A polydopamine hybrid agarose hydrogel, a preparation method and application thereof

Abstract

The present application relates to a kind of polydopamine hybridization agarose hydrogel and preparation method and application, preparation method includes: by dissolving dopamine hydrochloride in NaOH aqueous solution, oxidation reaction is carried out under air condition to prepare polydopamine solution, then, agarose is added to mixture, using microwave heating to dissolve the added agarose, the resulting hot thick uniform solution is converted into gel in 10 minutes under natural cooling, such hydrogel support prepared by the fast and economic efficient method of the present application only has excellent biocompatibility and cell adhesion, has wide application prospect in 3D cell culture and tissue engineering field.

Description

Technical Field

[0001] This invention relates to the field of biological scaffolds, and in particular to a polydopamine hybrid agarose hydrogel, its preparation method, and its application. Background Technology

[0002] Hydrogels with high-water-content three-dimensional networks have demonstrated excellent extracellular matrix (ECM)-like properties. Due to their ability to promote cell proliferation, encapsulate cells, and isolate biomolecules without affecting their biological activity, hydrogels play a crucial role in the development of biotechnology. In particular, these characteristics endow hydrogels with the potential to serve as cell culture scaffolds. Compared to traditional polystyrene cell culture flasks or dishes, the main advantages of hydrogels lie in their inherent protein-loading capacity and biocompatibility, allowing for direct implantation into organisms to promote tissue regeneration. The physical parameters of hydrogels, including stiffness, pore size, and hydrophilicity, have been shown to influence the interaction between cells and hydrogel scaffolds, as well as the corresponding cell proliferation, migration, and differentiation. However, the design of hydrogel scaffolds that meet all the requirements of tissue engineering remains far from being realized.

[0003] Agarose is a highly attractive natural polysaccharide, often used in the manufacture of hydrogel scaffolds due to its inherent biocompatibility and bioactivity. Currently, a major limitation of agarose-derived hydrogel scaffolds is the lack of surfaces that support cell adhesion and spreading, a prerequisite for tissue engineering applications. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a polydopamine hybrid agarose hydrogel, its preparation method, and its applications.

[0005] To achieve the above objectives, the present invention provides a method for preparing polydopamine hybrid agarose hydrogel. Solid dopamine hydrochloride is dissolved in an aqueous sodium hydroxide solution with pH 8-10. An oxidation reaction is carried out under stirring at room temperature to prepare an aqueous solution containing 0.02-2 g / L dopamine hydrochloride and sodium hydroxide. An aqueous agarose solution of 1-50 g / L is added, and the mixture is heated to dissolve. The product is then obtained by cooling. The volume ratio of the aqueous dopamine hydrochloride and sodium hydroxide solution to the aqueous agarose solution is 1:1.

[0006] Furthermore, the concentration of the sodium hydroxide hydrochloride-dopamine hydrochloride aqueous solution is 0.1-0.4 g / L, and the concentration of the agarose aqueous solution is 40 g / L.

[0007] The present invention also provides a polydopamine hybrid agarose hydrogel prepared by the above-mentioned method for preparing polydopamine hybrid agarose hydrogel.

[0008] The present invention also provides the application of the above-mentioned polydopamine hybridization agarose hydrogel, specifically its application in the preparation of cell scaffolds.

[0009] As one application of the present invention, the cell scaffold is a 3D lung fibroblast culture scaffold.

[0010] The present invention also provides the application of the above-mentioned polydopamine hybrid agarose hydrogel, specifically its use in preparing skin repair gels.

[0011] As one application of the present invention, the skin repair gel is loaded with one or more of the following: cell growth factors, antibacterial agents, moisturizers, preservatives, antioxidants, emulsifiers, thickeners, sweeteners, gelling agents, fragrances, and flavoring agents.

