A multivalent bonded gel, its preparation method and application

Abstract

This invention relates to a multivalently bonded hydrogel, its preparation method, and its applications, belonging to the field of biomedical technology. The multivalently bonded hydrogel of this invention is composed of acrylamide-acrylic acid-2-acrylamide-2-methylpropanesulfonic acid (AM-AA-AMPS) copolymer grafted with adamantane and dendritic polyamide-amine polymer (PAMAM-G1) grafted with cyclodextrin through host-guest interactions. Compared with existing technologies, the multivalently bonded hydrogel of this invention has a simple preparation method, can be formed in situ, and the prepared hydrogel exhibits excellent cell adhesion, which is beneficial to cell growth.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a multivalent bonded gel, its preparation method, and its application. Background Technology

[0002] Hydrogels, hydrophilic polymer networks, are widely considered ideal candidates for extracellular matrix (ECM) in two-dimensional or three-dimensional cell culture. This material has attracted significant attention due to its structural similarity to in vivo tissues, excellent biocompatibility, and structural diversity, enabling it to meet diverse biophysical and biochemical needs. In tissue engineering, to effectively regulate cell behavior and mimic the properties of the natural ECM, hydrogels must be carefully designed to introduce biosignaling molecules with specific biophysical and biochemical properties to precisely manipulate cell adhesion and behavior. Therefore, hydrogels need to be functionalized with biosignaling molecules (such as proteins, sugars, or enzymes) whose properties change over time in response to the physiological needs of tissues. This strategy aims to achieve highly precise control over cell-material interactions to advance further research and applications in tissue engineering and regenerative medicine.

[0003] Sulfonate-containing hydrogels have attracted considerable attention due to their tunable swelling and mechanical properties. Important natural sulfonate polysaccharides such as chondroitin sulfate and dermatan sulfate have been widely used in cartilage tissue engineering, skin replacements, and the treatment of joint diseases. The introduction of chondroitin sulfate allows for precise control over the swelling properties (RSCAdvance, 2012, 2, 11095-11103), elasticity (Macromolecules, 2013, 46, 9609-9617), and drug release rate (Carbohydratepolymers, 2012, 87, 721-729) of hydrogels, while also promoting cell adhesion. This strategy provides a potentially powerful tool for further research and application of hydrogels, particularly in tissue engineering and regenerative medicine. However, the complex and uncontrollable preparation methods and the inability to form hydrogels in situ limit their application in cell adhesion. Therefore, designing a stable hydrogel that is simple to synthesize and can be formed in situ to support cell adhesion and growth is expected to drive progress in biomedical science and engineering and provide more effective solutions to various medical challenges. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a multivalent bonded hydrogel, its preparation method, and its applications. The gel material provided by this invention forms in situ through multiple host-guest interactions, which facilitates the formation of a more stable hydrogel structure. Simultaneously, the amide and sulfonic acid groups in the gel polymer backbone also promote cell adhesion and growth, thus solving the problems of complex preparation and limited cell adhesion commonly found in existing tissue engineering hydrogels.

[0005] This invention is achieved through the following technical solution:

[0006] The first objective of this invention is to provide a multivalent bonded gel, which is composed of AM-AA-AMPS polymer grafted with adamantane and PAMAM-G1 grafted with cyclodextrin through host-guest interactions.

[0007] In one embodiment of the present invention, the AM-AA-AMPS polymer grafted with adamantane has the structural formula shown in Formula I:

[0008]

[0009] The structural formula of the PAMAM-G1 grafted cyclodextrin is shown in Formula II:

[0010]

[0011] In one embodiment of the present invention, the molar ratio of AM-AA-AMPS polymer grafted with adamantane to PAMAM-G1 grafted with cyclodextrin is 20:1 to 10:1.

[0012] The molar ratio of cyclodextrin to adamantane in the gel is 1.2:1 to 1:0.8.

[0013] In one embodiment of the present invention, the AM-AA-AMPS polymer grafted with adamantane is prepared by the following method:

[0014] The AM-AA-AMPS polymer was dispersed in an organic solvent and condensed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride under ice bath conditions. Then, adamantane and N-hydroxysuccinimide were added and reacted. After dialysis, the AM-AA-AMPS polymer was grafted with adamantane.

[0015] In one embodiment of the present invention, the weight-average molecular weight of the AM-AA-AMPS polymer is 100,000 to 300,000.

