Micro-patterned hydrogel film as well as preparation method and application thereof

Abstract

The invention provides a micro-patterned hydrogel film and a preparation method thereof, and belongs to the field of polymer functional materials. The preparation method comprises the following steps: dispersing sea squirt cellulose in an acid solution for hydrolysis reaction, then diluting and performing centrifugal treatment to take supernate, so as to obtain sea squirt cellulose nanocrystal suspension; adding methacrylic acid esterified gelatin and a photoinitiator into the sea squirt cellulose nanocrystal suspension, and mixing to obtain a pre-polymerized solution; and injecting the pre-polymerized liquid into a mold, placing a photomask with a pattern right above the mold, and carrying out photo-initiation polymerization under ultraviolet light to obtain the micro-patterned hydrogel film. The preparation method provided by the invention is simple in process and wide in raw material source, the prepared micro-patterned hydrogel film has good mechanical property, biocompatibility and biodegradability, clear and fine micro-patterns can be formed on the surface of the micro-patterned hydrogel film, and the micro-patterned hydrogel film has good application prospects in the fields of biomedicine, tissue engineering, flexible electronics and the like.

Description

Technical Field

[0001] The present invention belongs to the fields of chemical engineering and polymer functional materials, and particularly relates to a micropatterned hydrogel film, a preparation method thereof, and an application thereof. Background Art

[0002] Hydrogel is a kind of polymer functional material with a three-dimensional network structure, which can absorb and retain a large amount of water and has broad application prospects in the fields of biomedicine, drug carriers, tissue engineering, sensors, flexible electronics, etc. Hydrogel micropatterns are material patterns presenting complex structures and shapes at the micron scale. In recent years, with the rapid development of micro-nano processing technologies, micropatterned hydrogel films have attracted extensive attention from researchers due to their unique physical and chemical properties and potential application values.

[0003] Currently, the preparation methods of micropatterned hydrogel films mainly include template method, photolithography, 3D printing method, microcontact printing method, etc. Although these methods can prepare various patterned surfaces, such as groove-ridge structures, nanopores, nanorods, and other topological structures, there are still some limitations. For example, the complexity of high-precision manufacturing processes, the compatibility of materials and structures, high costs, poor scalability, limited pattern accuracy and resolution, and poor mechanical properties of some hydrogels. These factors all limit their wide use in practical applications.

[0004] Therefore, developing a preparation method for micropatterned hydrogel films with simple preparation processes, high pattern accuracy, excellent biocompatibility, and good mechanical properties is still an urgent technical challenge to be solved. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a preparation method for a micropatterned hydrogel film to solve the problems of complex processes, high costs, limited pattern accuracy, etc. existing in the prior art.

[0006] Another purpose of the present invention is to provide a micropatterned hydrogel film with high surface micropattern accuracy, simple preparation process, low cost, good biocompatibility, and good mechanical properties.

[0007] The third purpose of the present invention is to provide an application of the micropatterned hydrogel film.

[0008] The technical solution adopted by the present invention to achieve the first purpose is: to provide a preparation method for a micropatterned hydrogel film, including the following steps: S1. Dispersing ascidian cellulose in an acidic solution for hydrolysis reaction, then diluting and centrifuging to take the supernatant to obtain an ascidian cellulose nanocrystal suspension; S2. Add methacrylated gelatin and an initiator into the suspension of ascidian cellulose nanocrystals, and mix to obtain a prepolymer solution; in the prepolymer solution, the content of methacrylated gelatin is 5 wt.% - 10 wt.%. S3. Inject the prepolymer solution into a mold, place a photomask with a pattern directly above the mold, and perform photoinitiated polymerization under ultraviolet light for 0.5 - 3 min to obtain a micro-patterned hydrogel film.

[0009] The general idea and invention principle of the present invention are as follows: In order to improve the pattern accuracy of the micro-patterned hydrogel film and achieve the preparation of micron-scale patterns, the preparation method provided by the present invention mainly improves the preparation method of the hydrogel film from the following aspects: First, in the selection of the base material of the hydrogel, on the one hand, the present invention uses methacrylated gelatin, which has the advantage of rapid polymerization under ultraviolet light, facilitating patterning using a photomask; on the other hand, ascidian cellulose nanocrystals are added, which have high crystallinity and high strength rigidity, can reduce the swelling degree of the hydrogel, and can better maintain the micro-patterns on the hydrogel surface. In addition, ascidian cellulose nanocrystals also have a higher aspect ratio, high Young's modulus and high strength rigidity. Compared with raw materials such as cotton linter cellulose nanocrystals and bacterial cellulose nanocrystals, they can better enhance the mechanical properties of the micro-patterned hydrogel, further solving the problems of high swelling degree and low mechanical strength of the hydrogel prepared from single methacrylated gelatin.