[0012] The present invention has the following advantages: Polydopamine (PDA) has functional groups (catechol groups) similar to those in mussel foot muscle proteins, which can significantly enhance the adhesive properties of materials. The catechol groups of PDA can interact with various surfaces through hydrogen bonding, electrostatic interactions, and π-π stacking, giving the gel significant tissue adhesion properties. This application presents a polydopamine-doped agarose-based composite hydrogel, which provides the three-dimensional environment required for cell culture and can be used as a tissue engineering scaffold in tissue repair and tissue engineering. Attached Figure Description

[0013] Figure 1 This is a diagram showing the preparation of polydopamine in situ hybridization agarose hydrogel;

[0014] Figure 2 The results of LIVE / DEAD staining analysis of human embryonic lung fibroblasts on the cell scaffold material provided in the embodiments of the present invention;

[0015] Figure 3 SEM images of rat lung H&E staining and hydrogel scaffold;

[0016] Figure 4 The results of LIVE / DEAD staining analysis of rat skin fibroblasts on the cell scaffold material provided in the embodiments of the present invention. Detailed Implementation

[0017] The present invention will now be described in further detail with reference to embodiments and effect examples, but this is not intended to limit the scope of the invention.

[0018] Example 1: Preparation of dopamine in situ hybridization agarose hydrogel

[0019] The preparation method steps are as follows:

[0020] 1) The sodium hydroxide aqueous solution of dopamine hydrochloride is prepared by dissolving 0.2g of solid dopamine hydrochloride in 1L of sodium hydroxide aqueous solution (pH=8) and carrying out an oxidation reaction under air conditions (25℃, magnetic stirring for 24h).

[0021] 2) Take 2.5 mL of the dopamine hydrochloride sodium hydroxide aqueous solution from step 1 and mix them together with 7.5 mL of deionized water. Then, add 0.2 g of agarose to each mixture and dissolve the added agarose using microwave heating.

[0022] 3) Take the hot, thick, and homogeneous solution obtained in step 2 and allow it to cool naturally (25°C) for 10 minutes to transform into a gel, thus obtaining the final hydrogel.

[0023] Example 2: Preparation of dopamine in situ hybridization agarose hydrogel

[0024] The preparation method steps are as follows:

[0025] 1) The sodium hydroxide aqueous solution of dopamine hydrochloride is prepared by dissolving 0.2g of solid dopamine hydrochloride in 1L of sodium hydroxide aqueous solution (pH=8) and carrying out an oxidation reaction under air conditions (25℃, magnetic stirring for 24h).

[0026] 2) Take 5 mL of the dopamine hydrochloride sodium hydroxide aqueous solution from step 1 and mix it with 5 mL of deionized water. Then, add 0.2 g of agarose to each mixture and dissolve the added agarose using microwave heating.

[0027] 3) Take the hot, thick, and homogeneous solution obtained in step 2 and allow it to cool naturally (25°C) for 10 minutes to transform into a gel, thus obtaining the final hydrogel.

[0028] Example 3: Preparation of dopamine in situ hybridization agarose hydrogel

[0029] The preparation method steps are as follows:

[0030] 1) The sodium hydroxide aqueous solution of dopamine hydrochloride is prepared by dissolving 0.2g of solid dopamine hydrochloride in 1L of sodium hydroxide aqueous solution (pH=8) and carrying out an oxidation reaction under air conditions (25℃, magnetic stirring for 24h).

[0031] 2) Take 10 mL of the sodium hydroxide hydrochloride solution from step 1, and then add 0.2 g of agarose to each mixture. Dissolve the added agarose by microwave heating.

[0032] 3) Take the hot, thick, and homogeneous solution obtained in step 2 and allow it to cool naturally (25°C) for 10 minutes to transform into a gel, thus obtaining the final hydrogel.

[0033] Example 4: Evaluation of 3D Culture of Human Lung Fibroblasts (MRC-5 Cells)

[0034] The evaluation steps are as follows:

[0035] A control group was set up by adding 0.2g of agarose to 10ml of deionized water and dissolving it using microwave heating. The agarose was then naturally cooled (25℃) and converted into a control gel within 10 minutes, denoted as A0. The hydrogels prepared in Examples 1-3 were denoted as A1, A2, and A3, respectively. A schematic diagram of their preparation process is shown below. Figure 1 As shown.