[0016] In one embodiment of the present invention, the molar ratio of the carboxyl group to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in the AM-AA-AMPS polymer is 1:1 to 1:1.5; the molar ratio of the carboxyl group to adamantane in the AM-AA-AMPS polymer is 15:1 to 10:1; the molar ratio of the carboxyl group to N-hydroxysuccinimide in the AM-AA-AMPS polymer is 1:1 to 1:1.5; and the organic solvent is anhydrous N,N-dimethylformamide and / or anhydrous dimethyl sulfoxide.

[0017] In one embodiment of the present invention, the PAMAM-G1 grafted cyclodextrin is prepared by the following method:

[0018] The PAMAM-G1 solution was mixed with mono-6-O-(p-toluenesulfonyl)-β-cyclodextrin and reacted, precipitated in ethanol, dialyzed, and lyophilized to obtain the PAMAM-G1 grafted cyclodextrin.

[0019] In one embodiment of the present invention, the reaction conditions are: reaction at 60℃-80℃ for 12h to 72h.

[0020] In one embodiment of the present invention, the molecular weight of the PAMAM-G1 grafted cyclodextrin is 10,000 to 30,000.

[0021] The second objective of this invention is to provide a method for preparing a multivalent bonded gel, comprising the following steps:

[0022] AM-AA-AMPS polymer grafted with adamantane and PAMAM-G1 grafted with cyclodextrin were ultrasonically mixed in water to form a multivalent bonded gel through host-guest interaction.

[0023] In one embodiment of the present invention, the conditions for the ultrasound are: the power of the ultrasound is 100W to 500W; and the duration of the ultrasound is ≤1h.

[0024] A third objective of this invention is to provide the application of the multivalent hydrogel in cell adhesion.

[0025] Mechanism of the invention:

[0026] This invention uses a copolymer of acrylamide, acrylic acid, and 2-acrylamide-2-methylpropanesulfonic acid (AM-AA-AMPS) as a backbone, and crosslinks it with cyclodextrin and adamantane to form a gel. PAMAM, grafted with cyclodextrin, acts as a crosslinking agent for AM-AA-AMPS grafted with adamantane, forming a relatively stable hydrogel in situ through multiple host-guest interactions. The sulfonic acid and amide groups in the gel backbone enhance the interaction between cells and the gel through electrostatic interactions and hydrogen bonds, respectively, creating conditions for cell adhesion and growth of the hydrogel.

[0027] The technical solution of the present invention has the following advantages compared with the prior art:

[0028] 1. This invention provides a multivalent bonded hydrogel, its preparation method, and its application. The multivalent bonded gel of this invention is composed of acrylamide-acrylic acid-2-acrylamide-2-methylpropanesulfonic acid (AM-AA-AMPS) copolymer grafted with adamantane and dendritic polyamide-amine polymer (PAMAM-G1) grafted with cyclodextrin through host-guest interactions. The preparation method of this hydrogel is simple and can be formed in situ.

[0029] 2. The multivalent bonded hydrogel prepared by this invention has excellent cell adhesion and is suitable for cell growth. Attached Figure Description

[0030] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0031] Figure 1 This is a reaction route diagram of PAMAM-G1 grafted cyclodextrin in Example 1 of the present invention;

[0032] Figure 2 The above is the 1H NMR spectrum of AM-AA-AMPS polymer grafted with adamantane in Example 1 of this invention;

[0033] Figure 3 This is a reaction route diagram of AM-AA-AMPS polymer grafted with adamantane in Example 2 of the present invention;

[0034] Figure 4 This is a scanning electron microscope image of the hydrogel assembled in Example 3 of the present invention;

[0035] Figure 5 This is a schematic diagram of the assembly and formation of the gel in Embodiment 3 of the present invention;

[0036] Figure 6 A schematic diagram of the growth status of the U14 tumor cell line, labeled with green fluorescent dye, on a multivalently bound hydrogel. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0038] Example 1

[0039] This embodiment provides a method for preparing AM-AA-AMPS polymer grafted with adamantane, such as... Figure 1 As shown, the specific steps include the following:

[0040] Weigh 0.0842g of AM-AA-AMPS polymer (M w =200,000, Synthesis method reference Biomacromolecules 2016, 17, 4064-4074) dissolved in 20 mL of anhydrous N,N-dimethylformamide, added 0.0157 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and reacted in an ice bath for 1 hour. Then, 0.02 g of N-hydroxysuccinimide and 0.1 g of adamantane were added sequentially. After stirring at room temperature for 72 hours, dialyzed in dimethyl sulfoxide for 72 hours (M W =1,4000), continue dialyzing with deionized water for another 72 hours, and finally freeze-dry to obtain the product, namely AM-AA-AMPS polymer grafted adamantane.

