阅读说明：本技术 碱性磷酸酶诱导蚕丝蛋白溶液凝胶化和仿生矿化的方法 (Method for inducing gelation and biomimetic mineralization of fibroin solution by alkaline phosphatase ) 是由 李新明 李航 于 2020-07-29 设计创作，主要内容包括：本发明公开了一种碱性磷酸酶诱导蚕丝蛋白溶液凝胶化和仿生矿化的方法,本发明引入了对ALP敏感且具有良好生物相容性和自组装特性的小分子多肽作为凝胶因子前驱体,其在ALP的催化作用下能够脱除分子上的磷酸基团生成NY,触发超分子自组装,从而协同诱导SF共自组装,最终导致SF水凝胶的快速形成。SF-NY水凝胶网络中包裹的ALP仍然保持着其催化活性,并通过催化β-甘油磷酸酯释放出游离的磷酸根离子,从而在凝胶中诱导磷灰石矿物质的形成。由于温和的凝胶化过程以及在凝胶基质中形成了磷灰石矿物质,仿生矿化后的SF凝胶不仅可以用作仿生支架,在体外促进大鼠骨髓间充质干细胞的粘附,增殖和成骨分化,而且还可以在大鼠模型中促进股骨缺损的自然愈合。(The invention discloses a method for inducing gelation and biomimetic mineralization of a fibroin solution by alkaline phosphatase, which introduces micromolecule polypeptide which is sensitive to ALP and has good biocompatibility and self-assembly characteristics as a gel factor precursor, can remove phosphate groups on molecules under the catalytic action of the ALP to generate NY, triggers supermolecule self-assembly, and further synergistically induces SF co-self-assembly, so that SF hydrogel is rapidly formed. ALP encapsulated in the SF-NY hydrogel network still maintains its catalytic activity and induces the formation of apatite minerals in the gel by catalyzing beta-glycerophosphate to release free phosphate ions. Due to the mild gelation process and the formation of apatite minerals in the gel matrix, the biomimetic mineralized SF gel can be used not only as a biomimetic scaffold to promote the adhesion, proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells in vitro, but also to promote the natural healing of femoral defects in rat models.)

1. A method for inducing the gelation of a fibroin solution by alkaline phosphatase comprises the following steps: adding self-assembly small molecular polypeptide into a fibroin solution to serve as a gel factor precursor to obtain a fibroin and self-assembly small molecular polypeptide mixed solution, adding alkaline phosphatase into the mixed solution, removing phosphate groups on self-assembly small molecular polypeptide molecules through the alkaline phosphatase, triggering supermolecule self-assembly, and inducing the fibroin to perform self-assembly together to form the fibroin gel material.

2. The method of claim 1, wherein the self-assembling small molecule polypeptide is one or more of 2-naphthylacetic acid-glycine-phenylalanine-phosphotyrosine, 2-naphthylacetic acid-phenylalanine-lysine-phosphotyrosine, or 2-naphthylacetic acid-phenylalanine-phosphotyrosine.

3. The method according to claim 1, wherein the concentration of fibroin in the mixed solution is 0.1% -2.0%.

4. The method according to claim 1, wherein the concentration of the self-assembled small molecule polypeptide in the mixed solution is 0.05 wt% to 0.3 wt%.

5. The method according to claim 1, wherein the alkaline phosphatase is added in an amount of 10 to 40U/mL.

6. The method according to claim 1, wherein the pH of the mixed solution is 7 to 8.

7. A fibroin gel material produced by the method of any one of claims 1-6.

8. A method of biomimetically mineralising the fibroin gel material of claim 7, comprising the steps of: adding the fibroin gel material into mineralized liquid to culture for 5-10 days to obtain the biomimetic mineralized hydrogel, wherein the mineralized liquid comprises 10-40mM CaCl₂And 6-20mM beta-glycerophosphate.

