Nanofiber composite DNA gel ink, and preparation method and application thereof

Abstract

The application belongs to the technical field of hydrogel ink, and discloses a nanofiber composite DNA gel ink as well as a preparation method and application thereof. The method comprises the following steps: 1) carboxyl of amyloid fiber is activated, then reacted with 2-(2-pyridyl disulfide)ethylamine hydrochloride, and finally reacted with angiogenic polypeptide QK-SH to obtain AF-QK; 2) DNA is dissolved in a PBS solution or water, AF-QK and nanomaterials are added and uniformly mixed to obtain a pre-solution; and 3) the pre-solution is treated at a high temperature of 90-95 DEG C for 1-2 min, and is placed at room temperature to obtain a pre-polymer, i.e., the gel ink. The gel ink has good mechanical properties, and has functions of promoting osteogenesis and angiogenesis, and effectively improves the osteogenic activity of the ink. The gel ink is used for preparing bone repair materials.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of hydrogel ink, and particularly relates to a nanofiber composite DNA gel ink and a preparation method and application thereof. BACKGROUND

[0002] Hydrogels based on biological polymer networks can be used as an organic matrix to simulate bone tissue due to their good biocompatibility, extracellular matrix-like properties and adjustable performance, and provide a template for biomineralization. However, most hydrogels are statically cross-linked, and lack the dynamic activity of natural extracellular matrix. Therefore, an injectable hydrogel as a bone tissue engineering scaffold can better simulate the dynamic microenvironment of the extracellular matrix. It can be used by minimally invasive injection, and the precursor solution can be filled in irregular tissue defects, etc., has 3D printing forming property, and realizes efficient drug wrapping and release. However, most injectable hydrogels based on molecular networks have the disadvantages of weak mechanical strength, relatively complex molecular design, lack of nanofiber structure similar to the extracellular matrix, and relatively single function regulation of stem cells. Therefore, the behavior of stem cells can be regulated by introducing a functional nanofiber matrix into the DNA gel, and the effect of bone tissue repair can be further promoted.

[0003] Amyloid fibrils, as a kind of nanomaterial, have an extracellular matrix mimetic surface topography. Based on the highly repetitive arrangement of charged and uncharged residues on the surface, they can bind various molecules / biomolecules, such as proteins, DNA / RNA, biological membranes and small molecules [Das S, Jacob RS, Patel K, Singh N, Maji SK. Amyloid Fibrils: Versatile Biomaterials for Cell Adhesion and Tissue Engineering Applications [J]. Biomacromolecules. 2018, 19(6): 1826-1839].

[0004] During the healing of bone defects, the expression level of endogenous cytokines related to angiogenesis is relatively low, which largely limits the regeneration and repair of bone tissue [Einhom TA, Gerstenfeld LC. Fracture healing: mechanisms and interventions [J]. Nat Rev Rheumatol. 2015, 11(1): 45-54.].

[0005] How to obtain a dynamic gel 3D printing ink that can promote stem cell angiogenesis and osteogenic differentiation has become one of the problems to be solved. SUMMARY

[0006] To solve the problems of traditional gel ink, such as lack of dynamic mechanical properties, weak mechanical strength, relatively complex molecular design, lack of nanofiber structure similar to extracellular matrix, and biological properties such as promoting stem cell angiogenesis and osteogenic differentiation, the present application provides a nanofiber composite DNA gel ink with angiogenic and osteogenic functions, and a preparation method and application thereof. The present application is based on multiple physical crosslinking, and physically embeds a nanofiber network in the hydrogel internal network. Further, the fibers are functionally modified by QK polypeptide, and active silicate nanoparticles are introduced, thereby constructing a nanocolloid composite DNA gel ink with angiogenic and osteogenic functions. The physical crosslinking includes electrostatic interaction between DNA molecules, AF and silicate nanosheets, and the molecular network formed by hydrogen bonding between DNA molecules and DNA and AF. The gel ink of the present application is a nanofiber composite DNA gel 3D printing ink with angiogenic and osteogenic functions.

