Dynamic crosslinkers, in situ viscoelasticity-tunable hydrogels, and methods of making and using the same

Abstract

The present application provides a double salicylaldehyde dynamic crosslinking agent containing a visible light responsive thiuram group; also provides a preparation method of the double salicylaldehyde dynamic crosslinking agent, comprising: mixing and reacting chloromethyl salicylaldehyde and methyl piperazine thiuram to obtain the double salicylaldehyde dynamic crosslinking agent; the present application also provides a hydrogel capable of in-situ regulating viscoelasticity under visible light, which has low phototoxicity and high efficiency of reversibility, and has the advantages of in-situ time and space regulation and cell spreading, and is obtained by crosslinking reaction of the double salicylaldehyde dynamic crosslinking agent and a natural polymer as described above, and has the advantages of simple operation process and low raw material cost; the present application also provides a preparation method and application of the hydrogel capable of in-situ regulating viscoelasticity under visible light.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological materials, and particularly relates to a dynamic crosslinking agent, a hydrogel for in-situ regulation of viscoelasticity, and a preparation method and application thereof. BACKGROUND

[0002] Utilizing hydrogels to artificially construct a biomimetic extracellular matrix (ECM) is one of the research focuses in the field of biomaterials. In recent years, it has been shown that mechanical signals transformed from the ECM can regulate cell shape and affect physiological activities such as cell growth, metabolism, differentiation, and migration. Due to the diversity of chemical structures and mechanical characteristics, hydrogels can highly mimic the mechanical microenvironment of biological tissues, and are one of the most ideal synthetic materials for constructing an artificial ECM with precisely controllable mechanical environment. Therefore, the preparation of hydrogel materials with controllable mechanical properties and the exploration of the mechanism of cell behavior regulation have attracted more and more attention. At present, such a mechanical model of hydrogel has become one of the most effective tools for studying the mechanical interaction mechanism between some cells and the ECM, such as the regulation of cell adhesion, the migration pattern of cancer cells, and the induction of stem cell differentiation.

[0003] For a long time, people mainly use common elastic hydrogels and polystyrene (PS) plates as cell culture materials and models, including polyacrylamide (PAAM), gelatin, glyceride, and Matrigel. However, in recent years, it has been realized that the viscous characteristics and spatiotemporal heterogeneity of the ECM have an important influence on the behavior and function of cells. In fact, viscoelasticity is a common characteristic of living tissues and ECMs, and a viscoelastic material exhibits a transient elastic response, which is similar to that of a pure elastic solid, and then presents a time-varying mechanical response and energy dissipation, which is similar to that of a viscous liquid. Many studies have found that some important pathological problems, such as cancer, cardiovascular disease, and regional identification of the cerebral cortex, are closely related to the changes in the viscoelasticity of the ECM. For example, soft tissues have a higher viscoelasticity, and the viscous modulus thereof is usually 10%-20% of the storage modulus thereof.

[0004] At present, the development of some viscoelastic materials mainly depends on dynamic covalent bonds and physically weak cross-linking dynamic bonds to construct hydrogels for cell culture. For example, calcium ion cross-linking of sodium alginate, thioester bonds, and Schiff base bonds. By using these viscoelastic materials, people have begun to study the relationship between behaviors such as stem cell differentiation and cancer cell migration and the base viscoelasticity.

[0005] However, in the past viscoelastic materials, the viscoelasticity is usually regulated as a whole when the initial gel is formed. But for the study of pathological problems, the matrix of cell culture is best to be regulated in situ to simulate the spatiotemporal heterogeneity of ECM in the real mechanical environment. The spatiotemporal heterogeneity of ECM plays an important role in the problems such as the proliferation and differentiation of cancer cells, epithelial-mesenchymal transition, and tumor stemness and drug resistance.

[0006] It is a common method to regulate the mechanical properties of hydrogel by light because light stimulation has high spatiotemporal resolution, and can change the mechanical properties of hydrogel in time, point and quantity. However, in the past light-regulated materials, ultraviolet light is used, and small molecules such as initiators need to be introduced, which is not conducive to the application of viscoelastic light-regulated model to practice. Therefore, it is an urgent problem to develop a more integrated, completely in situ regulated and non-toxic hydrogel material. SUMMARY

[0007] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide a dynamic crosslinking agent, an in situ regulated viscoelastic hydrogel and a preparation method and application, so as to obtain an in situ regulated, non-toxic and integrated hydrogel.

[0008] To achieve the above-mentioned purposes and other related purposes, the present application provides a bis-salicylaldehyde dynamic crosslinking agent, whose structural formula is as follows:

[0009]

[0010] The present application also provides a preparation method of the bis-salicylaldehyde dynamic crosslinking agent as described above, which comprises: mixing and reacting chloromethyl salicylaldehyde and methyl piperazine thiuram to obtain the bis-salicylaldehyde dynamic crosslinking agent, and the reaction formula is as follows:

[0011]

[0012] In some embodiments of the present application, the mass ratio of the chloromethyl salicylaldehyde and the methyl piperazine thiuram is (1.0-1.2):1.

[0013] The present application also provides a visible light in situ regulated viscoelastic hydrogel, which is obtained by crosslinking reaction of the bis-salicylaldehyde dynamic crosslinking agent as described above and a natural polymer.

[0014] In some embodiments of the present application, the natural polymer is carboxymethyl chitosan (CMSC).

[0015] In some embodiments of the present application, the crosslinking reaction is carried out in PBS buffer.

[0016] The present application also provides a preparation method of the visible light in situ regulated viscoelastic hydrogel as described above, which comprises the following steps:

[0017] a. obtaining a solution of the bis-salicylaldehyde dynamic crosslinker as claimed in claim 1;

[0018] b. obtaining a solution of the natural high molecular polymer;

[0019] c. mixing the solution of the bis-salicylaldehyde dynamic crosslinker and the solution of the natural high molecular polymer in a certain concentration ratio to prepare the in-situ viscoelasticity-controllable hydrogel under visible light.

[0020] The application further provides an application of the in-situ viscoelasticity-controllable hydrogel under visible light as described above, which is used in the field of constructing a two-dimensional and three-dimensional model of cancer metastasis and proliferation, or in the field of in-vivo degradable drug delivery and controlled release hydrogel, or in the field of wound dressing, or in the field of constructing a biomimetic extracellular matrix.

[0021] As described above, the dynamic crosslinker, the in-situ viscoelasticity-controllable hydrogel and the preparation method and application thereof have the following beneficial effects:

[0022] 1. The bis-salicylaldehyde dynamic crosslinker is prepared by quaternary ammonium salt reaction of chloromethyl salicylaldehyde and methyl piperazine thiuram, the salicylaldehyde structure in the dynamic crosslinker can react with the amino group of carboxymethyl chitosan to form a Schiff base, so that the polymer is crosslinked to form a gel, and then the visible light corresponding thiuram structure in the dynamic crosslinker promotes free radical transfer and exchange under the action of visible light, and initiates exchange of the polymer main chain, so as to change the viscoelasticity of the hydrogel matrix.

