A polydopamine functionalized gelatin microgel and a preparation method thereof

Abstract

The application belongs to the field of biomedical materials, and discloses a kind of polydopamine functionalized gelatin microgel and its preparation method.The method: with anhydrous ethanol to construct emulsion phase, by introducing poloxamer and gum arabic in emulsion system to form stable gelatin droplets, the size of emulsion droplets is controlled to control the particle size of gelatin microgel.By introducing dopamine and sodium periodate in gelatin microgel dispersion, dopamine is oxidized by sodium periodate, and the oxidized dopamine can form polydopamine through self-polymerization reaction, and form a dynamic crosslinking network with the amine group on the gelatin microgel skeleton through Schiff base reaction.The gelatin microgel crosslinked by polydopamine is stable under physiological conditions, and has good biocompatibility, antioxidant activity and tissue adhesion performance, and can be used in the fields of tissue engineering, drug delivery, regenerative medicine, etc.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and specifically relates to a polydopamine-functionalized gelatin microgel and its preparation method. In particular, it describes the preparation of gelatin microgels with antioxidant activity and bioadhesion properties via an emulsion method combined with Schiff base dynamic chemical crosslinking. Background Technology

[0002] Gelatin, a hydrolyzed product of collagen, is a natural biomaterial with excellent biocompatibility, biodegradability, and non-immunogenicity. Furthermore, due to the presence of RGD (arginine-glycine-aspartic acid) recognition sites in its molecular structure, gelatin can promote cell proliferation and adhesion. Compared to traditional chemically cross-linked bulk hydrogels, gelatin microgels have a larger specific surface area, allowing for efficient material exchange between the substances within the microgel spheres and the environment, and exhibiting a degree of flowability and injectability. Moreover, physical or chemical cross-linking reactions can endow microgel systems with multifunctionality, such as bioadhesion properties, antioxidant activity, and photothermal response. Therefore, gelatin microgels have significant application value in regenerative medicine fields, including drug and bioactive molecule protection and loading, and tissue repair.

[0003] Emulsion-solvent extraction technology has been widely used in the preparation of gelatin microgels. However, the emulsion system typically requires the introduction of paraffin oil and isopropanol to form stable gelatin droplets, followed by washing with the organic solvent acetone to obtain the final gelatin microgel. The chemical reagents introduced in this preparation process may cause biocompatibility issues with the microgel. Therefore, it is essential to employ an emulsion system with better biocompatibility and to impart multifunctionality to the gelatin microgel in situ. Summary of the Invention

[0004] To address the shortcomings and deficiencies of existing technologies, this invention aims to provide a polydopamine-functionalized gelatin microgel and its preparation method. An emulsion phase is constructed using anhydrous ethanol. Stable gelatin droplets are formed by introducing poloxamer and gum arabic into the emulsion system. The particle size of the gelatin microgel is controlled by using stabilizers to control the droplet size. Poloxamer and gum arabic, as natural emulsion stabilizers, possess good biocompatibility. Based on this, dopamine and sodium periodate are introduced into the gelatin microgel dispersion solution. Dopamine is oxidized by sodium periodate, and the oxidized dopamine can self-polymerize into oligomers, which then form a dynamic chemical cross-linking network with the amine groups on the gelatin microgel backbone through a Schiff base reaction. The presence of catechol groups imparts tissue adhesion. Furthermore, the introduction of polydopamine also endows the microgel with antioxidant activity and photothermal intelligent responsive activity.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for preparing an injectable dopamine-gelatin microgel includes the following steps:

[0007] (1) Add gelatin to deionized water and stir until completely dissolved to obtain a gelatin solution.

[0008] The gelatin is derived from pigskin;

[0009] The stirring conditions are 300–500 rpm;

[0010] The stirring temperature is 40–60°C, and the reaction time is 0.5–1 hour.

[0011] The gelatin solution has a mass percentage of 3% to 5%;

[0012] (2) Anhydrous ethanol, poloxamer and gum arabic were added to the product and stirred overnight. After subsequent processing, gelatin microgels were obtained.

[0013] The volume ratio of anhydrous ethanol to deionized water is 3:5 to 1:1.

[0014] The volume ratio of gum arabic to gelatin is 0.05 to 0.1.

[0015] The stirring temperature is 20℃~25℃;

[0016] The mass ratio of poloxamer to gelatin is 0.125 to 0.25.

[0017] The subsequent processing involves centrifuging at 300g for 5 minutes to remove the emulsion phase, resuspending in PBS buffer (pH=7.4, 0.1M), centrifuging at 1000g for 5 minutes, discarding the supernatant, and repeating three times.

[0018] (3) Add dopamine hydrochloride and sodium periodate to deionized water and stir until completely dissolved.

[0019] The molar ratio of sodium periodate to dopamine hydrochloride is 0.5:1;

[0020] The concentration of the dopamine hydrochloride solution is 5–15 mg / mL;

[0021] The stirring conditions are 100–200 rpm;

[0022] The reaction time is 5 to 10 minutes;

[0023] The reaction temperature is 20–25°C.

