CN111701074A

CN111701074A - Polydopamine hybrid agarose hydrogel, and preparation method and application thereof

Info

Publication number: CN111701074A
Application number: CN202010487175.5A
Authority: CN
Inventors: 齐晓亮
Original assignee: Wenzhou Medical University
Current assignee: Wenzhou Medical University
Priority date: 2020-06-01
Filing date: 2020-06-01
Publication date: 2020-09-25

Abstract

The invention relates to polydopamine hybrid agarose hydrogel and a preparation method and application thereof, wherein the preparation method comprises the following steps: the dopamine hydrochloride is dissolved in NaOH aqueous solution, oxidation reaction is carried out under the air condition to prepare polydopamine solution, then agarose is added into the mixture, the added agarose is heated and dissolved by microwaves, and the obtained hot and thick uniform solution is converted into gel within 10 minutes under natural cooling.

Description

Polydopamine hybrid agarose hydrogel, and preparation method and application thereof

Technical Field

The invention relates to the field of biological scaffolds, in particular to a polydopamine hybrid agarose hydrogel, and a preparation method and application thereof.

Background

Hydrogels with high water content three-dimensional networks have shown excellent extracellular matrix (ECM) -like properties. Hydrogels play a crucial role in the development of biotechnology due to their ability to promote cell proliferation, to encapsulate and isolate biomolecules from cells without affecting their biological activity. In particular, these features confer the hydrogel the potential to act as a scaffold for cell culture. The main advantages of hydrogels over traditional polystyrene cell culture flasks or dishes are their inherent protein loading capacity and biocompatibility, which can be directly implanted into the organism to promote tissue regeneration. Physical parameters of hydrogels, including stiffness, pore size and hydrophilicity, have been shown to affect cell-hydrogel scaffold interactions and corresponding cell proliferation, migration, and differentiation. However, the design of hydrogel scaffolds to meet all the requirements of tissue engineering is still far from being realized.

Agarose is an attractive natural polysaccharide that is commonly used to make hydrogel scaffolds due to its inherent biocompatibility and bioactivity. Currently, the major limitation of agarose-derived hydrogel scaffolds is the inability to provide a surface that supports cell adhesion and spreading, a prerequisite for tissue engineering applications.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a polydopamine hybrid agarose hydrogel, a preparation method and application thereof.

In order to achieve the purpose, the invention provides a preparation method of a polydopamine hybrid agarose hydrogel, which is characterized in that solid dopamine hydrochloride is dissolved in a sodium hydroxide aqueous solution with the pH value of 8-10, the solid dopamine hydrochloride is stirred under the condition of normal temperature to carry out oxidation reaction to prepare a dopamine hydrochloride sodium hydroxide aqueous solution containing 0.02-2g/L, 1-50g/L agarose aqueous solution is added, the mixture is heated, mixed and dissolved, and the product is obtained by cooling, wherein the volume ratio of the dopamine hydrochloride sodium hydroxide aqueous solution to the agarose aqueous solution is 1: 1.

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

The invention also provides the polydopamine hybrid agarose hydrogel prepared by the preparation method of the polydopamine hybrid agarose hydrogel.

The invention also provides application of the polydopamine hybrid agarose hydrogel and application of the polydopamine hybrid agarose hydrogel in preparation of cell scaffolds.

As an application mode of the invention, the cell scaffold is a 3D lung fibroblast culture scaffold.

The invention also provides application of the polydopamine hybrid agarose hydrogel and application of the polydopamine hybrid agarose hydrogel in preparing skin repair gel.

As an application mode of the present invention, the skin repair gel is loaded with one or more of a cell growth factor, an antibacterial agent, a humectant, a preservative, an antioxidant, an emulsifier, a thickener, a sweetener, a mucilage, an aromatic and a flavoring agent.

The invention has the following advantages: the Polydopamine (PDA) has functional groups (catechol groups) similar to foot muscle protein of mussel, so that the adhesion performance of the material can be obviously enhanced, the catechol groups of the polydopamine can interact with various surfaces through hydrogen bonds, electrostatic interaction and pi-pi accumulation, so that the gel has obvious tissue adhesion characteristics.

Drawings

FIG. 1 is a diagram of the preparation of polydopamine in situ hybridization agarose hydrogel;

FIG. 2 shows LIVE/DEAD staining analysis results of human embryonic lung fibroblasts on a cell scaffold material provided in an embodiment of the present invention;

FIG. 3 is an electron SEM photograph of H & E staining of rat lungs and a hydrogel scaffold;

FIG. 4 shows LIVE/DEAD staining analysis results of rat skin fibroblast cells on cell scaffold materials provided in the examples of the present invention.

Detailed Description

The present invention will be further described in detail with reference to examples and effect examples, but the scope of the present invention is not limited thereto.

Example 1: preparation of dopamine in situ hybridization agarose hydrogel

The preparation method comprises the following steps:

1) the aqueous dopamine hydrochloride solution was prepared by dissolving 0.2g of solid dopamine hydrochloride in 1L of aqueous sodium hydroxide solution (pH 8) and carrying out oxidation reaction under air conditions (25 ℃, 24h with magnetic stirring).

2) 2.5mL of dopamine-sodium hydroxide hydrochloride aqueous solution obtained in step 1 and 7.5mL of deionized water were mixed together. Then, 0.2g of agarose was added to each mixture, and the added agarose was dissolved using microwave heating.

3) And (3) taking the hot thick uniform solution finally obtained in the step (2), and converting the hot thick uniform solution into gel in 10 minutes under natural cooling (25 ℃) to obtain the final hydrogel.