[0023] 2. The in-situ viscoelasticity-controllable hydrogel under visible light is prepared by introducing the visible light responsive thiuram group in the biocompatible natural high molecular polymer through the reaction of the bis-salicylaldehyde dynamic crosslinker and the biocompatible natural high molecular polymer, and a non-toxic and efficient spatiotemporal controllable dynamic hydrogel is prepared.

[0024] 3. In the preparation method of the in-situ viscoelasticity-controllable hydrogel under visible light, the hydrogel with different mechanical strengths of 1kPa-30kPa is obtained by controlling the content of the thiuram group; during use, the reconstruction rate of the polymer network can be accurately controlled by controlling the irradiation intensity of the light source, so that the stress relaxation speed of the hydrogel is systematically adjusted, the stress relaxation speed of the in-situ viscoelasticity-controllable hydrogel under visible light is between 1500 seconds and 6500 seconds, and the process is reversible after the light source is removed, so it is suitable for the regulation of any time scale of the whole life cycle of cells.

[0025] 4. The viscoelastic hydrogel of the present invention, which can be regulated in situ under visible light, has a wide range of raw material sources, is natural and has good biocompatibility, low phototoxicity and high reversibility, and has the advantages of in situ temporal and spatial control and cell spreading. At the same time, the operation process is simple and the raw material cost is low. Attached Figure Description

[0026] Figure 1 The diagram shows the network structure and chemical composition of the hydrogel structure with in-situ tunable viscoelasticity under visible light, as presented in this invention.

[0027] Figure 2 The diagram shows the photo-viscoelastic mechanism of the hydrogel with in-situ controllable viscoelasticity under visible light according to the present invention.

[0028] Figure 3 The diagram shows the storage modulus and loss modulus of the viscoelastic hydrogel that can be in situ controlled under visible light under different conditions. Figure 3 a shows the change of hydrogels with different crosslinking agent concentrations over time; G' represents the storage modulus and G” represents the loss modulus. Figure 3 b shows the change in storage modulus of G' for hydrogels with different polymer concentrations; Figure 3 c represents the stability change of the hydrogel after direct exposure to light under low stress.

[0029] Figure 4 a shows the strain scanning curve of the hydrogel of the present invention; Figure 4 b shows a statistical diagram of the strain locations of the hydrogel of this invention.

[0030] Figure 5 a shows the curves of stress change over time after the hydrogel is exposed to different light intensities; Figure 5 b represents the stress relaxation rate τ of the hydrogel under different light intensities. 1 / 2 ; Figure 5 c represents the stress relaxation rate τ of hydrogels with different crosslinking agent concentrations. 1 / 2 .

[0031] Figure 6 The expression τ represents the stress relaxation rate of the hydrogel under different temperature conditions. 1 / 2 .

[0032] Figure 7 a shows the flexibility curves of the hydrogel under different light intensities; Figure 7 b shows the creep rate of the hydrogel under different light intensities; Figure 7 c represents the creep behavior of strain increasing over time under different initial stresses and the same illumination intensity; Figure 7 d shows the frequency scanning curves of the hydrogel under different light intensities; Figure 7e shows the change in the loss angle of the hydrogel as light intensity increases under low-frequency conditions; Figure 7 f shows the change in the loss angle of the hydrogel as light intensity increases under high-frequency conditions.

[0033] Figure 8 A shows a schematic diagram of the stress relaxation cycle of the hydrogel under repeated light and dark conditions, with blue representing dark conditions and red representing light conditions; Figure 8 b shows the light-switching cycle where the viscoelastic hydrogel's stress relaxes to 1 / 5 of its original value; Figure 8 c represents the light-switching cycle in which the stress of the viscoelastic hydrogel relaxes to half of its original value.

[0034] Figure 9 a shows a schematic diagram of the preparation process of the hydrogel base; Figure 9 b shows a schematic diagram of grafting modification on the surface of the hydrogel.

[0035] Figure 10 a shows a schematic diagram illustrating the change in the number of cells spreading out as the RGD content on the hydrogel base increases; Figure 10 b shows the changes in cells fully cultured on a hydrogel substrate over time.

[0036] Figure 11 a shows the staining pattern of Hey cells after 24 hours; Figure 11 b shows the live / dead staining pattern of HeLa cells 24 hours later.

[0037] Figure 12 This is a statistical graph showing the phototoxicity of cell proliferation after exposure to light of different intensities on a hydrogel substrate using Almar blue staining.

[0038] Figure 13 This image shows the liveness and death staining of Hey cells under the same light intensity at different times on an elastic base.

[0039] Figure 14 The image shows an immunofluorescence pattern illustrating the spatiotemporal viscoelastic regulation of cell spreading by a dynamic hydrogel.

[0040] Figure 15 a shows the changes in cell area on a dynamic hydrogel as light intensity changes; Figure 15 b shows the changes in cell roundness on a dynamic hydrogel as light intensity changes.

[0041] Figure 16 This image shows the fluorescence of Yap cells after they have been subjected to light regulation.

[0042] Figure 17 The image shows immunofluorescence staining of adhesion spots on cells at different viscoelasticity levels.

[0043] Figure 18 This is a statistical graph showing the total displacement of Hey cells after they were exposed to light on a gel substrate.

[0044] Figure 19 The image shows the cell spread of Hey cells on a non-photoresponsive dynamic hydrogel under different light conditions.

[0045] Figure 20 a shows the change in cell area on a non-photoresponsive dynamic hydrogel as light intensity changes; Figure 20 b shows the change in cell roundness on a non-dynamic hydrogel as light intensity changes.

[0046] Figure 21 The images show immunofluorescence patterns of cell spreading on dynamic hydrogels and ordinary elastic hydrogels.

[0047] Figure 22 The data shows the cell spreading area after 24 hours of cell culture on dynamic hydrogels and ordinary elastic hydrogels.

[0048] Figure 23 The image shown is an NMR spectrum of the dynamic crosslinking agent of this invention.

[0049] Figure 24 The image shown is a mass spectrum of the dynamic crosslinking agent of this invention. Detailed Implementation

[0050] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0051] Please see Figures 1 to 24 It should be noted that the illustrations provided in this embodiment are only schematic representations of the basic concept of the present invention. Therefore, the drawings only show the components related to the present invention and are not drawn according to the actual number, shape and size of the components in the actual implementation. In the actual implementation, the form, quantity and proportion of each component can be arbitrarily changed, and the layout of the components may also be more complex.

[0052] This invention provides a bis(salicylaldehyde) dynamic crosslinking agent, the structural formula of which is:

[0053]

[0054] A second aspect of this invention provides a method for preparing a bis-salicylaldehyde dynamic crosslinking agent, comprising: mixing and reacting chloromethylsalicylaldehyde and methylpiperazine thiuram to obtain a bis-salicylaldehyde dynamic crosslinking agent, the reaction formula of which is as follows:

[0055]

[0056] In this invention, a dynamic crosslinking agent of bis-salicylaldehyde is obtained by quaternization reaction of chloromethylsalicylaldehyde and methylpiperazine thiuram.