[0007] The gel ink of the present application is used for preparing a bone tissue engineering scaffold for bone repair or a material for promoting stem cell angiogenesis and osteogenic differentiation.

[0008] The object of the present application is achieved by the following scheme.

[0009] A preparation method of a nanofiber composite DNA gel ink with angiogenic and osteogenic functions, comprising the following steps:

[0010] 1) Carboxyl activation of amyloid fibers (AF) is carried out, then reacted with 2-(2-pyridyl disulfide) ethylamine hydrochloride, and finally reacted with QK-SH to obtain QK polypeptide modified protein fibers (AF-QK);

[0011] 2) Dissolve DNA in PBS solution or water;

[0012] 3) Add AF-QK and nanomaterials to the solution obtained in step 2) and mix uniformly to obtain a pre-solution;

[0013] 4) The pre-solution is treated at 90-95°C for 1-2 min, and then left at room temperature to obtain a prepolymer, i.e. a gel ink.

[0014] Stirring is carried out during the high-temperature treatment of step 4), and the stirring speed is 150-200 rpm.

[0015] The QK polypeptide modified protein fibers (AF-QK) in step 1) are prepared by the following method:

[0016] S1: dissolve egg white lysozyme in an acid solution with pH=2, incubate at 85-95 DEG C for 8-12 hours to obtain an AF solution; the mass-volume ratio of the egg white lysozyme to the acid solution with pH=2 is 0.5 g:(4-5) mL;

[0017] S2: activate the carboxyl group of the AF by using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), then react with 2-(2-pyridyl disulfide) ethylamine hydrochloride (PDEA) for 0.5-1.5 hours, finally add QK-SH and react for 3-5 hours to obtain a protein fiber modified by a QK polypeptide.

[0018] The final concentration of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in the system is 2-4 mg / mL, and the final concentration of the N-hydroxysuccinimide (NHS) in the system is 0.4-0.6 mg / mL,

[0019] The mass ratio of the EDC to the NHS is 3:0.5.

[0020] The concentration of the 2-(2-pyridyl disulfide) ethylamine hydrochloride (PDEA) in the system is 0.15 mg / mL.

[0021] The mass ratio of the QK-SH to the AF is 0.3 mg:(0.04-0.06) g.

[0022] Water is added to the system in the reaction of step S2.

[0023] The DNA is deoxyribonucleic acid sodium salt of herring testis, Sigma-Aldrich, and the molecular weight is about 6.0*10 5 g / mol.

[0024] The nanomaterial is a silicate nanosheet (Clay nanosheents).

[0025] The concentration of the DNA in the pre-solution is 5-40 mg / mL, the concentration of the Clay nanosheents in the pre-solution is 0.5-1.0%, and the concentration of the AF-QK in the pre-solution is 2-10 mg / mL.

[0026] The application constructs a nanofiber composite DNA molecule dynamic gel ink capable of promoting stem cell differentiation into blood vessels and bone by using DNA as an organic phase, using Clay nanosheents as a bone inductive active component, and using AF-QK as an active molecule for promoting vascular differentiation.

[0027] The application can effectively improve the mechanical properties of DNA gel ink through the physical interaction of AF with DNA molecules, including electrostatic adsorption and hydrogen bonding. 0.7 Si8Mg 5.5 Li 0.3 O20(OH)4) can further enhance the mechanical properties of the ink, and the released Si 4+ 、Mg 2+ and Li + have osteogenic function. At the same time, QK polypeptides are chemically grafted on AF to endow the ink with angiogenic function.

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

[0029] (1) The application is based on DNA molecule combined with polypeptide modified protein nanofiber and silicate nanosheet, and constructs a nanocomposite dynamic hydrogel through multiple physical synergistic interactions. The protein nanofiber (AF) is grafted with angiogenic polypeptide to endow the DNA gel with extracellular matrix fiber structure biomimetic and angiogenic properties. The silicate nanosheet is assembled with DNA molecules to regulate the gel printing formability and osteogenic properties.