[0010] Secondly, during the preparation process of the prepolymer solution, control the addition amount of methacrylated gelatin to be 5 wt.% - 10 wt.%. Through research, it is found that when the addition amount of methacrylated gelatin is less than 5 wt.%, the mechanical properties of the prepared hydrogel are weak, the swelling degree is too large, and it is impossible to form surface micron-scale groove patterns; while when the addition amount of methacrylated gelatin is higher than 10 wt.%, the overall viscosity of the prepolymer solution becomes larger, which is not conducive to injection into the mold, and the hydrogel prepared from high-concentration methacrylated gelatin will be harder, which is not conducive to better application.

[0011] Finally, during the polymerization reaction process, control the ultraviolet light-initiated polymerization time of the hydrogel to be 0.5 - 3 min, so as to ensure the formation of high-precision, high-resolution, regular and clear groove patterns. Through research, it is found that a shorter polymerization time will result in a lower degree of polymerization of the hydrogel, the internal network is not formed, and it is difficult to form patterns on the surface; on the contrary, when the polymerization time is longer, the crosslinking density of the hydrogel is too high, which will achieve complete internal polymerization, resulting in the photomask on the surface losing its function and it is difficult to form the expected patterns.

[0012] Based on the above-mentioned improvements in multiple aspects, the present invention solves the problems existing in the prior art, such as limited pattern accuracy and resolution, and poor mechanical properties of hydrogels.

[0013] Further, in step S1, the temperature of the hydrolysis reaction is 55 - 65°C, and the time is 5 - 7 h. The temperature of the above hydrolysis reaction is relatively mild, which helps to reduce the occurrence of side reactions. At the same time, the hydrolysis time of 5 - 7 h can also ensure the full progress of the reaction. Controlling the temperature and time of the hydrolysis reaction helps to improve the quality and yield of the product.

[0014] In some preferred embodiments, the preparation method of the ascidian cellulose nanocrystal suspension comprises the following steps: Mix ascidian cellulose with a sulfuric acid solution with a concentration of 65 wt% according to a mass - to - volume ratio of 1:100 g / mL, and hydrolyze with stirring at 55 - 65°C for 5 - 7 h. Dilute the reacted mixture with deionized water by 8 - 12 times to terminate the reaction, and take the supernatant by centrifugation at 8000 - 12000 rpm to obtain the ascidian cellulose nanocrystal suspension. Dialyze the suspension with deionized water until a constant pH value is reached for use.

[0015] Further, in step S2, the methacrylated gelatin (GelMA) is prepared by the esterification reaction of gelatin and methacrylic anhydride.

[0016] In some preferred embodiments, the preparation method of methacrylated gelatin comprises the following steps: In a water - bath environment at 50 - 60°C, dissolve gelatin according to a mass - to - volume ratio of 1:10 g / mL in a 0.25 M buffer solution, which is composed of a sodium carbonate solution and a sodium bicarbonate solution. After it is completely dissolved, adjust the pH to 9 - 10. According to the dosage ratio of gelatin to methacrylic anhydride of 100:9 g / mL, drop - wise add methacrylic anhydride to the solution and react for 1 - 2 h. Finally, adjust the pH to 7 - 8 to terminate the reaction. Load the reaction solution into a dialysis bag with a molecular weight of 8000 - 14000 and dialyze it in deionized water at 30 - 40°C for 5 - 7 days, changing the deionized water every 3 - 4 h. Finally, freeze - dry the purified solution to obtain a white porous foam, namely methacrylated gelatin, and store it in a - 40°C refrigerator.

[0017] In the above - mentioned preparation method, by adjusting the pH to 9 - 10 for the reaction, it is beneficial for methacrylic anhydride to react with the amino group of gelatin, improving the reaction efficiency and the degree of methacrylation of the product. And finally, adjusting the pH to 7 - 8 to terminate the reaction can effectively control the end - point of the reaction and avoid the generation of by - products due to over - reaction. The degree of substitution of the methacrylated gelatin prepared by the above method is close to 100%.

[0018] Further, in step S2, in the prepolymer solution, the content of ascidian cellulose nanocrystals is 0.2 wt.% - 1 wt.%, and the content of the initiator is 0.5 wt.% - 1 wt.%. Among them, the appropriate addition of ascidian cellulose nanocrystals can enhance the mechanical properties of the material. However, if the addition amount is too large, the ascidian cellulose nanocrystals are prone to aggregation, which will instead make the material brittle and is not conducive to the improvement of mechanical properties. Controlling the content of the photoinitiator within a suitable range is beneficial for the prepolymer solution to quickly polymerize to form a patterned hydrogel. If the content of the photoinitiator is too low, the polymerization process is slow, and it is difficult to prepare a hydrogel with high-precision and high-resolution patterns on the surface.