[0057] The mass ratio of chloromethyl salicylaldehyde to methylpiperazine thiuram is (1.0–1.2):1. For example, it can be (1.0–1.05):1, (1.05–1.1):1, (1.1–1.15):1, or (1.15–1.2):1.

[0058] The mixed reaction of chloromethyl salicylaldehyde and methylpiperazine thiuram was carried out at room temperature.

[0059] The reaction of chloromethyl salicylic acid aldehyde and methylpiperazine thiuram is carried out in an organic solvent. The organic solvent can be one or more of THF (tetrahydrofuran), DCM (dichloromethane), and toluene. In a preferred embodiment of the invention, the organic solvent is tetrahydrofuran. The volume-to-mass ratio of the organic solvent to methylpiperazine thiuram is 25–40 ml: 1 g. For example, 25–30 ml: 1 g, 30–35 ml: 1 g, or 35–40 ml: 1 g.

[0060] The preparation process of the bis-salicylaldehyde dynamic crosslinking agent specifically includes:

[0061] 1. Dissolve methylpiperazine thiuram (NMPTDS) in anhydrous tetrahydrofuran (THF) and stir. Then, add chloromethyl salicylic acid dissolved in THF dropwise to the reaction solution at room temperature while stirring. During the reaction, a yellow solid powder will gradually precipitate.

[0062] 2. After the reaction is complete, the reaction solution is centrifuged and the supernatant is removed. Then, the solid is washed twice with dry THF and dichloromethane (DCM), and the centrifugation process is repeated to obtain a yellow solid. Finally, the yellow solid powder is placed in a vacuum pump for degassing and drying to obtain the bis(salicylaldehyde) dynamic crosslinking agent.

[0063] In the preparation method of the bis(salicylaldehyde) dynamic crosslinking agent of the present invention, the chloromethylsalicylaldehyde is obtained by a mixed reaction of salicylaldehyde, hydrochloric acid, and paraformaldehyde. The reaction formula is as follows:

[0064]

[0065] Among them, salicylaldehyde is prepared as chloromethyl salicylaldehyde via the Blank chloromethyl reaction. The specific reaction process includes:

[0066] 1. Mix paraformaldehyde and concentrated hydrochloric acid and stir, then slowly add salicylaldehyde dropwise to allow it to react at room temperature. The volume-to-mass ratio of salicylaldehyde to paraformaldehyde and concentrated hydrochloric acid is (14-16) ml:(2.1-25.2) g:(70-85) ml; the reaction time at room temperature is 10-14 hours. In a preferred embodiment of this invention, the reaction time is 12 hours at room temperature.

[0067] 2. After the reaction, add water to the reaction flask, filter under vacuum, wash the filter cake with water, then dissolve the product with ether, filter under vacuum, and obtain the filtrate.

[0068] 3. The filtrate was evaporated to dryness and then recrystallized with petroleum ether to obtain chloromethyl salicylic acid.

[0069] In the preparation method of the bis(salicylaldehyde) dynamic crosslinking agent of the present invention, the methylpiperazine thiuram is obtained by a mixed reaction of carbon disulfide, methylpiperazine, and sodium nitrite. The reaction formula is as follows:

[0070]

[0071] The specific preparation process of the methylpiperazine thiuram includes:

[0072] 1. Dissolve methylpiperazine and potassium hydroxide in deionized water by stirring thoroughly. Add carbon disulfide dropwise to the solution and stir thoroughly overnight to transform the solution from dispersed oil droplets into a homogeneous solution. The mass-to-volume ratio of methylpiperazine, potassium hydroxide, and carbon disulfide is (1.58–1.75) g:(7.8–8.2) g:(1.5–2) ml.

[0073] 2. Add methanol, then add sodium nitrite powder in batches to the reaction solution. The solution changes from colorless to light yellow. Continue stirring the reaction.

[0074] 3. After reacting for 1–1.5 hours, the solution is adjusted to a pH of approximately 1 by adding concentrated hydrochloric acid, causing a large amount of solid to precipitate from the solution. The solid is then filtered, and the filter cake is washed with water and ethanol. Alternatively, the solid can be dissolved in dichloromethane and then precipitated with n-hexane to obtain methylpiperazine thiuram.

[0075] A third aspect of the present invention provides a hydrogel whose viscoelasticity can be regulated in situ under visible light, obtained by crosslinking a natural polymer with a dynamic crosslinking agent of salicylaldehyde as described above.

[0076] The natural polymer is carboxymethyl chitosan. The molecular weight of the carboxymethyl chitosan is 20,000–50,000 Da, for example, 20,000–25,000 Da, 25,000–30,000 Da, 30,000–35,000 Da, 35,000–40,000 Da, 40,000–45,000 Da, or 45,000–50,000 Da. In a preferred embodiment of the invention, the molecular weight of the carboxymethyl chitosan is 30,000 Da. The degree of acetylation of the carboxymethyl chitosan is >90%.

[0077] The crosslinking reaction was carried out in PBS buffer.

[0078] A fourth aspect of the present invention provides a method for preparing a hydrogel with in-situ modulated viscoelasticity under visible light as described above, comprising the following steps:

[0079] a. Obtaining the bis(salicylaldehyde) dynamic crosslinking agent solution as described in claim 1;

[0080] b. Obtain a natural polymer solution;

[0081] c. Thoroughly mix and react the bis(salicylaldehyde) dynamic crosslinking agent solution and the natural polymer solution at a certain concentration ratio.

[0082] A hydrogel with in-situ modulated viscoelasticity under visible light was prepared.

[0083] In the method for preparing a viscoelastic hydrogel that can be in situ controlled under visible light according to the present invention, in step a, the solution of the bis(salicylaldehyde) dynamic crosslinking agent is obtained by dissolving the bis(salicylaldehyde) dynamic crosslinking agent in dimethyl sulfoxide. The specific steps include: dissolving the dynamic crosslinking agent in dimethyl sulfoxide (DMSO), treating it with ultrasound until it is completely dissolved to prepare a 50 wt% crosslinking agent solution, and then adding an appropriate amount of PBS to prepare crosslinking agent precursor solutions of different concentrations.

[0084] The ultrasonic intensity can be between 180 and 240 watts. In a preferred embodiment of the invention, a 10-second cyclic ultrasonic method is used.

[0085] In the method for preparing a viscoelastic hydrogel with in-situ controllable viscoelasticity under visible light according to the present invention, step b, wherein the solution of the natural polymer is obtained by dissolving the natural polymer in PBS buffer. The specific steps include: dissolving the natural polymer powder in a certain amount of PBS buffer, treating with ultrasound while stirring; then placing the solution in a heated metal bath, heating to 50 degrees Celsius, and then allowing it to stand to allow the polymer to fully dissolve; and controlling the amount of PBS buffer to prepare solutions containing different polymer concentrations of natural polymer.

[0086] The pH of the PBS buffer is 7.4.

[0087] In the preparation method of the viscoelastic hydrogel that can be in situ controlled under visible light according to the present invention, in step c, the thorough mixing reaction is carried out under vortex action.