9. A biomimetic mineralized hydrogel prepared according to the method of claim 8.

10. Use of the biomimetic mineralized hydrogel according to claim 9 for preparation of a material for repair of body tissue.

Technical Field

The invention relates to a method for inducing gelation and biomimetic mineralization of a fibroin solution by alkaline phosphatase, belonging to the technical field of materials.

Background

In nature, the biomineralization process is influenced by a number of organic components, including proteins, polysaccharides and enzymes, which have an important role in regulating the growth of hydroxyapatite crystals. During natural bone formation, alkaline phosphatase (ALP) secreted from osteoblasts increases the local phosphate concentration by releasing inorganic phosphate ions from organophosphates, promoting Hydroxyapatite (HA) mineralization. In an aqueous environment, alkaline phosphatase (alkalinephosphinotase) catalyzes the removal of phosphate groups from the substrate molecule, resulting in enhanced hydrophobicity of the substrate. In 2004, Xu et al reported for the first time that, under the catalysis of alkaline phosphatase, a substrate molecule Fmoc-pY (Fmoc ═ fluorenylmethyloxycarbonyl, pY ═ phosphorylated tyrosine) removes a phosphate group to generate Fmoc-Y, hydrogel is formed under pi-pi interaction, and meanwhile, a nanofiber network structure is formed through self-assembly, and the storage modulus of the hydrogel is about 1000 Pa. Then, starting from this pioneering work, a number of peptide hydrogels constructed based on phosphatase catalysis have been reported in succession, including Fmoc-FpY, Ac-YYYpY-OMe (Ac ═ acyl), Nap-GFFpY-OMe (Nap ═ naphthyl), Nap-FFGEpY, napfffpy, and the like.

Fibroin (Silk fibrin) is also known: silk fibroin is a natural polymer fiber protein, in which glycine (Gly), alanine (Ala), and serine (Ser) account for about 80% or more of the total composition. The material has the advantages of excellent biocompatibility, controllable biodegradability, good flexibility and tensile strength, and the like, and is widely researched by scientists. A large number of biomaterials (such as nanofibers, sponges, films, microspheres, hydrogels, etc.) constructed by taking fibroin as a base material are reported in succession, and are widely applied to the repair of various body tissues including bone tissues, skin, blood vessels, nerves, tendons, ligaments, etc. The fibroin hydrogel is favored by researchers because of the advantages of similar fiber structure with natural extracellular matrix, high water content, adjustable porosity, good affinity with cells and the like. However, the gelling process of fibroin solutions is very slow under physiological conditions, for example, a 2.0% strength aqueous fibroin solution at room temperature requires more than 14 days to change from solution to gel under physiological conditions. Therefore, gelation generally needs to occur under acidic conditions (pH of about 4) or at a relatively high temperature (60 ℃). These factors greatly limit the wide use of fibroin hydrogels in the biomedical field. Many studies have been made by scientists to modify the low pH, high temperature and long time required for the gelation of fibroin. For example, the gelation process of fibroin is accelerated by inducing the transition of the secondary structure of fibroin from the random coil conformation in solution to the β -sheet conformation in the gel state by physical methods such as sonication, vortex shearing and energization. Scientists also add organic reagent, inorganic compound, ionic liquid, high-pressure carbon dioxide, surfactant and chemical reagents such as artificially synthesized macromolecules into the fibroin solution to adjust the interaction between the fibroin solution and fibroin chains, so that the gel property of the fibroin is changed, and the fibroin gel is rapidly formed. In addition, scientists have also used poly (ethylene glycol diglycidyl ether) (PGDE), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), genipin, chloroauric acid, etc. as chemical cross-linking agents to prepare fibroin gel materials with good mechanical strength and stability.