Example 2: preparation of dopamine in situ hybridization agarose hydrogel

The preparation method comprises the following steps:

2) 5mL of dopamine hydrochloride sodium hydroxide aqueous solution and 5mL of deionized water in the step 1 are mixed together. Then, 0.2g of agarose was added to each mixture, and the added agarose was dissolved using microwave heating.

Example 3: preparation of dopamine in situ hybridization agarose hydrogel

The preparation method comprises the following steps:

2) 10mL of the dopamine hydrochloride aqueous solution in step 1 was taken, 0.2g of agarose was then added to each mixture, and the added agarose was dissolved by microwave heating.

Example 4: human lung fibroblast 3D culture evaluation (MRC-5 cells)

The evaluation steps are as follows:

setting a control group, adding 0.2g of agarose into 10ml of deionized water, dissolving the added agarose by microwave heating, and converting the agarose into a control gel A0 in 10 minutes under natural cooling (25 ℃); the hydrogels prepared in examples 1-3 were designated as A1, A2, A3, respectively, and the preparation process is schematically shown in FIG. 1.

The cell adhesion ability of the material was assessed by plating cells on the surface of the polydopamine in situ hybridization agarose hydrogel and adding culture medium (PDA content and incubation time bivariate) after incubating for 1, 4 and 7 days, wherein the culture medium uses DMEM medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (P/S, Gibco) and α -DMEM medium (DPSC, Gibco) containing cells in humidified 5% CO₂The culture was carried out at 37 ℃ under an atmosphere. The medium was changed every two days to remove dead cells.

The results in FIG. 2 show that the cell scaffold material (A3) provided in examples 1-3 of the present invention enables MRC-5 cells to adhere, grow and proliferate well thereon, and the prepared hydrogel can be used as a scaffold for 3D culture of MRC-5 cells.

The main pathogenesis of the novel coronavirus pneumonia (COVID-19) is that a large amount of inflammatory secretion or exudate is filled in lung interstitium and alveoli, so that fresh air is blocked from entering the alveoli to exchange carbon dioxide in the body, and the body is lack of oxygen. In order to better simulate the physiological condition of a new coronary pneumonia patient and enable drug screening to be more accurate, the 3D lung cell culture evaluation stent constructed by the invention can effectively simulate the environment of an organism to culture cells, tissues and organs, the internal structure of the stent is shown in figure 3, the stent has pores of 200 plus 1000 mu m, the internal structure of the stent is a 3-dimensional structure, the internal environment of the lung of a mouse is highly simulated, the biological behavior of the cells in the in-vitro culture process is researched and controlled, and therefore, partial key functions of the lung organ are reproduced, and the stent can be applied to various aspects such as antiviral drug evaluation, disease models, basic medical fields and the like.

Example 5: construction of in vitro skin fibroblast scaffolds (RS-1 cells)

The evaluation steps are as follows:

Incubation with the polydopamine in situ hybridization agarose hydrogel for 1, 4 and 7 days followed by LIVE/DEAD staining analysis of the cells (both variable PDA content and incubation time), wherein the culture medium used was DMEM medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (PS, Gibco) and α -DMEM medium (DPSC, Gibco), cells were incubated in humidified 5% CO₂The culture was carried out at 37 ℃ under an atmosphere. The medium was changed every two days to remove dead cells.

The results in FIG. 4 show that the cell scaffold material (A3) provided by the embodiment of the present invention enables RS-1 cells to adhere, grow and proliferate better thereon. The cell scaffold constructed by a simple and convenient one-step mixing method can provide an ideal growth environment for skin fibroblasts. And the hydrogel scaffold can be further loaded with some growth factors, antibacterial drugs and the like, so that the material has potential application value in repairing skin wounds.

Finally, it must be said here that: the above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be understood as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art according to the above descriptions of the present invention are within the scope of the present invention.

Claims

1. A preparation method of polydopamine hybrid agarose hydrogel is characterized by comprising the following steps: dissolving dopamine hydrochloride solid in a sodium hydroxide aqueous solution with the pH value of 8-10, stirring at normal temperature to perform oxidation reaction to prepare a dopamine hydrochloride aqueous solution containing 0.02-2g/L, adding 1-50g/L agarose aqueous solution, heating, mixing and dissolving, and cooling to obtain a product, wherein the volume ratio of the dopamine hydrochloride aqueous solution to the agarose aqueous solution is 1: 1.

2. A preparation method of polydopamine hybrid agarose hydrogel is characterized by comprising the following steps: the concentration of the dopamine-sodium hydroxide hydrochloride aqueous solution is 0.1-0.4g/L, and the concentration of the agarose aqueous solution is 40 g/L.

3. A polydopamine hybrid agarose hydrogel, wherein the gel is prepared by the method for preparing the polydopamine hybrid agarose hydrogel according to claim 1 or 2.

4. Use of a polydopamine hybrid agarose hydrogel according to claim 3 for the preparation of a cytoskeleton.

5. The use of the polydopamine hybrid agarose hydrogel according to claim 4, wherein the cell scaffold is a 3D lung fibroblast culture scaffold.

6. Use of a polydopamine hybrid agarose hydrogel according to claim 3 for the preparation of a skin repair gel.

7. The use of a polydopamine hybrid agarose hydrogel according to claim 6, wherein the skin repair gel is loaded with one or more of a cell growth factor, an antibacterial agent, a humectant, a preservative, an antioxidant, an emulsifier, a thickener, a sweetener, a mucilage, an aromatic, and a flavoring agent.