[0088] In step c, the concentration ratio of the bis(salicylaldehyde) dynamic crosslinking agent solution to the natural polymer solution is (1–4 wt%):(3–7 wt%). For example, it can be (1–1.5 wt%):(3–7 wt%), (1.5–2 wt%):(3–7 wt%), (2–2.5 wt%):(3–7 wt%), (2.5–3 wt%):(3–7 wt%), (3–3.5 wt%):(3–7 wt%), (3.5–4 wt%):(3–7 wt%), (1–4 wt%):(3–4 wt%), (1–4 wt%):(4–5 wt%), (1–4 wt%):(5–6 wt%), or (1–4 wt%):(6–7 wt%).

[0089] Step c specifically includes: at room temperature, rapidly mixing the above-mentioned bis(salicylaldehyde) dynamic crosslinking agent solution and the natural polymer solution in a 1:1 ratio under vortex conditions, forming a hydrogel through a Schiff base reaction between the aldehyde groups on the crosslinking agent and the amino groups on carboxymethyl chitosan. After mixing and gelling, allowing it to stand at room temperature for 30 minutes, dynamic hydrogels containing different crosslinking agent concentrations and polymer hardness are obtained.

[0090] The fifth aspect of the present invention provides an application of a hydrogel whose viscoelasticity can be regulated in situ under visible light as described above. The hydrogel is used in the field of constructing two-dimensional and three-dimensional models of cancer metastasis and proliferation, or in the field of controlled-release hydrogels for in vivo degradable drug delivery, or in the field of wound dressings, or in the field of constructing biomimetic extracellular matrix.

[0091] Example 1: Preparation of dynamic crosslinking agents and non-photoresponsive dynamic crosslinking agents

[0092] Preparation of dynamic crosslinking agents:

[0093] 1. Mix 22.4 g of paraformaldehyde and 80 mL of concentrated hydrochloric acid in a reaction flask and stir for 10 minutes. Then, slowly add 14 mL of salicylaldehyde dropwise and allow the mixture to react at room temperature for 12 hours. After the reaction is complete, add water to the reaction flask, filter under vacuum, wash the filter cake with water, dissolve the product in diethyl ether, filter under vacuum, and obtain the filtrate. Dry the filtrate by rotary evaporation, and then recrystallize it with petroleum ether to obtain chloromethyl salicylaldehyde with a yield of 76%. The reaction equation is as follows:

[0094]

[0095] 2. Take 1.6 g of methylpiperazine and 8 g of potassium hydroxide, and add 60 mL of deionized water. Add excess carbon disulfide (1.5 mL) to the solution and stir thoroughly overnight to transform the solution from dispersed oil droplets into a homogeneous solution. Then add 3 mL of methanol, followed by 3.3 g of sodium nitrite in batches. The solution changes from colorless to pale yellow. Continue stirring for 1 hour, then add concentrated hydrochloric acid to adjust the pH of the solution to approximately 1. A large amount of solid precipitates from the solution. Filter the solid, then wash the filter cake with water and ethanol to obtain methylpiperazine thiuram. The reaction equation is as follows:

[0096]

[0097] 3. Dissolve 1 gram of methylpiperazine thiuram in 30 mL of anhydrous tetrahydrofuran and stir for 10 minutes. Then, at 25°C, add 1 gram of chloromethyl salicylic aldehyde dissolved in 5 mL of THF dropwise to the reaction solution and stir for 4 hours. During the reaction, a yellow solid powder will gradually precipitate. After the reaction is complete, centrifuge the reaction solution at 4000 rpm for 5 minutes and then remove the supernatant. Then, wash with dry THF and DCM, repeating the washing twice, and then repeat the centrifugation operation to obtain a yellow solid. Finally, place the yellow solid powder in a vacuum pump for degassing and drying to obtain a bis-salicylaldehyde dynamic crosslinking agent with a yield of 85%. The NMR spectrum is shown below. Figure 23 As shown, the mass spectrum is as follows Figure 24 As shown. The reaction equation is as follows:

[0098]

[0099] Preparation of non-photoresponsive dynamic crosslinking agents:

[0100] Take 1 gram of the prepared chloromethyl salicylic acid as described above, place it in a double-necked flask, dissolve it in 30 mL of ultra-dry THF, and then stir for ten minutes. Use a syringe to draw 0.25 mL of tetramethylpropanediamine, dissolve it in ultra-dry THF, and add it dropwise to the reaction flask under a nitrogen atmosphere. Stir the reaction solution for four hours, and a yellow solid powder gradually precipitates. After the reaction is complete, centrifuge the reaction solution at 4000 rpm for 5 minutes, and then remove the supernatant. Subsequently, wash with ultra-dry THF, repeat the washing twice, and then repeat the centrifugation operation to obtain a yellow solid. Finally, place the yellow solid powder in a vacuum pump for degassing and drying to obtain a non-dynamic crosslinking agent with a yield of 67%. The reaction equation is as follows:

[0101]

[0102] Example 2: Preparation of dynamic hydrogels and non-photoresponsive dynamic hydrogels

[0103] Preparation of dynamic hydrogels:

[0104] 1. Dissolve carboxymethyl chitosan powder with a molecular weight of 30 kDa in a certain amount of PBS buffer (pH 7.4), and sonicate for 5 minutes while stirring. Then, place the solution in a heated metal bath and heat to 50°C, then allow it to stand to allow the polymer to fully dissolve. This prepares carboxymethyl chitosan solutions with polymer concentrations of 7 wt%, 5 wt%, and 3 wt%.

[0105] 2. Dissolve the dynamic crosslinking agent in DMSO and treat with ultrasound for 2-3 minutes until completely dissolved. Use a 10-second cycle of ultrasound with an intensity between 180 and 240 watts to prepare a 50 wt% crosslinking agent solution. Then add an appropriate amount of PBS to prepare dynamic crosslinking agent solutions of different concentrations: 1 wt%, 1.5 wt%, 2 wt%, 3 wt%, and 4 wt%.

[0106] 3. At room temperature, the two solutions of various concentrations were rapidly mixed in a 1:1 volume ratio under vortexing. A Schiff base reaction was then carried out between the aldehyde groups on the crosslinking agent and the amino groups on the carboxymethyl chitosan. After standing for 30 minutes, dynamic hydrogels of different hardnesses were obtained. The network structure diagram and chemical composition of the dynamic hydrogel are shown below. Figure 1 As shown.

[0107] Preparation of non-dynamic hydrogels:

[0108] The non-dynamic crosslinking agent was dissolved in PBS buffer to prepare a 1.3 wt% crosslinking agent solution. At room temperature, a 7 wt% polymer solution was mixed with the crosslinking agent precursor solution at a 1:1 ratio, and the mixture was allowed to stand at room temperature for 30 minutes to prepare a non-photodynamic hydrogel.

[0109] Example 3 Performance Characterization

[0110] 31) Characterization of the mechanical properties of dynamic hydrogels at different concentrations: The mechanical properties of the hydrogels were tested using a rheometer, where G' is the storage modulus and G” is the loss modulus. Each set of data was tested in parallel three times.