[0019] Further, in step S2, the photoinitiator is selected from one or a combination of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959), lithium phenyl(2,4,6-trimethylbenzoyl)phosphate (LAP), 2-hydroxy-2-methyl-1-phenyl-1-propanone (HMPP).

[0020] Further, in step S2, the prepolymer solution is obtained by mixing in a stirring manner, the mixing temperature is 50 °C, and the mixing time is 20 - 30 min; in step S3, the prepolymer solution is centrifuged to remove air bubbles and then injected into the mold.

[0021] Further, in step S3, the mold is composed of two layers of glass slides and a silicone gasket placed between the two layers of glass slides, and the depth of the mold is 0.1 - 0.5 mm. Among them, the glass slides have high light transmittance, which is convenient for the ultraviolet light to pass through, and the thickness and size of the patterned hydrogel are changed by adjusting the size of the silicone gasket.

[0022] Further, in step S3, the pattern of the photomask includes black and white stripes with different widths. The ultraviolet light transmittance intensity in the white stripe area is relatively high, and the ultraviolet light transmittance intensity in the black stripe area is relatively low. An ultraviolet light intensity difference will be formed between the two areas. The degree of polymerization of the gel under the white area is relatively high, and the degree of polymerization of the gel under the black area is relatively low, forming a micro-groove structure, so that a micron-scale groove pattern with a height difference is formed on the surface of the prepared hydrogel. Preferably, the width of the black stripe is 10 - 60 μm, and the width of the white stripe is 10 - 20 μm. In addition, the pattern of the photomask can also be a triangle, a square, a pentagram, etc., which can be customized according to specific requirements.

[0023] Further, in step S3, the wavelength of the ultraviolet light is 300 - 400 nm.

[0024] The second technical solution adopted to achieve the second object of the present invention is: to provide a micro-patterned hydrogel film, which is prepared by the preparation method according to the first object of the present invention. The pattern on the surface of the micro-patterned hydrogel film has a micro-groove structure, the groove width is 10 - 60 μm, the groove depth is 0.6 - 1.5 μm, and the ridge width is 10 - 20 μm.

[0025] The micro-patterned hydrogel film prepared by the present invention has good mechanical properties, with an elastic modulus of 50 - 56.5 kPa and a toughness of 263.3 - 320.5 kJ / m 3 。

[0026] The third technical solution adopted to achieve the third object of the present invention is: to provide an application of the micro-patterned hydrogel film according to the second object of the present invention in the fields of tissue engineering and regenerative medicine, bioimaging and diagnosis, or flexible electronics.

[0027] Furthermore, the micro-patterned hydrogel film provided by the present invention is applied to tissue engineering. The patterned hydrogel can be used to construct a cell scaffold with a specific structure. The present invention forms a groove structure on the surface of the hydrogel through a photomask technology, which can promote cell adhesion, proliferation, and differentiation. High-precision patterning can achieve precise regulation of the cell microenvironment, and the enhanced mechanical properties ensure the stability of the scaffold under physiological conditions.

[0028] Furthermore, the micro-patterned hydrogel film provided by the present invention is applied to cell imaging. By precisely controlling the shape and size of the pattern, a specific growth environment is provided for cells, thereby achieving more efficient imaging and analysis.

[0029] Furthermore, the micro-patterned hydrogel film provided by the present invention is applied to flexible electronic devices, such as wearable sensors, electronic skin, etc. The patterned hydrogel can be used as a flexible substrate or a functional layer. Its high-precision patterning ability can achieve complex circuit designs, and the enhanced mechanical properties ensure the stability of the device under deformation conditions such as bending and stretching.

[0030] Compared with the prior art, the beneficial effects of the present invention are: (1) Wide source of raw materials: The cellulose nanocrystals from ascidians and methacrylated gelatin used in the present invention are both natural biomaterials, having good biodegradability and biocompatibility. Methacrylated gelatin has the advantage of rapid photoinitiated polymerization, and at the same time, the addition of cellulose nanocrystals from ascidians further reduces the swelling degree of the hydrogel and improves the mechanical properties, solving the problem that in some existing studies, due to the slow photoinitiated polymerization of some substrate materials or the excessive swelling degree of the prepared hydrogel, it is difficult to maintain the micron-scale pattern on the surface.

[0031] (2) Simple process and low cost: The preparation process of the present invention does not require complex equipment and cumbersome steps, and is suitable for large-scale production and application. The present invention selects a photomask that can be mass-produced and can customize different patterns as the patterning mold, and the selected substrate materials, methacrylated gelatin and ascidian cellulose nanocrystals, are both natural biological materials, which are widely sourced and low in cost, solving the problems of high cost, low efficiency, and insufficient scalability in existing high-precision patterning technologies.

