阅读说明：本技术 一种果胶基双物理交联水凝胶及制备方法和应用 (Pectin-based double-physical crosslinked hydrogel and preparation method and application thereof ) 是由 姚芳莲 李俊杰 吴晓军 秦志辉 于 2019-08-13 设计创作，主要内容包括：本发明涉及一种果胶基双物理交联水凝胶及制备方法和应用,该凝胶制备过程分为三步：首先制备凝胶前驱液：将疏水单体增溶到十二烷基硫酸钠胶束溶液,果胶粉末、丙烯酰胺及光引发剂均匀分散在胶束溶液后获得前驱液；其次紫外光引发自由基聚合使凝胶前驱液转变为单交联水凝胶；最后是浸泡处理：将单交联水凝胶浸泡于Fe<Sup>3+</Sup>溶液中后,取出再置于去离子水中浸泡,得到具有高强韧性的果胶基双物理交联水凝胶。该水凝胶具有可调节的力学性能：拉伸强度0.60-2.0MPa,断裂应变130-1500％,弹性模量0.3-2.0MPa,韧性1.0-12.0MJ m<Sup>-3</Sup>。该水凝胶具有良好的生物相容性,可用于3D细胞培养。(The invention relates to a pectin-based dual-physical cross-linked hydrogel and a preparation method and application thereof, wherein the preparation process of the gel comprises the three steps of preparing a gel precursor solution, solubilizing a hydrophobic monomer into a lauryl sodium sulfate micelle solution, uniformly dispersing pectin powder, acrylamide and a photoinitiator in the micelle solution to obtain the precursor solution, initiating free radical polymerization by ultraviolet light to convert the gel precursor solution into the single cross-linked hydrogel, and finally soaking the single cross-linked hydrogel into a Fe 3+ solution, taking out the single cross-linked hydrogel and soaking the single cross-linked hydrogel in deionized water to obtain the pectin-based dual-physical cross-linked hydrogel with high toughness.)

1. A pectin-based dual physically crosslinked hydrogel; the gel is characterized in that the gel is determined to be physically cross-linked acrylamide and pectin by analyzing Fourier infrared spectrum, the total solid content is 12-25%, and the mass ratio of the pectin to the acrylamide is 1:1.5-1: 20.

2. A method of preparing the pectin-based dual physically crosslinked hydrogel of claim 1 comprising the steps of:

(1) Preparing 7 mass percent of anionic surfactant lauryl sodium sulfate micellar solution;

(2) adding a hydrophobic monomer octadecyl methacrylate into the micellar solution obtained in the step (1), and magnetically stirring at 25-70 ℃ to obtain a transparent mixed solution;

(3) Adding pectin powder into the mixed solution obtained in the step (2), and magnetically stirring at 25-70 ℃ to obtain a uniform mixed solution;

(4) Adding acrylamide and a photoinitiator I2959 into the solution obtained in the step (3), and magnetically stirring at 25-70 ℃ to obtain a uniform solution;

(5) injecting the uniform solution obtained in the step (4) into a mold, and initiating by ultraviolet light to obtain single-crosslinking single-network hydrogel;

(6) And (4) immersing the single-crosslinking single-network hydrogel obtained in the step (5) into a ferric ion solution, taking out the single-crosslinking single-network hydrogel and immersing the single-crosslinking single-network hydrogel into deionized water to obtain the double-crosslinking double-network hydrogel.

3. the method as set forth in claim 2, wherein the molar ratio of stearyl methacrylate to acrylamide in said step (2) is 1 to 4 mol%.

4. the method as set forth in claim 2, characterized in that the mass ratio of pectin to acrylamide in the step (3) is 1:1.5 to 1: 20.

5. The method of claim 2, wherein in step (4), the photoinitiator I2959 is 1 mol% of the total amount of acrylamide and the hydrophobic monomer octadecyl methacrylate.

6. the method as set forth in claim 2, wherein the photoinitiation time in said step (5) is 1-5 h; the ultraviolet light wavelength is 250-420nm, and the light intensity is 5-8W.

7. The method as set forth in claim 2, characterized in that the molar concentration of the solution of iron ions in said soaking in step (6) is 0.02-0.2M.