[0111] a. Dynamic crosslinking agent solutions with concentrations of 1 wt%, 2 wt%, 3 wt%, and 4 wt% were crosslinked with a carboxymethyl chitosan solution with a concentration of 7 wt% to obtain a series of CMSC-TDS hydrogels with different concentrations of crosslinking agent.

[0112] like Figure 3 As shown in a, Figure 3 a shows the changes in G' (storage modulus) and G” (loss modulus) of the hydrogel over time under different conditions. Figure 3It can be seen that under any conditions, G' is greater than G in the first 10 seconds, which means that the Schiff base reaction is very fast and the gel formation time is within 5 minutes.

[0113] Among them, a soft hydrogel was obtained by cross-linking reaction of 7 wt% carboxymethyl chitosan solution and 1 wt% dynamic cross-linking agent solution, with a hardness of 1000 Pa and a stress relaxation rate of 6500 s.

[0114] A soft hydrogel with a hardness of 5000 Pa and a stress relaxation rate of 3000 s was obtained by crosslinking a 7 wt% carboxymethyl chitosan solution with a 2 wt% dynamic crosslinking agent solution.

[0115] A soft hydrogel with a hardness of 8000 Pa and a stress relaxation rate of 2500 s was obtained by crosslinking a 7 wt% carboxymethyl chitosan solution with a 3 wt% dynamic crosslinking agent solution.

[0116] A soft hydrogel with a hardness of 10000 Pa and a stress relaxation rate of 2000 s was obtained by crosslinking a 7 wt% carboxymethyl chitosan solution with a 4 wt% dynamic crosslinking agent solution.

[0117] It can be seen that, without changing the polymer concentration, increasing the crosslinking agent content by 1 wt% to 4 wt% will significantly increase the storage modulus, from 1000 to 10000.

[0118] b. Carboxymethyl chitosan solutions with concentrations of 3 wt%, 5 wt%, and 7 wt% were crosslinked with a 1% dynamic crosslinking agent solution to obtain a series of CMSC-TDS hydrogels with different concentrations of polymers. For example... Figure 3 As shown in b, increasing the polymer concentration by 3wt% to 7wt% without changing the crosslinking agent content will also increase the gel hardness (storage modulus) from 1000 to 3000.

[0119] As can be seen from the above, the hardness and mechanical properties of dynamic hydrogels can be changed by adjusting the crosslinking agent content and polymer concentration.

[0120] Figure 3 c represents a hydrogel obtained by crosslinking a 7 wt% carboxymethyl chitosan solution with a 1 wt% dynamic crosslinking agent solution, demonstrating the stability of the dynamic hydrogel after direct light exposure. (The text then abruptly shifts to a seemingly unrelated topic: "from...") Figure 3 As shown in c, after prolonged testing, the gel modulus tends to stabilize, indicating that the formed gel network is stable and will not degrade due to dynamic covalent bonds. This is an important characteristic, demonstrating that the dynamic hydrogel of this invention can maintain its mechanical properties during long-term use.

[0121] 32) Photodegradation stability of dynamic hydrogels

[0122] To confirm that the gel would not degrade due to photoinduced side reactions (such as photoexchange reactions) during disulfide crosslinking, a photodegradation stability test was conducted. The specific steps were as follows: After the dynamic hydrogel stabilized, a small strain stress was applied, followed immediately by light irradiation, to detect changes in the storage modulus and loss modulus of the gel. It was found that even under prolonged high light intensity irradiation (40 mW / cm²), the gel remained stable. 2 Even under these conditions, the modulus of the gel did not change significantly, proving that the gel did not suffer from photodegradation.

[0123] The strain scan curves of hydrogels are tested, and the locations where irreversible deformation occurs can be determined by the intersection of the curves. For example... Figure 4 As shown in figure a, the storage modulus and loss modulus curves of the hydrogel intersect only at strains close to 100%, indicating that the hydrogel can maintain stable properties under cell culture conditions (strain scale between 15% and 30%).

[0124] Test the changes in hydrogel under different light intensities, such as Figure 4 As shown in b, illumination does not affect the position of the intersection points in the hydrogel, indicating that changes in light intensity do not affect the basic structure and properties of the gel.

[0125] The above tests show that the dynamic hydrogel of the present invention has photodegradation stability, and the controllability of the hydrogel within the strain range has been determined, and changes in light intensity do not affect the basic structure and properties of the gel.

[0126] 33) Photoviscoelasticity of dynamic hydrogels

[0127] To accurately characterize the photoviscoelastic properties of the hydrogel, the following steps were taken to eliminate the interference of the pH dependence of the Schiff base itself on the test results. First, the prepared photocontrolled dynamic hydrogel under the same conditions was soaked in PBS buffer (pH=7.4) overnight to maintain the pH consistency of the system and remove any trace amounts of DMSO that may have been introduced during the gel preparation process.

[0128] Then, stress relaxation experiments are used to describe the viscoelastic properties of the material. For example... Figure 5 Figure a shows the stress-time curves of the hydrogel after exposure to different light intensities. For ease of discussion, this invention uses normalized stress (σ / σ). max =e (-(t / tκ)β) The relationship between light intensity and time is used to represent the magnitude of stress relaxation rate. It can be observed from the figure that as light intensity increases (from 0 mW / cm²), the rate of stress relaxation increases. 2 Up to 40mW / cm 2The rate of decrease in the stress relaxation curve also increases. This indicates that the rate of stress relaxation increases with increasing light intensity, suggesting that the viscoelastic properties of the dynamic hydrogel are enhanced with increasing light intensity. Simultaneously, this also indicates that the photoinduced exchange and recombination rate of SS bonds accelerates with increasing light intensity. Figure 2 This invention describes the photo-viscoelastic mechanism of the hydrogel whose viscoelasticity can be situ controlled under visible light.

[0129] To more accurately describe the relaxation rate, this invention employs the half-life (τ) method. Wherein, τ... 1 / 2 This represents the time required for the stress to relax to half its maximum; this value is used to measure the rate of relaxation. For example... Figure 5 As shown in b, it represents the stress relaxation rate τ under different light intensities. 1 / 2 As light intensity increases (from 0 mW / cm²), 2 Up to 40mW / cm 2 The stress relaxation timescale was reduced from 2000 seconds to 400 seconds, showing an order-of-magnitude acceleration. Figure 5 c represents the stress relaxation rate τ of hydrogels obtained by crosslinking 1wt%, 2wt%, 3wt%, and 4wt% dynamic crosslinking agent solutions with a 7wt% carboxymethyl chitosan solution, as the crosslinking agent content changes. 1 / 2 As the concentration of the dynamic crosslinking agent increased from 1 wt% to 4 wt%, the relaxation rate of the hydrogel also increased significantly, decreasing from 4500 seconds to 1200 seconds.

[0130] This invention also investigated the temperature response of viscoelastic hydrogels, such as Figure 6 Temperature changes can affect the chain exchange rate, which in turn affects the gel relaxation rate. Figure 6 The results showed that as the temperature increased from 25°C to 60°C, the relaxation speed of the hydrogel also increased significantly, decreasing from 3000 seconds to 500 seconds.