[0030] (2) The application controls the reaction conditions (DNA concentration, fiber concentration and silicate nanosheet concentration, etc.) to make the prepared nanofiber composite DNA gel capable of promoting osteogenic differentiation of stem cells have good cell compatibility while having good cell angiogenic and osteogenic differentiation effects. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 TEM image of AF in Example 1;

[0032] Figure 2 SEM image of DNA / AF composite gel in Example 1;

[0033] Figure 3 Yield stress test result graph of DNA / AF composite gel in Example 1;

[0034] Figure 4 SEM image of DNA / AF / Clay nanosheets composite gel in Example 1;

[0035] Figure 5 Compression modulus test result graph of DNA / AF / Clay nanosheets composite gel in Example 1;

[0036] Figure 6Figure for the 3D printed scaffold of DNA / AF / Clay nanosheets composite gel ink in Example 2;

[0037] Figure 7 Figure for the time-modulus scan test results of DNA / AF-QK and DNA / AF-QK / Clay nanosheets composite gels in Example 3 and Example 4;

[0038] Figure 8 Figure for the cell live and dead staining fluorescence of HUVECs cultured in DNA / AF-QK composite gel for 1, 3, 5 days in Example 3;

[0039] Figure 9 Figure for the VEGF gene expression level of rBMSCs cultured in angiogenic peptide functionalized DNA / AF-QK / Clay nanosheets composite gel for 7 days and 14 days in Example 4;

[0040] Figure 10 Figure for the alkaline phosphatase activity expression level of rBMSCs cultured in angiogenic peptide functionalized DNA / AF-QK / Clay nanosheets composite gel for 7 days and 14 days in Example 4. DETAILED DESCRIPTION

[0041] For a better understanding of the present application, the following will describe the present application in conjunction with examples, but the embodiments of the present application are not limited thereto.

[0042] The following examples use Anton Paar rheometer to analyze the rheological properties of hydrogels, and use dynamic mechanical analyzer to test the mechanical properties of hydrogel scaffolds.

[0043] Example 1

[0044] (1): Preparation of amyloid fibrils (AF):

[0045] (1-1) 0.5 g of egg white lysozyme was added to 4.5 mL of aqueous solution (hydrochloric acid) with pH = 2, and stirred until dissolved;

[0046] (1-2) The above solution was incubated at 90°C for 10 h to obtain the required AF solution.

[0047] Figure 1 Figure for the TEM image taken after AF was dried.

[0048] (2): Preparation of deoxynucleotide / amyloid fibril / silicate nanosheet (DNA / AF / Clay nanosheets) composite gel

[0049] (2-1) 0.048 g of DNA (deoxyribonucleic acid sodium salt from herring testis, Sigma-Aldrich, molecular weight of about ~ 6.0 x 10 5 g / mol) was dissolved in 480 μL of deionized water at 40°C to obtain a DNA pre-solution;

[0050] (2-2) 0.04 g of silicate nanosheet was dissolved in 2 mL of deionized water to obtain a solution with a concentration of 2% (w / v%);

[0051] (2-3) 120 uL of AF solution (AF concentration of 10 wt%) and 600 uL of silicate nanosheet solution were added to (1), and stirred at 40°C until mixed evenly;

[0052] (2-4) The pre-solution was subjected to high-temperature treatment at 90°C for 2 min, and cooled at room temperature to obtain a pre-polymer;

[0053] (2-5) The physically cross-linked pre-polymer can be injected into a mold for molding to obtain a nanocolloid composite DNA hydrogel.