8. the method as set forth in claim 2, characterized in that the soaking time in the iron ion solution in the step (6) is 10min to 16 h; soaking in deionized water for 1-72 hr.

9. The pectin-based dual physically crosslinked hydrogel of claim 1 for use in 3D cell culture.

Technical Field

the invention relates to a pectin-based dual-physical cross-linked hydrogel as well as a preparation method and application thereof, in particular to a method for preparing the pectin-based hydrogel which has excellent biocompatibility and can be used for 3D cell culture through dual-physical cross-linking, and belongs to the field of functional materials.

Background

The hydrogel is a three-dimensional network structure formed by crosslinking various covalent bonds mainly through the physical or chemical crosslinking action of hydrophilic polymers, wherein the physical crosslinking action has the functions of hydrophobic association, coordination complexing, hydrogen bonds and the like, and water molecules are combined and retained in the network through free diffusion and attraction of hydrophilic groups, so that the hydrogel is soft and elastic in texture and has a certain shape. The unique 'soft and wet' property is similar to that of natural tissue extracellular matrix, and is an ideal material for developing a novel scaffold for repairing bone and cartilage tissues. The natural polysaccharide has good biocompatibility, degradability and immunogenicity, and becomes an excellent raw material for constructing hydrogel for tissue engineering. However, the inherent brittleness and poor stability of conventional polysaccharide-based hydrogels limit their application in the field of tissue engineering.

In view of the above problems, a series of high strength and high toughness hydrogels have been developed from the viewpoint of energy dissipation or stress dispersion: nano composite hydrogel, macromolecular microsphere hydrogel, slip ring structure hydrogel, double-network hydrogel, four-arm PEG hydrogel and the like. The high efficiency energy dissipation mechanism makes the double-network hydrogel a widely adopted strategy in recent years for constructing high-strength and high-toughness hydrogel. However, the double-network hydrogel formed by crosslinking through covalent bond action has the problem of poor fatigue resistance, and the covalent bond used for dissipating energy and playing the role of a 'sacrificial bond' is difficult to recover once being broken, while in a practical application scene, the hydrogel is generally required to endure repeated stretching or compression. The construction of the high-toughness double-network hydrogel based on pure physical crosslinking is one of effective means for solving the problems, and the dynamic property of the physical crosslinking effect can realize the recovery after the hydrogel is fractured. Therefore, the introduction of dynamic physical cross-linking bonds such as hydrogen bonds, ionic bonds, hydrophobic association and the like into the gel network to construct the high-toughness double-network hydrogel with self-recovery, anti-fatigue and even self-healing characteristics becomes a research hotspot. At present, physical crosslinked hydrogel with elastic modulus or tensile strength reaching MPa level is reported, but the physical crosslinked hydrogel mainly focuses on enhancing mechanical property and lacks of exploration in the aspect of biocompatibility, and the application field of the developed hydrogel is limited. The invention takes natural polymers with wide sources, low price and easy obtainment, biocompatibility and degradability as raw materials, adopts a 'double-network' strategy to develop high-strength and high-toughness hydrogel with excellent mechanical property and biocompatibility, and in vitro cell culture proves that the gel can be used as a 3D cell culture scaffold and has wide application prospect in the field of tissue engineering.

Disclosure of Invention

The invention aims to overcome the defects that the conventional polysaccharide-based dual-network high-toughness hydrogel is lack of mechanical self-recovery property, weak in fatigue resistance, poor in biocompatibility and the like, and provides the pectin-based physical cross-linked hydrogel which is simple in preparation process, has mechanical self-recovery property and fatigue resistance and has excellent biocompatibility and the preparation method thereof.