[0131] In this invention, a rheometer was used to test creep behavior and frequency sweeps. The preparation method of the experimental samples was the same as in Example 2. Figure 7 As can be observed in Figure a, the compliance (strain / stress) is very stable within the first 100 seconds of the creep test, indicating the first stage of creep. However, between 200 and 1000 seconds, the curve rises rapidly, and the creep curve gradually approaches a straight line, signifying that the gel has entered the constant creep stage. Figure 7 b shows that the creep rate increases with increasing light intensity (from 0.04 Pa). -1 s -1 up to 0.11 Pa -1 s -1 This indicates that the overall viscoelasticity of the gel does indeed increase with increasing light intensity. Figure 7As shown in Figure c, the hydrogel can withstand relatively high stress (1200 Pa; note: the stress of most collagen-based viscoelastic gels is relatively low, only about 60 to 100 Pa), and it can still exhibit viscoelastic uniform creep behavior, proving that the hydrogel has a wide range of applications and viscoelastic control capabilities. Figure 7 Figure d shows the frequency-dependent behavior of the hydrogel under different light intensities. As can be seen from the figure, at the same frequency, the storage modulus of the hydrogel is less affected by the light intensity, while the loss modulus is more affected by the light intensity, proving that the viscous mechanical behavior of the hydrogel is indeed controlled by the light intensity. Figure 7 As shown in Figure e, under low-frequency conditions (0.01 Hz), the loss angle of the CMCS-TDS hydrogel increases from 0.06 to 0.12 with increasing light intensity. Here, the low-frequency condition indicates that the gel exhibits light intensity-sensitive viscoelastic characteristics over a long timescale. This also means that over a longer timescale, the hydrogel is more elastic under low light intensity irradiation, while exhibiting more significant viscous characteristics under high light intensity. Meanwhile, under high-frequency conditions (10 Hz), such as... Figure 7 As shown in f, the gel did not exhibit significant light intensity dependence, indicating that on shorter timescales, the gel tends to be a rapidly deforming elastic material. This also emphasizes that viscoelastic changes require sustained stimulation over a longer period. These experimental results demonstrate that the system of this invention exhibits viscoelastic characteristics on long timescales, which is crucial for cell culture, as processes such as cell growth and proliferation typically occur over longer timescales.

[0132] 34) Light cycling test of dynamic hydrogels

[0133] First, the relaxation rate of the gel was tested under light-free conditions (see...). Figure 8 (a, blue represents dark conditions, red represents light conditions). Then, the gel was restored to twice its testing time to bring it back to its original state as much as possible. Next, light was applied (20mW / cm²). 2 The process was then repeated, and the stress relaxation of the gel was tested.

[0134] pass Figure 8 b leads to the conclusion that for τ 1 / 5 (τ 1 / 5 In terms of the time required for the temperature to decay to one-fifth, the hydrogel can provide stimulation for more than five cycles. For τ 1 / 2 ,like Figure 8 As shown in c, the stress relaxation of the gel can be performed in more than three cycles. (In the figure, "turn off" represents relaxation during the dark test, and "turn on" represents relaxation during the light test).

[0135] Depend on Figure 8As can be seen from a to c, the hydrogel of the present invention can repeatedly undergo relaxation behavior between turn on and turn off, indicating that the gel can be repeatedly subjected to light-controlled stimulation without changing its relaxation behavior, and also indicating its ability to be used repeatedly and precisely controlled in time and space.

[0136] 35) Stress relaxation test of non-photoresponsive dynamic hydrogels

[0137] The viscoelasticity of the non-photoresponsive dynamic hydrogel was evaluated using stress relaxation tests. The results showed that the mechanical strength and initial stress relaxation range of the non-photoresponsive dynamic hydrogel were comparable to those of the photoresponsive dynamic hydrogel.

[0138] Example 4: Preparation of dynamic hydrogel base for cell culture

[0139] 1. Treatment of hydrogel-based glass slides: Circular glass slides (14mm, CITOGLASS) were sequentially ultrasonically cleaned with dichloromethane, anhydrous ethanol, deionized water, anhydrous ethanol, and dichloromethane, 5 minutes each time. After cleaning, they were dried with N2 and placed in a glass dish. Plasma resonance was used to treat the glass slides for 15 minutes to activate the surface. The activated glass slides were then immersed overnight in a 5wt% APTES (3-aminopropyltriethoxysilane) / anhydrous ethanol solution for surface modification. The slides were washed three times with anhydrous ethanol and then baked in an 80℃ vacuum oven for 1 hour to stabilize the amino groups on the surface. The treated amino-modified glass slides can be stored for three weeks.

[0140] 2. Preparation of 2D cell culture hydrogel: Using the same method as in Example 2, prepare 7 wt% carboxymethyl chitosan solution and 1.5 wt% dynamic crosslinking agent solution. Mix the two solutions rapidly by vortexing at a 1:1 ratio. Quickly place the mixture onto the surface of a superhydrophobic film. Invert the amino-modified circular glass slide (i.e., the gel slide) from step 1 onto the solution. After standing for one hour, remove the gel slide and place it in a 24-well plate. Sterilize with 75% ethanol for 10 minutes, then soak in PBS three times for 10 minutes each time. Next, continuously soak the gel slide in PBS solution for seven days, changing the solution daily to ensure no residual small molecules remain in the gel. The preparation process for steps 1 and 2 is as follows: Figure 9 As shown in a.

[0141] 3. RGD Modification of Hydrogel Surface: Prepare 0.1M MES (2-(N-morpholino)ethanesulfonic acid) buffer. Dissolve 0.76g EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) in 1ml MES solution to prepare a 7.6wt% solution. Dissolve 0.36g NHS (N-hydroxysuccinimide) in 1ml MES solution to prepare a 3.6wt% solution. Mix the two solutions 1:1. Adjust the pH of the mixture to 5, soak the gel sheet obtained in step 2 for 15 minutes, wash the gel sheet three times with PBS, then react in RGDfk / PBS (0.1mg / ml) for 2 hours, wash the gel sheet three times, remove residual small molecules with 70% ethanol, and wash three times with PBS solution to obtain the RGD-modified gel sheet. The preparation process for step 3 is shown in 9b.

[0142] Example 5: Cell seeding on dynamic hydrogel

[0143] Hey or HeLa cells were cultured in DMEM medium containing 10 wt% fetal bovine serum (FBS) and 1 wt% penicillin-dextrose antibiotics in a 37°C incubator with 5% CO2. When the cells reached 80% confluence and were in good growth condition, the old medium was removed, the cells were washed twice with PBS, and then digested with 0.25 wt% trypsin for 1.5 minutes. DMEM was added to stop the digestion, the cell slurry was collected, the cells were counted, and 1×10⁶ cells were prepared. 5 Cell suspension of a certain concentration; 300 μL of diluted cell suspension was added to the surface RGD modified gel sheet prepared in Example 4 and cultured in a constant temperature incubator; after the cells were seeded onto the gel surface, the culture medium was changed every 24 hours.