[0024] (4) Add the gelatin microspheres prepared in step (2) to the polydopamine solution in step (3), then stir and react, and then process to obtain the dopamine-gelatin microspheres.

[0025] The reaction time is 5 to 10 minutes;

[0026] The stirring conditions are 100–300 rpm;

[0027] The subsequent treatment involves resuspending the dopamine-gelatin beads in PBS buffer, centrifuging at 1000g for 5 minutes, discarding the supernatant, and repeating the above operation 3 times.

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] This invention introduces dopamine and sodium periodate into a gelatin microgel dispersion solution. Dopamine is oxidized by sodium periodate, and the oxidized dopamine can self-polymerize into oligomers, which then form a dynamic chemical cross-linking network with the amine groups on the gelatin microgel backbone via a Schiff base reaction. The gelatin microgel cross-linked with polydopamine is stable under physiological conditions and exhibits good biocompatibility, antioxidant activity, and tissue adhesion properties, making it suitable for applications in tissue engineering, drug delivery, and regenerative medicine.

[0030] In this invention, the size of the gelatin microspheres is controlled by controlling the size of the emulsion droplets, resulting in uniform and controllable particle size of the gelatin microspheres. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0032] Figure 1 A macroscopic photograph of the polydopamine-functionalized gelatin microgel of this invention;

[0033] Figure 2 Morphology and particle size distribution of polydopamine-functionalized gelatin microgels provided by the present invention;

[0034] Figure 3 and Figure 4 Cytotoxicity evaluation of the polydopamine-functionalized gelatin microgel of this invention;

[0035] Figure 5 This invention evaluates the effect of polydopamine-functionalized gelatin microgels on promoting cell proliferation. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0037] Example 1

[0038] A method for preparing polydopamine-functionalized gelatin microgels includes the following steps:

[0039] (1) Add 0.4g of gelatin to 10mL of deionized water, keep the temperature of the constant temperature water bath at 50℃, and stir magnetically at 400rpm until completely dissolved.

[0040] (2) Add 6 mL of anhydrous ethanol to the reaction solution in step (1). After adding anhydrous ethanol, the reaction system changes from transparent to milky white.

[0041] (3) Weigh 0.05g of poloxamer and 0.02g of arabinose and add them to step (2). After they are completely dissolved, cool down to 24°C and continue stirring for 8 hours.

[0042] (4) Centrifuge 300g of the solution from step (3) for 5 minutes to remove the emulsion and obtain gelatin microgel;

[0043] (5) Resuspend the product obtained in step (4) with PBS, centrifuge at 1000g for 5 minutes, repeat three times to remove residual emulsion, and store the gelatin microgel precipitate at 4℃ after resuspending it with PBS.

[0044] (6) Weigh 0.04g of dopamine hydrochloride and 0.026g of sodium periodate into a glass bottle, add 4mL of deionized water, and stir magnetically at 200rpm for 10 minutes to ensure complete dissolution and obtain reaction system 1.

[0045] (7) Add the gelatin microgel obtained in step (5) to the reaction system 1, stir magnetically for 2 hours to ensure sufficient cross-linking, and obtain polydopamine-gelatin microgel solution;

[0046] (8) Centrifuge 1000g of the polydopamine-gelatin microgel solution from step (7) for 5 minutes to obtain polydopamine-gelatin microgel.

[0047] Example 2

[0048] A method for preparing polydopamine-functionalized gelatin microgels includes the following steps:

[0049] (1) Weigh 0.4g of gelatin into a condenser and add 10mL of deionized water. Control the temperature of the constant temperature water bath at 50℃ and stir magnetically at 400rpm until completely dissolved.

[0050] (2) Add 8 mL of anhydrous ethanol to the reaction solution in step (1). After adding anhydrous ethanol, the reaction system changes from transparent to milky white.

[0051] (3) Weigh 0.05g of poloxamer and 0.02g of arabinose and add them to step (2). After they are completely dissolved, cool down to 24°C and continue stirring for 8 hours.

[0052] (4) Centrifuge the solution from step (3) at 300g for 5 minutes to remove the emulsion and obtain gelatin microgel;

[0053] (5) Resuspend the product obtained in step (4) with PBS, centrifuge at 1000g for 5 minutes, repeat three times to remove residual emulsion, and store the gelatin microgel precipitate at 4℃ after resuspending it with PBS.

[0054] (6) Weigh 0.04g of dopamine hydrochloride and 0.026g of sodium periodate into a glass bottle, add 4mL of deionized water, and stir magnetically at 200rpm for 10 minutes to ensure complete dissolution and obtain reaction system 1.

[0055] (7) Add the gelatin microgel obtained in step (5) to the reaction system 1, stir magnetically for 2 hours to ensure sufficient cross-linking, and obtain polydopamine-gelatin microgel solution;

[0056] (8) Centrifuge 1000g of the polydopamine-gelatin microgel solution from step (7) for 5 minutes to obtain polydopamine-gelatin microgel.