[0036] Hydrogels A0, A1, A2, and A3 were used, and the fibroblast cell line (MRC-5) was selected. The in vitro cytotoxicity of the polydopamine in situ hybridization agarose hydrogels was evaluated using CCK-8 assay. Cell adhesion ability was assessed by seeding cells on the surface of the polydopamine in situ hybridization agarose hydrogels, adding culture medium, and incubating for 1, 4, and 7 days. Cell LIVE / DEAD staining results (bivariate analysis of PDA content and incubation time) were performed. The culture media used were DMEM (Gibco) and α-DMEM (DPSC, Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin / streptomycin (P / S, Gibco). Cells were cultured at 37°C in a humidified 5% CO2 atmosphere. The culture medium was changed every two days to remove dead cells.

[0037] Figure 2 The results showed that the cell scaffold material (A3) provided in Examples 1-3 of the present invention enabled MRC-5 cells to adhere, grow and proliferate well on it, and the prepared hydrogel could be used as a scaffold for 3D culture of MRC-5 cells.

[0038] The main pathogenesis of COVID-19 is the accumulation of inflammatory secretions or exudates in the pulmonary interstitium and alveoli, which blocks the exchange of fresh air with carbon dioxide, leading to hypoxia. To better mimic the physiological state of COVID-19 patients and improve the accuracy of drug screening, this invention constructs a 3D lung cell culture evaluation scaffold that effectively simulates the environment of a living organism for the culture of cells, tissues, and organs. Its internal structure is as follows... Figure 3 As shown, it has pores of 200-1000μm and an internal 3D structure, which highly simulates the internal environment of mouse lungs. It can be used to study and control the biological behavior of cells during in vitro culture, thereby reproducing some key functions of the lung organs and applying them in various fields such as antiviral drug evaluation, disease models and basic medicine.

[0039] Example 5: Construction of an in vitro skin fibroblast scaffold (RS-1 cells)

[0040] The evaluation steps are as follows:

[0041] A control group was set up by adding 0.2g of agarose to 10ml of deionized water and dissolving it using microwave heating. The agarose was then naturally cooled (25℃) and converted into a control gel within 10 minutes, denoted as A0. The hydrogels prepared in Examples 1-3 were denoted as A1, A2, and A3, respectively. A schematic diagram of their preparation process is shown below. Figure 1 As shown.

[0042] Hydrogels A0, A1, A2, and A3 were used, and the fibroblast cell line (RS-1) was selected. The in vitro cytotoxicity of the polydopamine in situ hybridization agarose hydrogels was evaluated using CCK-8 assay. Cell LIVE / DEAD staining analysis (bivariate analysis of PDA content and incubation time) was performed after incubation with the polydopamine in situ hybridization agarose hydrogels for 1, 4, and 7 days. The culture media used were DMEM medium (Gibco) and α-DMEM medium (DPSC, Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin / streptomycin (PS, Gibco). Cells were cultured at 37°C in a humidified 5% CO2 atmosphere. The culture medium was changed every two days to remove dead cells.

[0043] Figure 4 The results showed that the cell scaffold material (A3) provided in this embodiment of the invention enabled RS-1 cells to adhere, grow, and proliferate well. The cell scaffold constructed by this invention using a simple and convenient one-step mixing method can provide an ideal growth environment for skin fibroblasts. Furthermore, this hydrogel scaffold can be further loaded with growth factors and antibacterial drugs, giving this material potential application value in repairing skin wounds.

[0044] Finally, it is necessary to state that the above embodiments are only used to further illustrate the technical solution of the present invention in detail, and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention shall fall within the scope of protection of the present invention.

Claims

1. A polydopamine hybridization agarose hydrogel cell scaffold, characterized in that, The gel is prepared by the following method: Solid dopamine hydrochloride was dissolved in an aqueous sodium hydroxide solution with pH = 8-10, and an oxidation reaction was carried out under stirring at room temperature to prepare an aqueous solution of sodium hydroxide containing 0.1-0.4 g / L dopamine hydrochloride. 40 g / L agarose aqueous solution was added, and the mixture was heated to dissolve and then cooled to obtain the product. The volume ratio of the aqueous solution of sodium hydroxide containing dopamine hydrochloride to the aqueous solution of agarose was 1:

1.

2. A polydopamine hybrid agarose hydrogel cell scaffold, characterized in that, Application of the polydopamine hybrid agarose hydrogel cell scaffold according to claim 1 in the preparation of 3D lung fibroblast culture scaffolds.

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