[0144] Example 6: Detection of cell adhesion ability on dynamic hydrogels

[0145] Dynamic hydrogel bases were obtained using the same preparation method as in Example 4. Compared to the previous steps, the content of adhesion ligands (RGD) on the gel surface was varied to obtain gel bases with different RGD contents of 0, 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.25 mg / mL, and 0.5 mg / mL. Cells were then seeded onto different gels following the same steps as in Example 5, and the number of cells spread was observed under a microscope after 24 hours of culture. Figure 10 As shown in Figure a, the number of cells spreading on the gel increases with increasing RGD content. When the RGD content reaches 0.2 mg / ml, the number of cells spreading no longer increases, and excessively high concentrations of RGD may hinder sufficient cell spreading. Figure 10As shown in b, on a gel substrate with a ligand (RGD) content of 0.1 mg / mL, cells exhibited larger cell area and fully extended pseudopodia with increasing culture time, demonstrating that the cells possess good growth and proliferation capabilities on the hydrogel.

[0146] Example 7: Detection of cell biocompatibility on dynamic hydrogel

[0147] Use the live / dead cell staining method (propidium iodide and fluorescein) and follow the instructions in the live / dead cell staining kit: observe cell viability; live cells will emit green fluorescence, and dead cells will emit red fluorescence. Figure 11 As shown, after 24 hours, Hey( Figure 11 a) and Hela ( Figure 11 b) The cells in the hydrogel all exhibited good cell morphology and almost no red fluorescence, proving that the hydrogel has low toxicity and good biocompatibility; that is, the hydrogel will not cause any damage to the cells under normal culture conditions (without light).

[0148] Example 8: Detection of Cell Proliferation Activity on Dynamic Hydrogels

[0149] 1) Using the Alma staining method, cells were seeded onto a gel and allowed to grow under the same culture conditions. Cells were exposed to light of varying intensities (10-40 mW / cm²). 2 The culture medium was changed daily, and Alamar blue dye was added before each test. The cells were incubated for two hours, and the fluorescence intensity was measured. Figure 12 This is a statistical graph using Almar blue staining to show the phototoxicity of cell proliferation after exposure to different light intensities on a hydrogel substrate. Fluorescence intensity represents the number of viable cells. The graph shows that after light exposure, the fluorescence intensity steadily increased on days three and seven, indicating a steady increase in cell number, compared to the control group (0 mW / cm²). 2 The cells showed no significant change in proliferation capacity, further demonstrating the excellent biocompatibility of this dynamic hydrogel.

[0150] 2) Using the same method as in Example 5, Hey cells were seeded into 24-well plates and cultured for 24 hours to allow for full cell spread. Then, the plates were placed in a live cell culture apparatus. A collimated 445nm laser was installed, allowing the cells to be directly irradiated with 445nm light in the wells. After irradiation, the culture medium was immediately removed, and the cells were washed three times with PBS. Live / dead cell staining was performed using the same method as in Example 7. Immediately after staining, the cells were observed under a fluorescence microscope. Figure 13 As shown. This method is used to test the toxicity of light itself. Many existing studies indicate that cells irradiated with ultraviolet light undergo significant apoptosis within half an hour to one hour. However, this invention uses visible light. Figure 13Characterizing whether visible light itself affects cell activity: from Figure 13 It can be seen that even after prolonged exposure to visible light (0–8 h), almost no red fluorescence appeared in the cells, indicating no significant phototoxicity. This experiment verified the cells' tolerance to visible light, proving that even prolonged exposure to 445 nm visible light has almost no effect on cell activity.

[0151] Example 9: Effect of photosensitive viscoelasticity of dynamic hydrogels on cell spreading behavior

[0152] 1. Experiments on cells on light-controlled dynamic hydrogels: Cells on light-controlled dynamic hydrogels were irradiated with lasers of different intensities at 445 nm for half an hour; the culture medium was removed immediately after irradiation and the cells were washed with PBS; the cells were fixed on ice with 4% paraformaldehyde aqueous solution for 30 minutes; the gel slides were soaked in 0.1% Triton-X-100 for 10 minutes to increase the permeability of the cell membrane.

[0153] 2. Yap-related protein staining: Block the gel slides with 5% bovine serum albumin (BSA) solution for 30 minutes; add 2% BSA-dissolved YAP primary antibody (1:200) and Paxillin primary antibody (1:200) to the blocking solution and incubate overnight at 4°C; wash the gel slides with PBS; incubate the gel slides with 2% BSA-dissolved secondary antibody containing a 488nm fluorescent group (goat anti-mouse 1:500) for 30 minutes; wash three times with PBS.

[0154] 3. Actin and nuclear staining: After antibody staining, add Phalloidin solution (1:500) modified with Alexa 633nm fluorescent group and DAPI ready-to-use solution (1:200), and incubate for 20 minutes; wash three times with PBS; mount with anti-fluorescence quencher and store at 4°C.

[0155] 4. Use a fluorescence confocal microscope to observe the signals of DAPI, Yap / Paxillin, and Actin.

[0156] like Figure 14 Immunofluorescence image of cell spreading regulated by spatiotemporal viscoelasticity of dynamic hydrogel; red staining (Actin) indicates phalloidin, blue fluorescence (DAPI) indicates the cell nucleus, and so on. Figure 14 It can be seen that as viscoelasticity increases (stress relaxation speed increases), cells become rounder, cell area decreases, pseudopodia become more filamentous or even completely unable to extend pseudopodia to spread, cell spreading becomes difficult, which means that the cell responds to this in situ viscoelastic change.

[0157] like Figure 15a represents the change in cell area on the dynamic hydrogel as the stress relaxation rate changes; Figure 15 b represents the change in cell roundness on the dynamic hydrogel as the stress relaxation rate changes. As shown in the figure, after light exposure, the cell area and roundness decrease, indicating that the cell spreading is affected by changes in viscoelasticity.

[0158] like Figure 16 This is a fluorescence image of Yap in cells after they have been exposed to light, where actin is in red and Yap is in green. As can be seen from the image, the nuclear localization of Yap changes after the cells are exposed to light.

[0159] like Figure 17 Immunofluorescence staining images of cell adhesion plaques on different viscoelastic bases: dapi (blue) and Paxillin (green); As can be seen from the figure, the adhesion plaques on the viscoelastic bases become smaller but more numerous, showing good adhesion ability and the regulatory effect of hydrogel.

[0160] Example 10: Effect of light-controlled viscoelasticity of dynamic hydrogels on cell migration

[0161] 1. A dynamic hydrogel base was obtained using the same preparation method as in Example 4. Hey cells were seeded on the dynamic hydrogel base using the same method as in Example 5. After culturing for 24 hours to allow the cells to spread fully, the base was placed in a live cell culture device and placed under a fluorescence microscope for bright-field time-series imaging (one frame every 5 minutes, monitoring for four hours) to monitor cell migration behavior. For the experimental group, a 445nm laser fiber was installed with an optical collimator so that the diverging laser could be linearly and fully irradiated onto the gel sheet. The optically controlled viscoelastic migration was monitored without changing any microscope equipment.

[0162] 2. Cell migration data were analyzed in Imagej software using a migration plugin to determine the total migration distance of the cells.

