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Method for inducing gelation and biomimetic mineralization of fibroin solution by alkaline phosphatase

A biomimetic mineralization technology of silk protein, applied in the field of materials, can solve the problems of mismatching clinical and medical environments, cytotoxicity affecting the biocompatibility of silk protein gel materials, and poor biocompatibility of silk protein gel materials

Active Publication Date: 2020-11-06
SUZHOU UNIV
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Problems solved by technology

[0004] However, these traditional methods still face some challenges and deficiencies when they are applied in clinical medicine.
For example, when the rapid gelation of silk protein solution is induced by physical methods such as ultrasonic treatment, swirling shear and electrification, the gelation process under non-physiological conditions triggered by electronic instruments does not match the clinical medical environment
Although the gelation time of silk protein was shortened to a certain extent by adding organic reagents, inorganic compound, ionic liquid, high-pressure carbon dioxide, surfactants and synthetic polymers to the silk protein solution, this series The gelation process is incompatible with some clinical use environments, such as the potential cytotoxicity of organic molecules, the biological inertness of high molecular polymers, and the disadvantages of being difficult to degrade in vivo
In addition, although scientists also use poly(ethylene glycol diglycidyl ether) (PGDE), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), geni Ping, chloroauric acid, etc. are used as chemical cross-linking agents to obtain silk protein gel materials with good mechanical strength and stability, but the potential cytotoxicity of the remaining chemical cross-linking agents in the system affects the biological properties of silk protein gel materials. compatibility
In summary, although these measures can shorten the gelation time of silk protein to a certain extent, the obtained silk protein gel material has poor biocompatibility and high cytotoxicity. These problems lead to its application in biomedical materials. The application of the aspect is greatly limited

Method used

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  • Method for inducing gelation and biomimetic mineralization of fibroin solution by alkaline phosphatase
  • Method for inducing gelation and biomimetic mineralization of fibroin solution by alkaline phosphatase
  • Method for inducing gelation and biomimetic mineralization of fibroin solution by alkaline phosphatase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: Preparation and purification of silk protein solution

[0037] (1) Silk degumming

[0038] Weigh 4.24g of anhydrous sodium carbonate and dissolve in 2L of boiling deionized water (Na 2 CO 3 Concentration of 0.02M), add 5.0g silk and boil for 1 hour. During the boiling process, the silk should be often stripped to avoid entanglement into bundles. After the end, the silk was pulled out and scrubbed with deionized water for 3-4 times, then air-dried overnight at room temperature, and the weight of the degummed silk was 3.5g, accounting for about 70% of the total weight of the silk.

[0039] (2) silk dissolving

[0040] Weigh 32.3g of anhydrous LiBr to prepare 40mL of a solution with a concentration of 9.3M, and filter with filter paper. 2 g of degummed silk was weighed and added to 12 mL of LiBr solution, and heated and stirred slowly at 60° C. for 4 hours.

[0041] (3) Solution dialysis

[0042] First, wash the dialysis bag with deionized water for 2-3...

Embodiment 2

[0053] Example 2: Solid phase synthesis of phosphorylated small molecule polypeptide NYp

[0054] The synthesis process of polypeptide molecule NYp is as follows: figure 1 shown. According to the sequence of the designed target molecule, phosphorylated tyrosine (Fmoc-Tyr(H 2 PO 3 )-OH), phenylalanine (Fmoc-Phe-OH), phenylalanine (Fmoc-Phe-OH), glycine (Fmoc-Gly-OH) and dinaphthylacetic acid (Nap) structural units, specific synthesis Proceed as follows:

[0055] (1) Resin swelling

[0056] Take by weighing 0.5g 2-chlorotrityl resin and join in the solid-phase synthesis reactor, under the effect of nitrogen, add appropriate anhydrous dichloromethane (DCM) swelling resin 30 minutes, extrude anhydrous DCM then and use Washed three times with anhydrous N,N-dimethylformamide (DMF).

[0057] (2) connected to Fmoc-Tyr (H 2 PO 3 )-OH

[0058] Weigh 0.845g Fmoc-Tyr (H 2 PO 3 )-OH was dissolved in 8mL of anhydrous DMF, and 0.76mL of DIEA was added thereto, dissolved with the a...

Embodiment 3

[0084] (1) Weigh 10 mg NYp and dissolve it in a glass vial, adjust the pH by adding an appropriate amount of 1mol / L NaOH to completely dissolve NYp in ultrapure water to form a clear and transparent solution; then add an appropriate amount of 1mol / L HCl to make the system When the pH value is about 7.4, add deionized water to make the total volume 2mL to obtain NYp stock solution with a concentration of 0.5wt%.

[0085] (2) Take a certain amount of silk protein solution with a concentration of 7.9% and add it to a glass vial, adjust the pH value to about 7.4 by adding 1mol / L NaOH, and adjust the volume with ultrapure water to obtain a SF reserve with a concentration of 6.0% and pH=7.4 liquid.

[0086] (4) Pipette 5 μL of SF stock solution into a glass vial, then pipette 30, 48, 60, 120 and 180 μL of NYp stock solution into the SF solution, then add 3 μL of ALP, and finally make the volume to 300 μL. The concentration of SF is 0.1%, and the concentration of NYp is 0.05, 0.08, ...

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Abstract

The invention discloses a method for inducing gelation and biomimetic mineralization of a fibroin solution by alkaline phosphatase. According to the invention, the micromolecular polypeptide which issensitive to ALP and has good biocompatibility and self-assembly property is introduced as a gelator precursor; phosphoric acid groups on molecules can be removed under the catalytic action of ALP togenerate NY, supermolecule self-assembly is triggered, and therefore SF co-self-assembly is synergistically induced, and finally SF hydrogel is rapidly formed. ALP wrapped in an SF-NY hydrogel networkstill maintains the catalytic activity of the SF-NY hydrogel network, and free phosphate ions are released by catalyzing beta-glycerophosphate so that formation of apatite minerals is induced in thegel. Due to the mild gelation process and the formation of apatite minerals in the gel matrix, the biomimetic mineralized SF gel can be used as a biomimetic scaffold to promote the adhesion, proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells in vitro, and can also promote the natural healing of femoral defects in a rat model.

Description

technical field [0001] The invention relates to a method for inducing gelation and bionic mineralization of silk protein solution by alkaline phosphatase, which belongs to the field of material technology. Background technique [0002] In nature, the biomineralization process is affected by many organic components, including proteins, polysaccharides, and enzymes, which play an important role in regulating the growth of hydroxyapatite crystals. During natural bone formation, alkaline phosphatase (ALP) secreted by osteoblasts increases the local phosphate concentration by releasing inorganic phosphate ions from organophosphate, prompting hydroxyapatite (HA ) mineralization. In the water environment, alkaline phosphatase (alkalinephosphatase) can catalyze the removal of the phosphate group on the substrate molecule, so that the hydrophobicity of the substrate is enhanced. In 2004, Xu et al. reported for the first time that under the catalysis of alkaline phosphatase, the sub...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08J3/075C08L89/00C08K5/20C08K3/32A61L27/52A61L27/22
CPCC08J3/075A61L27/52A61L27/227A61L2430/02C08J2389/00C08K2003/325C08K5/20C08L89/00C12N9/18C12Y301/03001A61L27/3604
Inventor 李新明李航
Owner SUZHOU UNIV
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