[0041] The 1H NMR spectrum of the AM-AA-AMPS polymer grafted with adamantane obtained in this embodiment is shown below. Figure 2 As shown, by Figure 2 It can be seen that adamantane was successfully grafted.

[0042] Example 2

[0043] This embodiment provides a method for preparing PAMAM-G1 grafted cyclodextrin, such as... Figure 3 As shown, the specific steps include the following:

[0044] 0.05 g of PAMAM-G1 (purchased from Bailingwei, CAS: 142986-44-5) was dissolved in 20 mL of anhydrous dimethyl sulfoxide, and 0.045 g of mono-6-O-(p-toluenesulfonyl)-β-cyclodextrin was added to the solution. The mixture was reacted at 70 °C for 12 hours. After the reaction was complete, the mixture was precipitated in 200 mL of ethanol and washed three times with ethanol. The precipitate was collected by centrifugation. The precipitate was dissolved in water and dialyzed against a dialysis bag with a molecular weight cutoff of 14,000 for seven days. The product, PAMAM-G1 grafted cyclodextrin, was obtained by lyophilization.

[0045] Example 3

[0046] This embodiment provides a method for preparing a multivalent bonded hydrogel (a schematic diagram of the gel assembly is shown below). Figure 5 As shown), the specific steps include the following:

[0047] Weigh 9 mg of AM-AA-AMPS polymer grafted with adamantane prepared in Example 1 and 1 mg of PAMAM-G1 grafted with cyclodextrin prepared in Example 2 and place them in a 4 mL transparent sample bottle. Add 1 mL of deionized water and sonicate at room temperature for 1 h to form a hydrogel with multivalent bonds.

[0048] Scanning electron microscope (SEM) images of the multivalently bonded hydrogel prepared in this embodiment are shown below. Figure 4 As shown, by Figure 4 It can be seen that the hydrogel has a dense membrane structure with a certain degree of surface roughness, which is conducive to cell adhesion and growth.

[0049] Example 4

[0050] Matrigel is a multifunctional, highly biocompatible cell culture medium that mimics the natural growth environment of cells and is commonly used for the culture of stem cells and 3D cells. Therefore, this invention uses matrigel as a comparison to verify the cell adhesion properties of the hydrogel in this invention.

[0051] The multivalent bonded hydrogel and matrix gel (0.5 mL) prepared in Example 3 were spread evenly on the bottom of a 6-well plate and then mixed with U14 cells labeled with green fluorescent protein (purchased from Shanghai Chunmai Biotechnology, CL-0460) at a density of 5 × 10⁻⁶ m² / well. 5 After co-culturing at a density of cells / well for 24 and 48 hours, observation was performed under a fluorescence microscope, and the results are as follows: Figure 6 As shown in the figure. The group without hydrogel was set up as the control group. The results showed that U14 cells grew well on the hydrogel, and the number of cells was significantly increased after 48 hours compared with the control group and the matrix gel group, which proved that the hydrogel has good cell adhesion and is conducive to cell growth.

[0052] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A multivalent cross-linked gel, characterized in that, The gel is compounded by host-guest interaction of AM-AA-AMPS polymer graft adamantane and PAMAM-G1 graft cyclodextrin; The structural formula of the AM-AA-AMPS polymer graft adamantane is shown as formula I: I； The structural formula of the PAMAM-G1 graft cyclodextrin is shown as formula II: II.

2. The gel of claim 1, wherein, The molar ratio of the AM-AA-AMPS polymer graft adamantane to the PAMAM-G1 graft cyclodextrin is 20:1-10:

1.

3. The gel of claim 1, wherein, The PAMAM-G1 graft cyclodextrin is prepared by the following method: The PAMAM-G1 graft cyclodextrin is obtained by mixing and reacting PAMAM-G1 solution and mono-6-O-(p-toluenesulfonyl)-β-cyclodextrin, precipitating in ethanol, dialysis and freeze-drying.

4. The gel of claim 3, wherein, The reaction conditions are as follows: 60-80℃ for 12-72 h.

5. A method of preparing the multivalent bond-bound gel of claim 1, characterized by, The method comprises the following steps: The AM-AA-AMPS polymer graft adamantane and the PAMAM-G1 graft cyclodextrin are mixed in water by ultrasonic, and the gel combined by multiple valence bonds is formed by host-guest interaction.

6. The production method according to claim 5, wherein The ultrasonic conditions are as follows: the power is 100-500 W, and the time is ≤1 h.

7. Use of the gel combined by multiple valence bonds in any one of claims 1-4 in cell adhesion.

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