[0163] Depend on Figure 18 It was observed that the total migration distance of cells decreased after illumination. This suggests that changes in the viscoelasticity of the photodynamic hydrogel affect cell motility over long time scales. This reduced migration distance may be due to the rapidly increasing viscoelasticity causing cells to perceive viscous flow. This phenomenon is similar to that observed in conditions such as brain cell lesions, which may suggest that photocontrolled viscoelasticity has important biological significance for cell migration behavior.

[0164] These experimental results demonstrate that photodynamic hydrogels modulate cell migration behavior over long timescales. This modulation may affect cell motility by altering the viscoelasticity of the hydrogel, which could have important applications in cell research, particularly in understanding cell migration, tumor metastasis, and other biological processes.

[0165] Example 11 Cell spreading behavior on non-photoresponsive dynamic hydrogels and ordinary elastic hydrogels

[0166] The non-photoresponsive dynamic hydrogel base was obtained using the same preparation method as in Example 4. Hey cells were seeded on the non-photoresponsive dynamic hydrogel base using the same method as in Example 5. The cells were stained and observed using the same method as in Example 9.

[0167] Figure 19 This image shows the cell spread of Hey cells on a non-photoresponsive dynamic hydrogel under different light irradiations. The excellent cell adhesion observed on the non-photoresponsive dynamic hydrogel demonstrates its good biocompatibility.

[0168] Figure 20 'a' represents the change in cell area on a non-photoresponsive dynamic hydrogel as light intensity changes. Cells cultured on the non-photoresponsive dynamic hydrogel did not show a change in cell area after light exposure. Figure 20 b represents the change in cell roundness on a non-photoresponsive dynamic hydrogel as light intensity changes. Figure 20 The cells did not show significant changes in cell spreading on the non-photoresponsive dynamic hydrogel, indicating that the cells do not respond to light itself, but only to the changes in viscoelasticity caused by light.

[0169] Depend on Figure 21 For dynamic hydrogels ( Figure 21 (Right column) and ordinary elastic hydrogels (acrylamide gels, Figure 21 As shown in the immunofluorescence image of cell spreading in the left column, the cells spread more fully on the dynamic hydrogel base of the present invention. The common elastic hydrogels are elastic gels reported in other studies, such as acrylamide gels, which lack viscoelasticity and are not non-photoresponsive dynamic hydrogels.

[0170] Depend on Figure 22 On dynamic hydrogels ( Figure 22 (right column) and elastic hydrogel ( Figure 22 (Left column) A statistical chart of cell area after 24 hours of cell culture, by Figure 22 It can be seen that the cell spreading area on the viscoelastic gel of the present invention is 1.5 times that of ordinary elastic gel, indicating that the photodynamic hydrogel of the present invention has a better promoting effect on cell growth and reproduction than ordinary elastic matrix, demonstrating the great advantage of the dynamic hydrogel of the present invention for cell culture.

[0171] In summary, the viscoelastic hydrogel of this invention, which can be in situ modulated under visible light, regulates the viscoelasticity of cell culture hydrogels through its thiuram structure that responds under visible light. This method is non-toxic, highly efficient, and possesses high spatiotemporal resolution, enabling the modulation of cell responses to the mechanical properties of the substrate. The hydrogel of this invention provides possibilities for studying other problems in cell biology and for constructing more realistic in vitro cell culture models. Therefore, this invention effectively overcomes the various shortcomings of existing technologies and has high industrial applicability.

[0172] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A bis-salicylaldehyde dynamic crosslinker, characterized in that, The structural formula is:

2. A process for the preparation of the bis-salicylaldehyde dynamic crosslinker as claimed in claim 1, characterized in that, It comprises: The chloromethyl salicylaldehyde and the methyl piperazine thiuram are mixed to obtain a bis-salicylaldehyde dynamic crosslinking agent, and the reaction formula is as follows:

3. The preparation method of the bis(salicylaldehyde) dynamic crosslinking agent as described in claim 2, characterized in that, The mass ratio of the chloromethyl salicylaldehyde and the methyl piperazine thiuram is (1.0-1.2):

1.

4. The preparation method of the bis(salicylaldehyde) dynamic crosslinking agent as described in claim 2, characterized in that, The mixing reaction is carried out at room temperature; And / or, the mixing reaction is carried out in an organic solvent.

5. A hydrogel that can be in situ viscoelasticity modulated under visible light, characterized in that, The bis-salicylaldehyde dynamic crosslinking agent is obtained by crosslinking reaction of the bis-salicylaldehyde dynamic crosslinking agent and a natural polymer, and the method comprises the following steps: a. obtaining the bis-salicylaldehyde dynamic crosslinking agent solution as claimed in claim 1; b. obtaining a natural polymer solution; c. mixing the bis-salicylaldehyde dynamic crosslinking agent solution and the natural polymer solution at a certain concentration ratio to obtain a hydrogel capable of in-situ viscoelasticity regulation under visible light; the concentration ratio of the bis-salicylaldehyde dynamic crosslinking agent solution and the natural polymer solution is (1-4 wt%):(3-7 wt%).

6. The viscoelasticity in situ tunable hydrogel under visible light of claim 5, wherein, The natural polymer is selected from one or more of carboxymethyl chitosan, chitin, collagen, gelatin, and polylysine; And / or, the crosslinking reaction is carried out in a PBS buffer.

7. The in situ viscoelasticity-tunable hydrogel under visible light of claim 6, wherein, The carboxymethyl chitosan has a molecular weight of 20,000-50,000 Da; And / or, the degree of acetylation of the carboxymethyl chitosan is greater than 90%.

8. A method for preparing a hydrogel whose viscoelasticity is controllable in situ under visible light according to any one of claims 5 to 7, characterized by, The method comprises the following steps: a. obtaining the bis-salicylaldehyde dynamic crosslinking agent solution as claimed in claim 1; b. obtaining a natural polymer solution; c. mixing the bis-salicylaldehyde dynamic crosslinking agent solution and the natural polymer solution at a certain concentration ratio to obtain a hydrogel capable of in-situ viscoelasticity regulation under visible light; the concentration ratio of the bis-salicylaldehyde dynamic crosslinking agent solution and the natural polymer solution is (1-4 wt%):(3-7 wt%).

9. The production method according to claim 8, wherein In step a, the bis-salicylaldehyde dynamic crosslinking agent solution is obtained by dissolving the bis-salicylaldehyde dynamic crosslinking agent in dimethyl sulfoxide; And / or, in step b, the natural polymer solution is obtained by dissolving the natural polymer in a PBS buffer; And / or, in step c, the mixing is carried out under vortex action; And / or, in step c, the concentration ratio of the bis-salicylaldehyde dynamic crosslinking agent solution and the natural polymer solution is (1-4 wt%):(3-7 wt%).

10. Use of a hydrogel according to any one of claims 5 to 7 for in situ viscoelastic regulation under visible light, characterized in that, The hydrogel is used in the field of constructing two-dimensional and three-dimensional models of cancer metastasis and proliferation, or in the field of in-vivo degradable drug delivery and controlled-release hydrogels, or in the field of wound dressings, or in the field of constructing biomimetic extracellular matrix.

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