized water to obtain solution VI;

[0058] Solution III and solution VI are alternately added dropwise onto the upper surface of the composite hydrogel film obtained in step 2) until polyaniline is evenly coated on the surface. The obtained hydrogel film is immersed in deionized water to remove any impurities to obtain a polyaniline hydrogel film-based cell culture container, the surface of which is as follows: Figure 2 shown.

[0059] Example 2

[0060] 1) Dissolve 4 g of sodium alginate in 80 mL of deionized water and stir at room temperature for 60 min to obtain Solution I.

[0061] Take 10 mL of solution I, add 2.0 g of acrylamide, 0.35 g of acrylic acid, 0.0144 g of N,N-methylenebisacrylamide, and 0.0120 g of ammonium persulfate, and mix well to obtain solution II;

[0062] 2) pouring the solution II obtained in step 1) into a cell culture container with a hydrophilic surface treatment, and spin coating it at a speed of 500 rpm for 1 minute using a spin coater, and then irradiating it under ultraviolet light for 20 minutes to obtain a composite hydrogel film;

[0063] 3) Add 5 g of aniline hydrochloride to 10 mL of deionized water to obtain solution III;

[0064] Add 10 g of ammonium persulfate to 10 mL of deionized water to obtain solution VI;

[0065] Solution III and solution VI are alternately added dropwise onto the upper surface of the composite hydrogel film obtained in step 2) until polyaniline is evenly coated on its surface. The obtained hydrogel film is immersed in deionized water to remove any impurities to obtain a polyaniline hydrogel film-based cell culture container.

[0066] Example 3

[0067] 1) Dissolve 3 g of sodium alginate in 80 mL of deionized water and stir at room temperature for 50 min to obtain Solution I.

[0068] Take 10 mL of solution I, add 1.5 g of acrylamide, 0.25 g of acrylic acid, 0.0108 g of N,N-methylenebisacrylamide, and 0.0090 g of ammonium persulfate, and mix well to obtain solution II;

[0069] 2) pouring the solution II obtained in step 1) into a cell culture container with a hydrophilic surface treatment, and spin coating it at 3000 rpm for 1 minute using a spin coater, and then irradiating it under ultraviolet light for 15 minutes to obtain a composite hydrogel film;

[0070] 3) Add 4 g of aniline hydrochloride to 10 mL of deionized water to obtain Solution III;

[0071] Add 7 g of ammonium persulfate to 10 mL of deionized water to obtain solution VI;

[0072] Solution III and solution VI are alternately added dropwise onto the upper surface of the composite hydrogel film obtained in step 2) until polyaniline is evenly coated on its surface. The obtained hydrogel film is immersed in deionized water to remove any impurities to obtain a polyaniline hydrogel film-based cell culture container.

[0073] Example 4

[0074] 1) Dissolve 2.5 g of sodium alginate in 80 mL of deionized water and stir at room temperature for 60 min to obtain Solution I.

[0075] Take 10 mL of solution I, add 1.8 g of acrylamide, 0.3 g of acrylic acid, 0.0092 g of N,N-methylenebisacrylamide, and 0.0080 g of ammonium persulfate, and mix well to obtain solution II;

[0076] 2) Pour solution II obtained in step 1) into a cell culture container with a hydrophilic surface treatment, and spin-coat the container at a speed of 1500 rpm for 3 minutes using a spin coater, and then irradiate the container under ultraviolet light for 10 minutes to obtain a composite hydrogel film;

[0077] 3) Add 3 g of aniline hydrochloride to 10 mL of deionized water to obtain solution III;

[0078] Add 5 g of ammonium persulfate to 10 mL of deionized water to obtain solution VI;

[0079] Solution III and solution VI are alternately added dropwise onto the upper surface of the composite hydrogel film obtained in step 2) until polyaniline is evenly coated on its surface. The obtained hydrogel film is immersed in deionized water to remove any impurities to obtain a polyaniline hydrogel film-based cell culture container.

[0080] Example 5

[0081] 1) Dissolve 3.5 g of sodium alginate in 80 mL of deionized water and stir at room temperature for 60 min to obtain Solution I.

[0082] Take 10 mL of solution I, add 1.6 g of acrylamide, 0.25 g of acrylic acid, 0.0120 g of N,N-methylenebisacrylamide, and 0.0085 g of ammonium persulfate, and mix well to obtain solution II;

[0083] 2) Pour solution II obtained in step 1) into a cell culture container with a hydrophilic surface treatment, and spin-coat the container at 2000 rpm for 2 minutes using a spin coater, followed by irradiation under ultraviolet light for 15 minutes to obtain a composite hydrogel film;

[0084] 3) Add 4 g of aniline hydrochloride to 10 mL of deionized water to obtain Solution III;

[0085] Add 6 g of ammonium persulfate to 10 mL of deionized water to obtain solution VI;

[0086] Solution III and solution VI are alternately added dropwise onto the upper surface of the composite hydrogel film obtained in step 2) until polyaniline is evenly coated on its surface. The obtained hydrogel film is immersed in deionized water to remove any impurities to obtain a polyaniline hydrogel film-based cell culture container.