The technical scheme of the invention is as follows:

A pectin-based dual-physical cross-linked hydrogel is prepared through such steps as dissolving octadecyl methacrylate (SMA) in the solution of Sodium Dodecyl Sulfate (SDS) micelle, uniformly dispersing pectin powder in the solution of hydrophobic monomer, adding acrylamide and photo-trigger, photo-trigger converting the precursor liquid to single-cross-linked single-network hydrogel, ultraviolet radiating to trigger in-situ free radical polymerization to obtain the copolymer chain of acrylamide and methyl methacrylate (AM-co-SMA), which is prepared through the hydrophobic association between SMA to obtain hydrophobic micro-domain as cross-linking point to obtain single-network hydrogel, immersing the single-network hydrogel in trivalent Fe ion solution to obtain dual-network hydrogel, immersing the dual-network hydrogel in deionized water to remove Fe ³⁺, and removing Fe ³⁺.

the invention relates to a pectin-based dual-physical crosslinked hydrogel; the hydrogel with mechanical property self-recovery property, fatigue resistance and excellent biocompatibility and high toughness is obtained, and through analysis and comparison of raw materials and infrared spectra of various hydrogels, the gel is composed of a physically crosslinked acrylamide network and a pectin network, the total solid content is 12% -25%, the mass ratio of pectin to acrylamide is 1:1.5-1:20, and the rest components of the hydrogel except pectin and acrylamide are water.

The invention discloses a preparation method of pectin-based dual-physical crosslinked hydrogel, which comprises the following specific preparation steps:

(1) preparing 7 mass percent of anionic surfactant lauryl sodium sulfate micellar solution;

(2) adding a hydrophobic monomer octadecyl methacrylate into the micellar solution obtained in the step (1), and magnetically stirring at 25-70 ℃ to obtain a transparent mixed solution;

(3) adding pectin powder into the mixed solution obtained in the step (2), and magnetically stirring at 25-70 ℃ to obtain a uniform mixed solution

(4) adding acrylamide and a photoinitiator I2959 into the solution obtained in the step (3), and magnetically stirring at 25-70 ℃ to obtain a uniform solution;

(5) Injecting the uniform solution obtained in the step (4) into a mold, and initiating by ultraviolet light to obtain single-crosslinking single-network hydrogel;

(6) soaking the single-crosslinking single-network hydrogel obtained in the step (5) into a ferric ion solution, taking out the single-crosslinking single-network hydrogel and soaking the single-crosslinking single-network hydrogel into deionized water to obtain a double-crosslinking double-network hydrogel;

the double-network hydrogel obtained by the invention can be directly used for 3D cell culture after being sterilized by high pressure.

and (3) after each charging in the processes of the steps (2) to (4), magnetic stirring is adopted to obtain a uniform mixed solution, so that the uniformity of the finally obtained hydrogel structure is ensured, and the mechanical property loss caused by the non-uniform gel structure is avoided.

The preferred conditions for the hydrogel preparation process are as follows:

In the step (2), the molar ratio of the octadecyl methacrylate to the acrylamide is 1-4 mol%.

the mass ratio of pectin to acrylamide in the step (3) is 1:1.5-1: 20.

The pectin used in said step (3) is preferably added with a high methoxylated (> 65%) citrus or apple derived pectin.

in the step (4), the photoinitiation time in the step (5) is 1-5h relative to 1 mol% of the total amount of acrylamide and the hydrophobic monomer octadecyl methacrylate in the step (4).

The ultraviolet wavelength used in the steps (5) and (5) is 250-420nm, and the light intensity is 5-8W.

the molar concentration of the iron ion solution soaked in the step (6) is 0.02-0.2M.

And (3) soaking in the iron ion solution for 10min-16h in the step (6).

and (3) soaking in deionized water for 1-72h in the step (6).

The treatment scheme of the hydrogel applied to in vitro cell culture is as follows:

the pectin-based hydrogel can be directly used for 3D cell culture after being autoclaved; sterilizing at 121 deg.C for 30 min; the cell model used was mouse pre-chondroblast ATDC5, and ATDC5 cells were seeded on the hydrogel surface, showed normal proliferation behavior, and gradually infiltrated into the interior of the cells.

Compared with the prior art, the invention has the following outstanding characteristics:

(1) the preparation method has the advantages of low energy consumption, high biological safety, low price, simple and convenient operation, environmental friendliness and the like in the whole preparation process.