However, these conventional methods also have some challenges and deficiencies for clinical medical applications. For example, when the fibroin solution is induced to rapidly gel by physical methods such as ultrasonic treatment, rotational flow shearing and electrification, the gelation process under non-physiological conditions such as triggering by an electronic instrument is not matched with clinical medical environment. Although the gelation time of the fibroin is shortened to a certain extent by adding organic reagents, inorganic compound, ionic liquid, high-pressure carbon dioxide, surfactants, synthetic macromolecules and other chemical reagents into the fibroin solution, the series of gelation processes are incompatible with certain clinical use environments, such as potential cytotoxicity of organic molecules, biological inertia of macromolecular polymers, difficult degradation in organisms and the like. In addition, although scientists have also used poly (ethylene glycol diglycidyl ether) (PGDE), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), genipin, chloroauric acid, etc. as chemical cross-linking agents to obtain a fibroin gel material with good mechanical strength and stability, the potential cytotoxicity of the chemical cross-linking agents remaining in the system affects the biocompatibility of the fibroin gel material. In conclusion, although the measures can shorten the gelation time of the fibroin to a certain extent, the obtained fibroin gel material has poor biocompatibility and high cytotoxicity, and the application of the fibroin gel material in biomedical materials is greatly limited due to the problems.

Disclosure of Invention

To solve the above problems, the present invention induces gelation and biomimetic mineralization of fibroin (SF) by continuous catalytic reaction triggered by alkaline phosphatase (ALP). In the system, small molecular polypeptide (NYp) sensitive to ALP and having good biocompatibility and excellent self-assembly property is introduced as a gel factor precursor, and under the catalytic action of ALP, phosphate groups on molecules can be removed to generate NY, so that supermolecule self-assembly is triggered, SF co-self-assembly is synergistically induced, and SF hydrogel is rapidly formed. ALP encapsulated in the SF-NY hydrogel network still maintains its catalytic activity and induces the formation of apatite minerals in the gel by catalyzing beta-glycerophosphate to release free phosphate ions.

The first purpose of the invention is to provide a method for inducing the gelation of fibroin solution by alkaline phosphatase, which comprises the following steps: adding self-assembly small molecular polypeptide into a fibroin solution to serve as a gel factor precursor to obtain a fibroin and self-assembly small molecular polypeptide mixed solution, adding alkaline phosphatase into the mixed solution, removing phosphate groups on self-assembly small molecular polypeptide molecules through the alkaline phosphatase, triggering supermolecule self-assembly, and inducing the fibroin to perform self-assembly together to form the fibroin gel material.

Further, the self-assembly small molecule polypeptide is one or more of 2-naphthylacetic acid-glycine-phenylalanine-phosphorylated tyrosine (NYp), 2-naphthylacetic acid-phenylalanine-lysine-phosphorylated tyrosine (napfkyp) or 2-naphthylacetic acid-phenylalanine-phosphorylated tyrosine (NapFFYp).

Further, the concentration of the fibroin in the mixed solution is 0.1% -2.0%.

Further, the concentration of the self-assembly small molecule polypeptide in the mixed solution is 0.05 wt% -0.3 wt%.

Further, the amount of the alkaline phosphatase to be added is 10U/mL-40U/mL.

Further, the pH of the mixed solution is 7-8.

The second purpose of the invention is to provide a fibroin gel material prepared by the method.

The third purpose of the invention is to provide a method for biomimetically mineralizing the fibroin gel material, which comprises the following steps: adding the fibroin gel material into mineralized liquid to culture for 5-10 days to obtain the biomimetic mineralized hydrogel, wherein the mineralized liquid comprises 10-40mM CaCl₂And 6-20mM beta-glycerophosphate (. beta. -GP).

The fourth purpose of the invention is to provide the biomimetic mineralized hydrogel prepared by the method.

The fifth purpose of the invention is to provide the application of the biomimetic mineralized hydrogel in the preparation of a material for repairing body tissues.