[0032] (3) High pattern accuracy: The present invention can achieve high-precision micropatterning through photomask and ultraviolet light-induced polymerization technology, meeting the requirements of different application scenarios.

[0033] (4) Broad application prospects: The micropatterned hydrogel film prepared by the present invention has broad application potential in the fields of biomedicine, tissue engineering, and flexible electronics. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is a schematic diagram of a method for preparing a micropatterned hydrogel film provided by the present invention; Figure 2 are optical microscope images corresponding to the micropatterned hydrogel films prepared in Examples 1-4 of the present invention; in the figures, (a), (b), (c), and (d) correspond to Examples 1, 2, 3, and 4 respectively; Figure 3 are 3D optical profilometry images and their surface profile curves corresponding to the patterned hydrogel films prepared in Examples 1-4 of the present invention; in the figures, (a), (b), (c), and (d) correspond to Examples 1, 2, 3, and 4 respectively; Figure 4 are scanning electron microscope surface images corresponding to the micropatterned hydrogel films prepared in Examples 1-4 of the present invention; in the figures, (a), (b), (c), and (d) correspond to Examples 1, 2, 3, and 4 respectively; Figure 5 is a statistical graph of the groove width and interval width corresponding to the micropatterned hydrogel films prepared in Examples 1-4 of the present invention; Figure 6 are mechanical property characterization diagrams corresponding to the micropatterned hydrogel films prepared in Examples 1-4 of the present invention; among them, (a) is the tensile stress-strain curve diagram corresponding to the micropatterned hydrogel films in Examples 1-4; (b) is the elastic modulus and toughness corresponding to the micropatterned hydrogel films in Examples 1-4; Figure 7 are the optical microscope image (a) and scanning electron microscope surface image (b) corresponding to the micropatterned hydrogel film prepared in the comparative example of the present invention; Figure 8The figure shows the tensile stress-strain curve corresponding to the micro-patterned hydrogel film prepared as a comparative example of the present invention. Detailed implementation manners

[0035] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the protection scope of the present invention.

[0036] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0037] The embodiments of the present invention provide a method for preparing a micro-patterned hydrogel film, which includes the following steps: Step 1: Dissolve ascidian cellulose in an acidic solution for hydrolysis reaction, then dilute and centrifuge to obtain the supernatant, and obtain an ascidian cellulose nanocrystal suspension; wherein, the temperature of the hydrolysis reaction is 55-60 °C, and the time is 5-7 h.

[0038] The method for preparing the ascidian cellulose nanocrystal suspension includes the following steps: Mix ascidian cellulose with a sulfuric acid solution with a concentration of 65 wt% according to a mass-volume ratio of 1:100 g / mL, and hydrolyze with stirring at 55-65 °C for 5-7 h. Dilute the reacted mixture 8-12 times with deionized water to terminate the reaction, and obtain the supernatant by centrifugation at 8000-12000 rpm to obtain an ascidian cellulose nanocrystal suspension. Dialyze this suspension with deionized water until the pH value is constant for use.

[0039] Step 2: Add methacrylated gelatin and an initiator to the ascidian cellulose nanocrystal suspension to obtain a prepolymer solution; in the prepolymer solution, the content of methacrylated gelatin is 5 wt% - 10 wt%, the content of ascidian cellulose nanocrystals is 0.2 wt% - 1 wt%, and the content of the initiator is 0.5 wt% - 1 wt%; the photoinitiator is selected from one or a combination of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, 2-hydroxy-2-methyl-1-phenyl-1-propanone; The prepolymer solution is obtained by mixing with stirring, the mixing temperature is 50 °C, and the mixing time is 20-30 min.

[0040] The preparation method of the methacrylated gelatin comprises the following steps: in a water bath environment at 50-60 °C, dissolve gelatin in a 0.25 M sodium carbonate / sodium bicarbonate buffer solution according to a mass-volume ratio of 1:10 g / mL. After it is completely dissolved, adjust the pH to 9-10. According to the dosage ratio of gelatin to methacrylic anhydride of 100:9 g / mL, dropwise add methacrylic anhydride to the solution and react for 1-2 h. Finally, adjust the pH to 7-8 to terminate the reaction. Load the reaction solution into a dialysis bag with a molecular weight of 8000-14000 and dialyze it in deionized water at 30-40 °C for 5-7 days, changing the deionized water every 3-4 h. Finally, freeze-dry the purified solution to obtain a white porous foam, namely methacrylated gelatin, and store it in a refrigerator at -40 °C.