[0054] Preparation of a deoxynucleotide / protein fiber (DNA / AF) composite gel

[0055] (1) 0.04 g of DNA (deoxyribonucleic acid sodium salt from herring testis, Sigma-Aldrich, molecular weight of about ~ 6.0 x 10 5 g / mol) was dissolved in 480 μL of deionized water at 40°C to obtain a DNA pre-solution;

[0056] (2) 100 uL of AF solution (AF concentration of 10 wt%) was added, and stirred at 40°C until mixed evenly;

[0057] (3) The pre-solution was subjected to high-temperature treatment at 90°C for 2 min, and cooled at room temperature to obtain a pre-polymer;

[0058] (4) The physically cross-linked pre-polymer can be injected into a mold for molding to obtain a DNA / AF composite hydrogel.

[0059] Figure 2 The SEM image of the DNA / AF composite gel after freeze-drying, and the pore structure in the image is caused by freeze-drying and ice crystal evaporation. The obvious pore structure can be observed. Figure 3 The yield stress test result of the DNA / AF composite gel, and the yield stress is gradually increased with the increase of the AF content. DA0.2, DA0.5, and DA1.0 represent the AF mass in the DNA / AF composite gel of 0.2% g, 0.5% g, and 1% g.

[0060] Figure 4SEM image of the freeze-dried nanocolloidal DNA hydrogel. The pore size of the gel can be clearly seen to be reduced. Figure 5 Compression modulus test results of the nanocolloidal DNA hydrogel. The compression modulus gradually increases with increasing concentration of silicate nanosheets. DAC0.2, DAC0.5, DAC1.0 represent the mass of silicate nanosheets in the nanocolloidal DNA hydrogel is 0.2%, 0.5%, 1% g.

[0061] Example 2

[0062] (1) Preparation of amyloid fibrils (AF)

[0063] (1-1) 0.5 g of egg white lysozyme was added to 4.5 mL of aqueous solution (hydrochloric acid) with pH = 2, and stirred until dissolved;

[0064] (1-2) The above solution was incubated at 90°C for 10 h to obtain the required AF solution.

[0065] (2) Preparation of deoxynucleotide / protein fibril (DNA / AF / Clay nanosheets) composite gel

[0066] (2-1) 0.048 g of DNA (deoxyribonucleic acid sodium salt derived from herring testis, Sigma-Aldrich, molecular weight of about ~ 6.0 x 10 5 g / mol) was dissolved in 480 μL of deionized water at 40°C to obtain a DNA pre-solution;

[0067] (2-2) 0.04 g of silicate nanosheets was dissolved in 2 mL of deionized water to obtain a solution with a concentration of 2% (w / v%);

[0068] (2-3) 120 uL of AF solution (AF concentration of 10 wt%) and 300 μL of silicate nanosheet solution were added to (2-1), and stirred at 40°C until mixed evenly;

[0069] (2-4) The pre-solution was subjected to high-temperature treatment at 90°C for 2 min, and a rhodamine solution was added for staining during the heating process, and then cooled to room temperature to obtain a pre-polymer;

[0070] (2-5) The physically cross-linked pre-polymer can be subjected to printing performance research by an extrusion type 3D printer. The printing temperature was 60°C. Figure 6 Scaffold printed by the gel ink prepared in this example.

[0071] Example 3

[0072] (1) Preparation of angiogenic functionalized amyloid fibrils (AF-QK)

[0073] (1-1) 0.5 g of egg white lysozyme was added to 4.5 mL of water solution (hydrochloric acid) with pH = 2, and stirred until dissolved;

[0074] (1-2) The above solution was incubated at 90°C for 10 h to obtain the desired 10 wt% AF solution;

[0075] (1-3) The carboxyl group of AF (5 wt% in the system, water was added to the AF solution of step (1-2)) was functionalized with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, final concentration in the system was 3 mg / mL) and N-hydroxysuccinimide (NHS, final concentration in the system was 0.5 mg / mL) for 30 min, then reacted with 2-(2-pyridyl disulfide) ethylamine hydrochloride (PDEA, final concentration in the system was 0.15 mg / mL) for 1 h, and finally QK-SH (Shanghai Qiangyao Biotechnology Co., Ltd., final concentration in the system was 0.3 mg / mL) was added and stirred for 4 h to obtain blood vessel forming QK polypeptide modified protein fibers (AF-QK).