[0057] Example 3

[0058] A method for preparing polydopamine-functionalized gelatin microgels includes the following steps:

[0059] (1) Add 0.4g of gelatin to 10mL of deionized water, keep the temperature of the constant temperature water bath at 50℃, and stir magnetically at 400rpm until completely dissolved.

[0060] (2) Add 10 mL of anhydrous ethanol to the reaction solution in step (1). After adding anhydrous ethanol, the reaction system changes from transparent to milky white.

[0061] (3) Weigh 0.1g of poloxamer and 0.04g of arabinose and add them to step (2). After they are completely dissolved, cool down to 24°C and continue stirring for 8 hours.

[0062] (4) Centrifuge 300g of the solution from step (3) for 5 minutes to remove the emulsion and obtain gelatin microgel;

[0063] (5) Resuspend the product obtained in step (4) with PBS, centrifuge at 1000g for 5 minutes, repeat three times to remove residual emulsion, and store the gelatin microgel precipitate at 4℃ after resuspending it with PBS.

[0064] (6) Weigh 0.04g of dopamine hydrochloride and 0.026g of sodium periodate into a glass bottle, add 4mL of deionized water, and stir magnetically at 200rpm for 10 minutes to ensure complete dissolution and obtain reaction system 1.

[0065] (7) Add the gelatin microgel obtained in step (5) to the reaction system 1, stir magnetically for 2 hours to ensure sufficient cross-linking, and obtain polydopamine-gelatin microgel solution;

[0066] (8) Centrifuge 1000g of the polydopamine-gelatin microsphere solution from step (7) for 5 minutes to obtain polydopamine-gelatin microgel.

[0067] A physical image of the polydopamine-gelatin microgel prepared in this example is shown below. Figure 1 As shown, the gelatin microgels crosslinked with polydopamine changed from colorless to brownish-red, and the polydopamine-gelatin microspheres remained stable under physiological conditions, with the gel network not being disrupted. By introducing emulsion stabilizers (poloxam and gum arabic) into the emulsion system to control the size of the emulsion droplets, polydopamine-gelatin microspheres of three sizes were prepared. Figure 2 As shown, the particle sizes of the three sizes of polydopamine-gelatin microspheres are mainly distributed at 15 μm, 40 μm, and 100 μm. The cytotoxicity evaluation of the polydopamine-gelatin microgels prepared in this example is as follows: Figure 3 , Figure 4 As shown, after culturing L929 cells with polydopamine-gelatin microgel extract for 24 hours, the cell viability remained above 90%, and immunofluorescence results also demonstrated that the polydopamine-gelatin microgel exhibited good cell compatibility. Figure 5 As shown, after culturing cells for 72 hours, the cell number increased significantly over time, especially on the third day. Cells cultured in the polydopamine-gelatin microgel extract showed more significant proliferation, with OD values ​​higher than the positive control group. This indicates that polydopamine-gelatin microgel promotes cell proliferation.

[0068] Conclusion: We have successfully prepared a polydopamine-functionalized gelatin microgel with good biocompatibility, antioxidant activity and tissue adhesion properties, which can be used in tissue engineering, drug delivery, regenerative medicine and other fields.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered therein.

Claims

1. A method for preparing polydopamine-functionalized gelatin microgels, characterized in that: Includes the following steps: Using anhydrous ethanol as the emulsion phase, stable emulsion droplets are formed by introducing an emulsion stabilizer. Subsequently, dopamine and sodium periodate are introduced into the gelatin microgel dispersion. Dopamine is oxidized by sodium periodate, and the oxidized dopamine self-polymerizes into oligomers, which then form a dynamic cross-linked network with the amine groups on the gelatin microgel backbone through a Schiff base reaction. The preparation method of the polydopamine-functionalized gelatin microgel includes the following steps: (1) Add gelatin to deionized water and stir at 40~60℃ until completely dissolved to obtain solution 1; (2) Add anhydrous ethanol to solution 1 from step (1). After adding anhydrous ethanol, the reaction system changes from transparent to milky white, resulting in solution 2. The volume ratio of anhydrous ethanol to deionized water is 3:5 to 1:

1. (3) Add poloxamer and gum arabic to solution 2 in step (2), cool to 20℃~25℃ and stir. After subsequent processing, gelatin microgels are obtained; wherein the volume ratio of gum arabic to gelatin is 0.05~0.1 and the mass ratio of poloxamer to gelatin is 0.125~0.

25. (4) Add dopamine and sodium periodate to deionized water and stir for 5-10 minutes to obtain polydopamine solution; wherein the molar ratio of sodium periodate to dopamine is 0.5:1; and the concentration of dopamine solution is 5-15 mg / mL. (5) Add the gelatin microgel prepared in step (3) to the polydopamine solution in step (4), and then stir magnetically for 1 to 2 hours to obtain dopamine-gelatin microspheres; wherein the magnetic stirring speed is 100 to 300 rpm.

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