[0041] Step 3: After subjecting the prepolymer solution to centrifugal defoaming treatment, inject it into a mold. Place a photomask with a pattern directly above the mold and expose it to ultraviolet light for photoinitiated polymerization. The polymerization reaction time is 0.5-3 min to obtain a micro-patterned hydrogel film. Among them, the mold is composed of two upper and lower glass slides and a silicone gasket placed between the two glass slides, and the depth of the mold is 0.1-0.5 mm; the pattern of the photomask includes black and white stripes with different widths, the width of the black stripes is 10-60 μm, and the width of the white stripes is 10 μm; the wavelength of the ultraviolet light is 300-400 nm.

[0042] The present invention will be further described below in conjunction with specific embodiments, but it is not limited to the present invention.

[0043] The size information of the photomask and the obtained micro-patterned hydrogel in Examples 1-4 is shown in Table 1 below, and the component content and polymerization time of the hydrogel prepolymer solution in Examples 1, 5 and 6 are shown in Table 2 below.

[0044] Table 1

[0045] Table 2

[0046] Example 1 This example provides a preparation method of a micro-patterned hydrogel film, comprising the following steps: (1) The sea squirt cellulose nanocrystals (TCNC) were prepared by sulfuric acid hydrolysis method, and the specific process was as follows: 5 g of sea squirt cellulose was dispersed in 500 mL of 65 wt% sulfuric acid solution, and hydrolyzed with stirring at 60 °C for 6 h. The reaction mixture was diluted 10 times with deionized water to terminate the reaction, and the supernatant was obtained by centrifugation at 10000 rpm to obtain a suspension of sea squirt cellulose nanocrystals. The suspension was dialyzed with deionized water until a constant pH value was obtained and then could be used. The diameter of the sea squirt cellulose nanocrystals was 30±6 nm, the length was 2040±600 nm, and the aspect ratio was about 68.

[0047] (2) Gelatin methacrylate (GelMA) was prepared by the esterification reaction of gelatin and methacrylic anhydride. The specific process was as follows: 10 g of gelatin was dissolved in 100 mL of 0.25 M sodium carbonate / sodium bicarbonate buffer solution in a 60 °C water bath environment. After it was completely dissolved, the pH was adjusted to 9.4, and 0.9 mL of methacrylic anhydride was added dropwise and reacted for 1 h. Finally, the pH was adjusted to 7.4 to terminate the reaction. The reaction solution was filled into a dialysis bag with a molecular weight of 8000 - 14000 and dialyzed in deionized water at 30 - 40 °C for 5 - 7 days, and the deionized water was changed every 3 - 4 h. Finally, the purified solution was freeze-dried to obtain a white porous foam, that is, gelatin methacrylate, and stored in a -40 °C refrigerator. The degree of substitution of the prepared gelatin methacrylate was about 95%.

[0048] (3) In a 50 °C water bath environment, the monomer gelatin methacrylate and the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone were dissolved in the aqueous dispersion of sea squirt cellulose nanocrystals in a certain proportion to obtain a prepolymer solution; the addition amounts of the monomer, photoinitiator, and sea squirt cellulose nanocrystals in the prepolymer solution were 10 wt%, 1 wt%, and 0.2 wt% (calculated by the weight percentage of water), respectively. After centrifugation and degassing, the prepolymer solution was injected into a mold, a photomask was placed directly above the mold, and it was placed under ultraviolet light at 365 nm for photoinitiated polymerization for 2 min to obtain a surface micropatterned hydrogel film.

[0049] In this example, a photomask composed of striped microchannels with a width of 10 μm and a spacing of 10 μm was used, and a micropatterned hydrogel with a clear, fine, neat and regular striped groove structure on the surface was prepared (as shown in Figure 2 a), where the groove width was 10 μm, the depth was 0.6 μm, and the ridge width was 10 μm (as shown in Figure 3 a).

[0050] Example 2 In this example, the types of raw materials, their ratios, and the reaction conditions in steps (1)-(3) are the same as those in Example 1. The difference is that in this example, a photomask composed of striped microchannels with a width of 20 μm and a spacing of 10 μm is used to prepare a micropatterned hydrogel with a clearly defined, neat, and regular striped groove structure on its surface (as shown in Figure 2 as shown in b), where the groove width is 20 μm, the depth is 1.5 μm, and the ridge width is 10 μm (as shown in Figure 3 as shown in b).

[0051] Example 3 In this example, the types of raw materials, their ratios, and the reaction conditions in steps (1)-(3) are the same as those in Example 1. The difference is that in this example, a photomask composed of striped microchannels with a width of 40 μm and a spacing of 10 μm is used to prepare a micropatterned hydrogel with a clearly defined, neat, and regular striped groove structure on its surface (as shown in Figure 2 as shown in c), where the groove width is 40 μm, the depth is 1.5 μm, and the ridge width is 10 μm (as shown in Figure 3 as shown in c).