The invention has the beneficial effects that:

the present invention induces gelation and biomimetic mineralization of fibroin (SF) by continuous catalytic reaction triggered by alkaline phosphatase (ALP). In the system, small molecular polypeptide (NYp) sensitive to ALP and having good biocompatibility and excellent self-assembly property is introduced as a gel factor precursor, and under the catalytic action of ALP, phosphate groups on molecules can be removed to generate NY, so that supermolecule self-assembly is triggered, SF co-self-assembly is synergistically induced, and SF hydrogel is rapidly formed. ALP encapsulated in the SF-NY hydrogel network still maintains its catalytic activity and induces the formation of apatite minerals in the gel by catalyzing beta-glycerophosphate to release free phosphate ions. Due to the mild gelation process and the formation of apatite minerals in the gel matrix, the biomimetic mineralized SF gel can be used not only as a biomimetic scaffold to promote adhesion, proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells (rbmscs) in vitro, but also to promote the natural healing process of femoral defects in rat models.

Drawings

FIG. 1 shows a solid phase synthesis procedure for polypeptide molecule NYp;

fig. 2 is (a) a solution of gelator precursor NYp (0.08 wt%, pH 7.4); (b) catalyzing NYp solution by ALP (10U/mL) to perform supramolecular self-assembly to form supramolecular hydrogel; dynamic rheology testing of NY supramolecular hydrogels (NY 0.08 wt%, pH 7.4, ALP 10U/mL) (c) strain sweep and (d) frequency sweep;

FIG. 3 is (a) a 2.0% SF solution; (b) SF hydrogel with concentration of 2.0%; dynamic rheology testing of SF hydrogel (SF 2.0%, pH 7.4, ALP 10U/mL) (c) strain sweep and (d) frequency sweep;

FIG. 4 is the gelation process and the mechanical property characterization of the mixed Gel 1; (a) NYp solution (0.16 wt%, pH 7.4); (b) SF solution (0.2%, pH 7.4); (c) mixed hydrogel Gel 1 containing NY (0.08 wt%) and SF (0.1%) at pH 7.4 and ALP 10U/mL; dynamic rheological testing of Gel 1 hydrogels (d) strain sweep and (e) frequency sweep;

FIG. 5 is the gelation process of the mixed Gel 2 and the characterization of the mechanical properties thereof; (a) NYp solution (0.2 wt%, pH 7.4); (b) SF solution (0.2%, pH 7.4); (c) mixed hydrogel Gel 2 containing NY (0.1 wt%) and SF (0.1%) at pH 7.4 and ALP 10U/mL; dynamic rheological testing of Gel 2 hydrogels (d) strain sweep and (e) frequency sweep;

FIG. 6 is the gelation process and the mechanical property characterization of the mixed Gel 3; (a) NYp solution (0.4 wt%, pH 7.4); (b) SF solution (0.2%, pH 7.4); (c) mixed hydrogel Gel 3 containing NY (0.2 wt%) and SF (0.1%) at pH 7.4 and ALP 10U/mL; dynamic rheological testing of Gel 3 hydrogels (d) strain sweep and (e) frequency sweep;

FIG. 7 is a gelation process of mixed Gel 4 and its mechanical property characterization; (a) NYp solution (0.6 wt%, pH 7.4); (b) SF solution (0.2%, pH 7.4); (c) mixed hydrogel Gel 4 containing NY (0.3 wt%) and SF (0.1%) at pH 7.4 and ALP 10U/mL; dynamic rheological testing of Gel 4 hydrogels (d) strain sweep and (e) frequency sweep;

FIG. 8 is the gelation process of mixed Gel 5 and its mechanical property characterization; (a) NYp solution (0.6 wt%, pH 7.4); (b) SF solution (1.0%, pH 7.4); (c) mixed hydrogel Gel 5 containing NY (0.3 wt%) and SF (0.5%) at pH 7.4 and ALP 10U/mL; dynamic rheological testing of Gel 5 hydrogels (d) strain sweep and (e) frequency sweep;