[0076] (2): Preparation of deoxyribonucleotide / protein fiber (DNA / AF-QK) composite gel

[0077] (2-1) 0.048 g of DNA (deoxyribonucleic acid sodium salt derived from herring testis, Sigma-Aldrich, molecular weight about ~ 6.0 x 10 5 g / mol) was dissolved in 960 μL of deionized water at 40°C to obtain a DNA pre-solution;

[0078] (2-2) 240 uL of AF-QK solution (5 wt%) was added to (2-1) and stirred until mixed uniformly at 40°C;

[0079] (2-3) The pre-solution was subjected to high temperature treatment at 90°C for 2 min, and then cooled at room temperature to obtain a pre-polymer;

[0080] (2-4) The physically cross-linked pre-polymer can be injected into a mold to form a nano-colloid composite DNA hydrogel (DA-QK) with pro-angiogenic function.

[0081] Figure 7 Time scanning test results of the DA-QK gel prepared in Example 3 and the DAC1.0-QK gel prepared in Example 4. Both AF and AF-QK can significantly improve the modulus of the DNA gel, and after the composite silicate nanosheet, the modulus is further improved.

[0082] Figure 8 Live and dead staining fluorescence map of human vascular endothelial cell proliferation promoted by the DA-QK gel prepared in Example 3.

[0083] Example 4

[0084] (1) Preparation of Vascularization Peptide Functionalized Amyloid Fibrils (AF-QK)

[0085] (1-1) 0.5 g of hen egg white lysozyme was added to 4.5 mL of water solution with pH = 2 and stirred until dissolved;

[0086] (1-2) The above solution was incubated at 90°C for 10 h to obtain the AF solution with a desired concentration of 10 wt%;

[0087] (1-3) The carboxyl group of AF (5 wt%) was functionalized with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 3 mg / mL) and N-hydroxysuccinimide (NHS, 0.5 mg / mL) for 30 min, then reacted with 2-(2-pyridyldithio)ethylamine hydrochloride (PDEA, 0.15 mg / mL) for 1 h, and finally added QK-SH (0.3 mg / mL) and stirred for 4 h to obtain the desired vascularization peptide modified protein fibrils (AF-QK) (5 wt%).

[0088] (2) Preparation of Deoxyribonucleotide / Polypeptide Modified Protein Fibril / Silicate Nanosheet (DNA / AF-QK / Clay nanosheets, DAC1.0-QK) Composite Gel

[0089] (2-1) 0.048 g of DNA (deoxyribonucleic acid sodium salt derived from herring testis, Sigma-Aldrich, molecular weight of about ~6.0 x 10 5 g / mol) was dissolved in 360 μL of deionized water at 40°C to obtain a DNA pre-solution;

[0090] (2-2) 0.04 g of silicate nanosheet was dissolved in 2 mL of deionized water to obtain a solution with a concentration of 2% (w / v);

[0091] (2-3) 240 uL of AF-QK solution with a concentration of 5 wt% and 600 μL of silicate nanosheet solution were added to (2-1) and stirred at 40°C until mixed evenly;

[0092] (2-4) The pre-solution was subjected to high-temperature treatment at 90°C for 2 min and then cooled at room temperature to obtain a pre-polymer;

[0093] (2-5) The physically cross-linked pre-polymer can be injected into a mold to form a nanocolloid composite DNA hydrogel with the functions of promoting vascularization and osteogenesis.