(2) The pectin-based physically-crosslinked high-toughness hydrogel prepared by the method has good mechanical properties, and through the synergistic effect of the hydrophobically-associating acrylamide network and the pectin network in ion coordination and complexation, the pectin-based hydrogel has the tensile strength of 0.60-2.0MPa, the breaking strain of 130-1500%, the elastic modulus of 0.3-2.0MPa and the toughness of 1.0-12.0MJm ^-3, and can be adjusted.

(3) the pectin-based physically-crosslinked high-toughness hydrogel prepared by the method has good mechanical self-recovery property and fatigue resistance.

(4) the pectin-based physically-crosslinked high-toughness hydrogel prepared by the method has excellent cell compatibility, can be used for three-dimensional cell culture, and various cells adhered to the surface of the hydrogel can maintain normal proliferation behavior and can gradually infiltrate into the hydrogel.

Drawings

FIG. 1: a hydrogel preparation flow real object diagram and a corresponding cross-linking principle schematic diagram;

FIG. 2: hydrogel preparation raw materials (pectin; acrylamide) and single/double network hydrogel infrared spectra;

FIG. 3: tensile stress-strain curves of the pectin-based high-toughness hydrogel under different hydrophobic monomer concentrations;

FIG. 4: the tensile stress-strain curve of the pectin-based high-toughness hydrogel under different mass ratios of pectin and acrylamide;

FIG. 5: the pectin-based high-toughness hydrogel is used for 3D culture of ATDC5 cells in vitro, and the cells proliferate normally and show infiltration behavior.

Detailed Description

the technical scheme of the invention is further described by combining specific examples, and is shown in figure 1 of the specification, firstly, a gel precursor solution is prepared, namely, a hydrophobic monomer octadecyl methacrylate (SMA) is solubilized into a Sodium Dodecyl Sulfate (SDS) micelle solution, then pectin powder is uniformly dispersed into the micelle solution containing the hydrophobic monomer, acrylamide and a photoinitiator are added, secondly, the hydrogel precursor solution is converted into a single-crosslinked single-network hydrogel under the photoinitiation condition, ultraviolet light is irradiated to initiate in-situ free radical polymerization to form acrylamide and methyl methacrylate copolymer chain poly (AM-co-SMA), the copolymer chain takes a hydrophobic micro-domain formed by hydrophobic association between SMA as a crosslinking point to form the single-network hydrogel, then two-step soaking treatment is carried out, after the single-network hydrogel is soaked in a ferric ion solution, multi-dentate coordination complex is formed between-COO ^- and Fe ³⁺ on the pectin chain, a double-network toughness hydrogel is obtained, the double-network hydrogel is soaked in deionized water, and Fe ³⁺ which is unstable and crosslinked, and the high-performance fruit double-physical crosslinked hydrogel containing stable tridentate coordination is obtained.

the reagents used in the following examples include essentially the following: sodium dodecyl sulfate, pectin, ferric chloride hexahydrate, acrylamide and octadecyl methacrylate. The preferred conditions are as follows:

(1) Preparing 7 mass percent of anionic surfactant lauryl sodium sulfate micellar solution;

(2) Solubilizing hydrophobic monomers into the micelle solution obtained in the step (1) according to mol ratio of 1-4 mol%, and magnetically stirring at 25-70 ℃ until the solution is transparent;

(3) adding pectin powder into the solution obtained in the step (2) according to the mass ratio of 1:1.5-1:20, and magnetically stirring at 25-70 ℃ to obtain a uniform mixed solution

(4) Adding acrylamide into the solution obtained in the step (3) according to the proportion selected in the step (3), adding a photoinitiator according to 1 mol%, and magnetically stirring at 25-70 ℃ to obtain a uniform solution;

(5) Injecting the uniform solution obtained in the step (4) into a mold, and initiating for 1-5 hours under ultraviolet light with the light intensity of 5-8W and the light wavelength of 250-420nm to obtain single-network hydrogel;

(6) and (3) soaking the single-network hydrogel obtained in the step (5) in a ferric ion solution with the molar concentration of 0.02-0.2M for 10min-16h, taking out the single-network hydrogel and then soaking the single-network hydrogel in deionized water for 1-72h to obtain the high-strength and high-toughness double-network hydrogel with a uniform structure.