[0052] Example 4 In this example, the types of raw materials, their ratios, and the reaction conditions in steps (1)-(3) are the same as those in Example 1. The difference is that in this example, a photomask composed of striped microchannels with a width of 60 μm and a spacing of 10 μm is used to prepare a micropatterned hydrogel with a clearly defined, neat, and regular striped groove structure on its surface (as shown in Figure 2 as shown in d), where the groove width is 60 μm, the depth is 1.5 μm, and the ridge width is 10 μm (as shown in Figure 3 as shown in d).

[0053] Figure 4 Figure shows the scanning electron microscope surface images corresponding to the micropatterned hydrogel films prepared in Examples 1-4. This figure shows that the dried samples obtained after freeze-drying the micropatterned hydrogel films can still maintain a clear and regular groove distribution on the surface, and at the same time, it also proves that the micropatterned hydrogel films prepared by this method achieve high-precision and high-resolution patterning.

[0054] Figure 5 Figure shows the statistical chart of the groove width and the interval width corresponding to the micropatterned hydrogel films prepared in Examples 1-4. This figure is based on Figure 4 the corresponding scanning electron microscope images in, and the corresponding groove width and interval width are statistically analyzed. From Figure 5It can be seen that the surface microstructure of the micro-patterned hydrogel film prepared by the photomask basically conforms to the set value. The average spacing widths of the surfaces of the micro-patterned hydrogel films prepared by the 10×10, 10×20, 10×40, and 10×60 photomasks are 9.23 μm, 6.59 μm, 9.98 μm, and 8.01 μm respectively, and the groove widths are distributed as 11.25 μm, 22.07 μm, 40.32 μm, and 55.45 μm. Among them, some hydrogel films will shrink slightly during freeze-drying, resulting in the width being slightly smaller than the set value. The patterns of the hydrogel films obtained by the 10×10 and 10×40 photomasks maintain better consistency with the size of the photomask, and the size of the pattern of the hydrogel film prepared by the 10×40 photomask is the most accurate.

[0055] Figure 6 It is a mechanical property characterization diagram corresponding to the micro-patterned hydrogel films prepared in Examples 1-4. It can be seen from this figure that the hydrogel with a groove pattern with a width of 10 μm and a spacing of 10 μm on the surface has an elastic modulus of 51.67 kPa and a toughness of 263.3 kJ / m 3 ; the hydrogel with a groove pattern with a width of 20 μm and a spacing of 10 μm on the surface has an elastic modulus of 50 kPa and a toughness of 270.3 kJ / m 3 ; the hydrogel with a groove pattern with a width of 40 μm and a spacing of 10 μm on the surface has an elastic modulus of 52.33 kPa and a toughness of 276.1 kJ / m 3 ; the hydrogel with a groove pattern with a width of 60 μm and a spacing of 10 μm on the surface has an elastic modulus of 56.5 kPa and a toughness of 320.5 kJ / m 3 . This figure also shows that the prepared micro-patterned hydrogel film has good mechanical properties.

[0056] Example 5 This example provides a preparation method of a micro-patterned hydrogel film, including the following steps: (1) Prepare tunicate cellulose nanocrystals (TCNC) by the sulfuric acid hydrolysis method. The specific process is as follows: Disperse 5 g of tunicate cellulose in 500 mL of 65 wt% sulfuric acid solution, and hydrolyze it with stirring at 60 °C for 6 h. Dilute the reacted mixture 10 times with deionized water to terminate the reaction, and take the supernatant by centrifugation at 10000 rpm to obtain a tunicate cellulose nanocrystal suspension. Dialyze this suspension with deionized water until a constant pH value is obtained for use. The diameter of the tunicate cellulose nanocrystals is 30±6 nm, the length is 2040±600 nm, and the aspect ratio is about 68.

[0057] (2) Gelatin methacrylate (GelMA) was prepared by the esterification reaction of gelatin and methacrylic anhydride. The specific process was as follows: 10 g of gelatin was dissolved in 100 mL of 0.25 M sodium carbonate / sodium bicarbonate buffer solution in a 60 °C water bath. After complete dissolution, the pH was adjusted to 9.4. 0.9 mL of methacrylic anhydride was added dropwise and reacted for 1 h. Finally, the pH was adjusted to 7.4 to terminate the reaction. The reaction solution was filled into a dialysis bag with a molecular weight of 8000 - 14000 and dialyzed in deionized water at 30 - 40 °C for 5 - 7 days, and the deionized water was changed every 3 - 4 h. Finally, the purified solution was freeze-dried to obtain a white porous foam, namely gelatin methacrylate, which was stored in a -40 °C refrigerator. The degree of substitution of the prepared gelatin methacrylate was about 95%.