FIG. 9 is the gelation process of mixed Gel 6 and its mechanical property characterization; (a) NYp solution (0.6 wt%, pH 7.4); (b) SF solution (2.0%, pH 7.4); (c) mixed hydrogel Gel 6 containing NY (0.3 wt%) and SF (1.0%) at pH 7.4 and ALP 10U/mL; dynamic rheological testing of Gel 6 hydrogels (d) strain sweep and (e) frequency sweep;

FIG. 10 is the gelation process of mixed Gel 7 and its mechanical property characterization; (a) NYp solution (0.6 wt%, pH 7.4); (b) SF solution (4.0%, pH 7.4); (c) mixed hydrogel Gel 7 containing NY (0.3 wt%) and SF (2.0%) at pH 7.4 and ALP 10U/mL; dynamic rheological testing of Gel 7 hydrogels (d) strain sweep and (e) frequency sweep;

FIG. 11 is the gelation process and its mechanical property characterization of mixed Gel 8; (a) NYp solution (0.6 wt%, pH 7.4); (b) SF solution (4.0%, pH 7.4); (c) mixed hydrogel Gel 8 containing NY (0.3 wt%) and SF (2.0%) at pH 7.4 and ALP 20U/mL; dynamic rheological testing of Gel 8 hydrogels (d) strain sweep and (e) frequency sweep;

FIG. 12 is the gelation process and its mechanical property characterization of mixed Gel 9; (a) NYp solution (0.6 wt%, pH 7.4); (b) SF solution (4.0%, pH 7.4); (c) mixed hydrogel Gel 9 containing NY (0.3 wt%) and SF (2.0%) at pH 7.4 and ALP 40U/mL; dynamic rheological testing of Gel 9 hydrogels (d) strain sweep and (e) frequency sweep;

FIG. 13 shows different calcium ions (Ca)²⁺) Scanning Electron Microscope (SEM) images and Energy Dispersive Spectroscopy (EDS) data of the concentration biomimetically mineralized hydrogel material; (a) and (d) a calcium ion concentration of 10 mM; (b) and (e) a calcium ion concentration of 20 mM; (c) and (f) a calcium ion concentration of 50 mM. HA and SF-NY gel (SF 2.0%, NY 0.3 wt%, ALP 10U/mL) and Ca-20gel (SF 2.0%, NY 0.3 wt%, ALP 10U/mL, Ca before and after biomimetic mineralization²⁺20mM) X-ray diffraction analysis of the hydrogel, (h) fourier infrared spectroscopy and (i) X-ray photoelectron spectroscopy.

FIG. 14 is (a) fluorescence images of dead and live staining of rat bone marrow mesenchymal stem cells (rBMSCs) after 1, 4, 7 days of surface culture in blank plates, SF-NY gel and Ca-20gel and (b) corresponding cell density statistics; (c) cytotoxicity assays for SF-NY gel and Ca-20gel (CCK8 method);

FIG. 15 shows the qRT-PCR detection of osteogenesis-related gene and protein expression (a) Runx2, (b) Col1 α, (c) OCN, (d) OPN;

FIG. 16 is (a) two-dimensional Micro-CT images at 4 and 8 weeks post-operative of rat femur and (b) three-dimensionally reconstructed Micro-CT images; (c) quantitative analysis results 4 and 8 weeks after femoral surgery in rats: bone density (BMD), bone volume to total tissue volume ratio (BV/TV), trabecular thickness (tb.th), trabecular gap (tb.sp).

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

2-chlorotrityl chloride resin (100-200 mesh, 0.3-0.8 mmol/g), Fmoc-Tyr (H)₂PO₃) -OH, Fmoc-Gly-OH, Fmoc-Phe-OH and HBTU (benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate) were purchased from Gill Biochemical company, Shanghai; DIEA (N, N-diisopropylethylamine) was purchased from Annagel corporation; 2-naphaleneactic acid purchased from national drug company; the other organic solvent isOrdered from Jiangsu prosperous company.