[0094] The present application takes DNA molecules as a building block, combines polypeptide modified protein nanofibers and silicate nanosheets to construct a self-assembled dynamic physical gel, regulates directional differentiation of stem cells and promotes vascularized bone regeneration repair. Figure 9 The VEGF gene mRNA expression level results after the DAC1.0-QK gel extraction liquid and rBMSCs cells are co-cultured for 7 and 14 days. Figure 10 The alkaline phosphatase expression level results after the DAC1.0-QK gel and rBMSCs cells are co-cultured for 7 and 14 days.The above research results show that the AF-QK and the silicate nanosheet simultaneously up-regulate the alkaline phosphatase expression level and the angiogenesis gene expression, thereby further showing a synergistic bone effect.

Claims

1. A nanofiber composite DNA gel ink having a function of promoting stem cell angiogenic and osteogenic differentiation, characterized by: The nanofiber composite DNA gel ink with the function of promoting stem cell angiogenesis and osteogenesis differentiation is a nanofiber composite DNA gel 3D printing ink with the function of promoting angiogenesis and osteogenesis; The preparation method of the nanofiber composite DNA gel ink with the function of promoting stem cell angiogenesis and osteogenesis differentiation comprises the following steps: 1) First, dissolve egg white lysozyme in an acid solution with pH = 2, incubate at 85-95 ℃ for 8-12 h to obtain an AF solution; the mass-volume ratio of the egg white lysozyme to the acid solution with pH = 2 is 0.5 g:(4-5) mL; then activate the carboxyl group of AF by using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and react with 2-(2-pyridyl disulfide) ethylamine hydrochloride for 0.5-1.5 h, and finally add angiogenic polypeptide QK-SH and react for 3-5 h to obtain a QK polypeptide-modified protein fiber, which is denoted as AF-QK; the mass ratio of QK-SH to AF is 0.3 mg:(0.04-0.06) g; 2) Dissolve DNA in a PBS solution or water; 3) Add AF-QK and nanomaterials to the solution obtained in step 2) and mix uniformly to obtain a pre-solution; 4) Perform high-temperature treatment on the pre-solution at 90 ℃-95 ℃ for 1-2 min, and stand at room temperature to obtain a prepolymer, i.e., a gel ink; The nanomaterial is silicate nanosheet Na 0.7 Si8Mg 5.5 Li 0.3 O 20 (OH)4; The concentration of DNA in the pre-solution is 5-40 mg / mL, the concentration of silicate nanosheet in the pre-solution is 0.5-1 wt%, and the concentration of AF-QK in the pre-solution is 2-10 mg / mL.

2. The nanofiber composite DNA gel ink with the function of promoting stem cell angiogenesis and osteogenesis differentiation according to claim 1, characterized in that: In the preparation method, the DNA is deoxyribonucleic acid sodium salt from the testes of herring, having a molecular weight of ~6.0 x 10 5 g / mol.

3. The nanofiber composite DNA gel ink with the function of promoting stem cell angiogenesis and osteogenesis differentiation according to claim 1, characterized in that: The final concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in the system is 2-4 mg / mL, and the final concentration of N-hydroxysuccinimide (NHS) in the system is 0.4-0.6 mg / mL; The mass ratio of EDC to NHS is 3:0.5; The concentration of 2-(2-pyridyl disulfide) ethylamine hydrochloride in the system is 0.15 mg / mL.

4. The nanofiber composite DNA gel ink with the function of promoting stem cell angiogenesis and osteogenesis differentiation according to claim 1, characterized in that: Stir during the high-temperature treatment of step 4), and the stirring speed is 150-200 rpm.

5. The use of the nanofiber composite DNA gel ink having a function of promoting stem cell hemogenic and osteogenic differentiation according to any one of claims 1 to 4, characterized by: The gel ink is used for preparing a bone tissue engineering scaffold for bone repair or for preparing a material for promoting stem cell angiogenesis and osteogenesis differentiation.

6. Use according to claim 5, characterized in that: The gel ink is used for preparing a bone tissue engineering scaffold for bone repair or for preparing a material for promoting stem cell angiogenesis and osteogenesis differentiation by 3D printing.

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