[0058] (3) In a 50 °C water bath, the monomer gelatin methacrylate and the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone were dissolved in the aqueous dispersion of ascidian cellulose nanocrystals in a certain proportion to obtain a prepolymer solution; in the prepolymer solution, the addition amounts of the monomer, the photoinitiator, and the ascidian cellulose nanocrystals were 5 wt%, 1 wt%, and 0.7 wt% (calculated by weight percentage of water), respectively. After centrifugation and degassing, the prepolymer solution was injected into a mold, and a photomask was placed directly above the mold. It was placed under ultraviolet light at 365 nm and polymerized by photoinitiation for 3 min to obtain a surface micropatterned hydrogel film.

[0059] In this example, a photomask composed of striped microchannels with a width of 10 μm and a spacing of 10 μm was used to prepare a micropatterned hydrogel with a clearly fine, neat, and regular striped groove structure on the surface, where the groove width was 10 μm, the depth was 0.6 μm, and the ridge width was 10 μm.

[0060] Example 6 This example provides a method for preparing a micropatterned hydrogel film, including the following steps: (1) Ascidian cellulose nanocrystals (TCNC) were prepared by the sulfuric acid hydrolysis method. The specific process was as follows: 5 g of ascidian cellulose was dispersed in 500 mL of 65 wt% sulfuric acid solution, and hydrolyzed with stirring at 60 °C for 6 h. The reacted mixture was diluted 10 times with deionized water to terminate the reaction, and the supernatant was taken by centrifugation at 10000 rpm to obtain an ascidian cellulose nanocrystal suspension. The suspension was dialyzed with deionized water until a constant pH value was obtained and could be used. The diameter of the ascidian cellulose nanocrystals was 30 ± 6 nm, the length was 2040 ± 600 nm, and the aspect ratio was about 68.

[0061] (2)Gelatin was used to react with methacrylic anhydride to prepare methacrylated gelatin (GelMA). The specific process was as follows: 10 g of gelatin was dissolved in 100 mL of 0.25 M sodium carbonate / sodium bicarbonate buffer solution in a 60 °C water bath. After complete dissolution, the pH was adjusted to 9.4, and 0.9 mL of methacrylic anhydride was added dropwise and reacted for 1 h. Finally, the pH was adjusted to 7.4 to terminate the reaction. The reaction solution was placed in a dialysis bag with a molecular weight of 8000 - 14000 and dialyzed in deionized water at 30 - 40 °C for 5 - 7 days, and the deionized water was changed every 3 - 4 h. Finally, the purified solution was freeze-dried to obtain a white porous foam, namely methacrylated gelatin, which was stored in a -40 °C refrigerator. The degree of substitution of the prepared methacrylated gelatin was about 95%.

[0062] (3)In a 50 °C water bath environment, the monomer methacrylated gelatin and the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone were dissolved in the aqueous dispersion of ascidian cellulose nanocrystals in a certain proportion to obtain a prepolymer solution; in the prepolymer solution, the addition amounts of the monomer, photoinitiator, and ascidian cellulose nanocrystals were 8 wt%, 1 wt%, and 1.0 wt% (calculated based on the weight percentage of water), respectively. After centrifugation and degassing, the prepolymer solution was injected into a mold, and a photomask was placed directly above the mold and irradiated with ultraviolet light at 365 nm for 0.5 min through photoinitiated polymerization to obtain a surface micropatterned hydrogel film.

[0063] In this example, a photomask composed of striped microchannels with a width of 10 μm and a spacing of 10 μm was used to prepare a micropatterned hydrogel with a clear, fine, neat, and regular striped groove structure on the surface, where the groove width was 10 μm, the depth was 0.6 μm, and the ridge width was 10 μm.

[0064] Comparative Example The main difference between this example and Example 1 was that ascidian cellulose nanocrystals were not added to the prepolymer solution.

[0065] A method for preparing a micropatterned hydrogel film provided by the comparative example included the following steps: (1) Esterified gelatin (GelMA) was prepared by the esterification reaction of gelatin and methacrylic anhydride. The specific process is as follows: At 60 °C in a water bath environment, 10 g of gelatin was dissolved in 100 mL of 0.25 M sodium carbonate / sodium bicarbonate buffer solution. After it was completely dissolved, the pH was adjusted to 9.4, and 0.9 mL of methacrylic anhydride was added dropwise and reacted for 1 h. Finally, the pH was adjusted to 7.4 to terminate the reaction. The reaction solution was filled into a dialysis bag with a molecular weight of 8000 - 14000 and dialyzed in deionized water at 30 - 40 °C for 5 - 7 days, and the deionized water was changed every 3 - 4 h. Finally, the purified solution was freeze-dried to obtain a white porous foam, namely esterified gelatin, and stored in a -40 °C refrigerator. The degree of substitution of the prepared esterified gelatin was about 95%.