[0087] Figure 3 The optical image of the polyaniline hydrogel film-based cell culture dish (i.e., near-infrared light-responsive smart cell culture dish) produced in Example 1 clearly shows that there is an opaque film at the bottom of the cell culture dish. Figure 3 Human chronic myeloid leukemia cells were cultured on the cell culture dish shown to obtain a cell sheet, such as Figure 4 As shown, it can be determined that the polyaniline hydrogel film-based cell culture dish prepared in the present invention can successfully carry out cell culture.

[0088] In order to verify the near-infrared light response ability and stability (reusability) of the polyaniline hydrogel film-based cell culture dish prepared by the present invention, the present invention also uses near-infrared light to irradiate the culture dish to perform a desorption test, and the desorption efficiency is as follows: Figure 5As shown in the figure, it can be seen that the polyaniline hydrogel film-based cell culture dish prepared by the present invention has a good near-infrared light response ability, converts light energy into heat energy, and desorbs the cell sheet; then the culture dish with the desorbed cell sheet is washed and the cell sheet is continued to be cultured on it and the desorption process is carried out. The desorption efficiency of the second desorption cell sheet is shown in FIG. Figure 6 , it can be seen that it can still maintain a high desorption capacity after repeated use, and has good stability. In addition, the biocompatibility of the polyaniline hydrogel film-based cell culture dish prepared by the present invention was tested, and the cell activity was tested for 24h and 72h. The test results are as follows Figure 7 As shown, it can be seen that the cell culture dish produced by the present invention has good biocompatibility.

[0089] In summary, the cell culture dish produced by the present invention can respond intelligently under near-infrared light irradiation, has a simple production process, low cost, high efficiency, and good biocompatibility and stability.

[0090] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with this technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed in the present invention, and these modifications or replacements should all be included in the scope of protection of the present invention.

Claims

1. A method for manufacturing a near-infrared light-responsive intelligent cell culture container, characterized by: A near-infrared light-responsive polyaniline hydrogel film is prepared on a cell culture container with a hydrophilic surface treatment to obtain a near-infrared light-responsive smart cell culture container; The near-infrared light-responsive polyaniline hydrogel film is prepared according to the following method: 1) Dissolve sodium alginate in water and stir evenly at room temperature to obtain solution I; Add acrylamide, acrylic acid, N,N-methylenebisacrylamide and ammonium persulfate to solution I and mix well to obtain solution II; 2) pouring solution II obtained in step 1) onto a cell culture container with a hydrophilic surface treatment and performing spin coating, and then irradiating the cell culture container with an ultraviolet lamp to obtain a composite hydrogel film; 3) Add aniline hydrochloride to water to obtain solution III; Add ammonium persulfate to water to obtain solution VI; Solution III and solution VI are alternately added dropwise to the upper surface of the composite hydrogel film obtained in step 2) until polyaniline grows evenly on the composite hydrogel film. After soaking in water to remove impurities, a polyaniline hydrogel film is obtained on the cell culture container.

2. The production method according to claim 1, characterized in that: In step 1), the mass volume ratio of sodium alginate to water is 1:20-40 g / mL, and the mixture is stirred at room temperature for 30-60 minutes to obtain solution I; The mass ratio of sodium alginate, acrylamide, acrylic acid, N,N-methylenebisacrylamide and ammonium persulfate is 25-60:1000-3000:200-500:1-10:1-10.

3. The production method according to claim 1 or 2, characterized in that: Step 2) is as follows: Solution II obtained in step 1) is poured onto a cell culture container with a hydrophilic surface treatment, and spin-coated at a speed of 500-3000 r / min for 1-5 minutes using a spin coater. The cell culture container is then irradiated with an ultraviolet lamp for 5-20 minutes to obtain a composite hydrogel film.

4. The production method according to claim 3, characterized in that: In step 3), the mass volume ratio of the aniline hydrochloride to water is 0.1-0.5 g / ml; The mass volume ratio of ammonium persulfate to water is 0.2~1g / ml.

5. A near-infrared light-responsive intelligent cell culture container, characterized in that: The method is obtained by any one of claims 1 to 4.

6. Use of the near-infrared light-responsive intelligent cell culture container according to claim 5 in the desorption and separation of cell sheets.

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