[0066] (2) At 50 °C in a water bath environment, the monomer esterified gelatin and the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone were dissolved in deionized water in a certain proportion. The addition amounts of the monomer and the photoinitiator were 10 wt% and 1 wt% respectively (calculated as the weight percentage of water). After centrifugation and defoaming, the prepolymer solution was injected into a mold, and a photomask was placed directly above the mold. It was placed under ultraviolet light at 365 nm and polymerized by photoinitiation for 2 min to obtain a surface micropatterned hydrogel film.

[0067] In this comparative example, a photomask composed of striped microchannels with a width of 60 μm and a spacing of 10 μm was used. The groove pattern on the surface of the prepared hydrogel was not regular enough (as Figure 7 shown). As can be seen from Figure 7 a, the swelling degree of the hydrogel in the wet state was too large, resulting in a large deviation between the width of the surface grooves and the spacing width and the set values. At the same time, the surface grooves may be too shallow in depth, resulting in the surface groove pattern not looking clear and obvious enough. As can be seen from Figure 7 b, the distribution of the surface grooves of the freeze-dried hydrogel was not very regular, with some places wide and some places narrow. At the same time, due to the too large swelling degree of the hydrogel, the surface microstructure of the obtained micropatterned hydrogel film deviated from the set value. The average width and spacing width of the surface grooves of the micropatterned hydrogel film prepared in this example were 73.21 μm and 22.13 μm respectively, which was significantly deviated from the set value of the photomask.

[0068] Further, the mechanical properties of the micropatterned hydrogel film prepared in the comparative example are as Figure 8 shown. Its elastic modulus was 36.72 kPa, and its toughness was 71.03 kJ / m 3 . Compared with the micropatterned hydrogel prepared by adding cellulose nanocrystals from ascidians, its mechanical properties decreased significantly.

[0069] The above are only the preferred embodiments of the present invention, and thus do not limit the implementation manners and protection scope of the present invention. For those skilled in the art, it should be realized that all the equivalent replacements and obvious changes made by using the content of the specification of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a micro-patterned hydrogel film, characterized in that: The following steps are involved: S1, dispersing the ascidian cellulose in an acidic solution for hydrolysis reaction, and then diluting and centrifuging to obtain the supernatant to obtain an ascidian cellulose nanocrystal suspension; S2, adding methacrylated gelatin and a photoinitiator to the suspension of ascidian cellulose nanocrystals, and mixing to obtain a prepolymer solution; in the prepolymer solution, the content of methacrylated gelatin is 5wt.%-10wt.%; S3, injecting the prepolymer solution into a mold, placing a photomask with a pattern just above the mold, placing it under ultraviolet light to initiate polymerization, the polymerization reaction time is 0.5-3 minutes, and obtaining a micro-patterned hydrogel film.

2. The preparation method according to claim 1, characterized in that: In step S1, the hydrolysis reaction temperature is 55-65°C and the time is 5-7h.

3. The preparation method according to claim 1, characterized in that: In step S2, in the prepolymer solution, the content of ascidian cellulose nanocrystals is 0.2wt.%-1wt.%, and the content of initiator is 0.5wt.%-1wt.%.

4. The preparation method according to claim 1, characterized in that: In step S2, the photoinitiator is selected from one or more combinations of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, and 2-hydroxy-2-methyl-1-phenyl-1-propanone.

5. The preparation method according to claim 1, characterized in that: In step S2, the prepolymer liquid is mixed by stirring, the mixing temperature is 45-55° C., and the mixing time is 20-30 min. In step S3, the prepolymer liquid is centrifugally degassed and then injected into the mold.

6. The preparation method according to claim 1, characterized in that: In step S3, the mold is composed of two layers of glass sheets, an upper layer and a lower layer, and a silicone gasket placed between the two layers of glass sheets. The depth of the mold is 0.1-0.5 mm.

7. The preparation method according to claim 1, characterized in that: In step S3, the pattern of the photomask includes black and white stripes of different widths, the width of the black stripes is 10-60 μm, and the width of the white stripes is 10 μm.

8. The preparation method according to claim 1, characterized in that: In step S3, the wavelength of the ultraviolet light is 300-400 nm.

9. A micro-patterned hydrogel film, prepared by the preparation method according to any one of claims 1 to 8, characterized in that: The pattern on the surface of the micro-patterned hydrogel film is a micro-groove structure, with a groove width of 10-60 μm, a groove depth of 0.6-1.5 μm, and a ridge width of 10 μm.

10. Use of the micropatterned hydrogel film according to claim 9 in the fields of tissue engineering and regenerative medicine, bioimaging and diagnosis, or flexible electronics.

Images