CN105363069B - An intelligent polypeptide hydrogel regulated by FeCl3, its preparation method and its application - Google Patents

Abstract

The invention discloses an intelligent polypeptide hydrogel regulated by FeCl ₃ , a preparation method and an application thereof. The polypeptide is selected from a Tris-NH ₂ -VdopaVKVKVK-VDPPT-KVKVKVKV-NH ₂ polypeptide with a pH of 9-10. Formed by self-assembly in HCl solution, the obtained hydrogel can collapse into a liquid state under the action of FeCl ₃ , and the hydrogel prepared by the present invention can be used as a cell culture scaffold. The hydrogel formed by the self-assembly of polypeptide molecules proposed in the present invention can be collapsed into a solution state by adding Fe ³⁺ , which is an intelligent hydrogel regulated by Fe ³⁺ , and is an ideal nano Tissue engineering materials are of great significance to human life and health.

Description

An intelligent polypeptide hydrogel regulated by FeCl3, its preparation method and its application

technical field

The invention relates to the technical field _of hydrogel preparation, in particular to an intelligent polypeptide hydrogel regulated by FeCl3, a preparation method and an application thereof.

Background technique

Ideal tissue engineering materials not only need to have a grid structure that supports and connects tissues, but also need to have advantages in regulating tissue generation and physiological activities of cells. At present, most scaffold materials use biomacromolecules (chitosan, alginate, collagen) or synthetic macromolecules (polyethylene glycol), etc., to provide a microenvironment to support cell growth. However, problems such as chemical residues, disease source transmission and synthesis cost limit its in-depth application.

The preparation methods of hydrogels in the prior art are generally two types: small molecule self-assembly and polymer crosslinking. At present, small molecules are easy to synthesize and low in cost, but their structures are artificially designed and difficult to achieve degradation; polymer gels are easy to prepare and have controllable structures, but it is difficult to achieve reasonable insertion and degradation of biological response sites. In this study, the corresponding sites were rationally embedded into the small molecule, which is a convenient synthetic peptide, to achieve a definite degradation ability. In the tissue engineering materials used for cell culture, when the cells grow to a certain number and do not need scaffold support, it is hoped that the tissue engineering materials can be effectively degraded, so that the cells can be better integrated into the body tissue.

Contents of the invention

The object of the present invention is to provide an intelligent polypeptide hydrogel and its preparation method, the hydrogel can collapse into a liquid state under the action of FeCl ₃ .

One of the tasks of the present invention is to provide a method for preparing the hydrogel.

A kind of preparation method of hydrogel, it is to select structural formula to be:

The NH ₂ -VdopaVKVKVK-VDPPT-KVKVKVKV-NH ₂ polypeptide is self-assembled in a Tris-HCl solution with a pH of 9-10, and the hydrogel can collapse into a liquid state under the action of FeCl ₃ .

The beneficial technical effects directly brought by the above-mentioned technical solutions are: first, the selection of short peptides that can be conveniently synthesized by solid-phase synthesis technology makes the synthesis cost low and the purification is convenient; at the same time, the designed and synthesized polypeptide adopts a mature self-assembly model, It has a good ability to assemble and form hydrogels; secondly, the hydrogels are prepared by physical methods of adjusting pH at room temperature, which avoids the damage to tissues caused by external chemical agents and ultraviolet light. Compared with the existing technology The risk of causing tissue damage is reduced; thirdly, by adding FeCl ₃ for degradation, the function of degrading the gel through the physical action of simple inorganic ions is realized, and the potential biological toxicity and immunogenicity caused by the introduction of chemical reactions are avoided.

As a preferred version of the present invention, the volume ratio of hydrogel to FeCl is ₃ :1.

The concentration of the polypeptide in Tris-HCl solution was 4 mM.

Another task of the present invention is to provide the hydrogel prepared by the above preparation method.

The above-mentioned hydrogel is a nanofibrous structure.

Application of the above-mentioned hydrogel in a cell culture scaffold.

Beneficial technical effects brought by the present invention:

The invention provides a method for preparing hydrogel. The biggest difference between the prepared hydrogel and the prior art is that it can be degraded under the action of FeCl ₃ ; from the aspect of raw material selection, the present invention selects polypeptide in Tris -HCl solution, the pH is controlled to be 9 self-assembled, and the nanofibers formed by self-assembled peptides are used as scaffolds to prepare stable hydrogels, so that they have corresponding biological functions and enrich the peptide hydrogel formation. type.

The hydrogel formed by the self-assembly of polypeptide molecules proposed in the present invention can be collapsed into a solution state by adding Fe ³⁺ , which is an intelligent hydrogel regulated by Fe ³⁺ , and is an ideal nano Tissue engineering materials are of great significance to human life and health.

Description of drawings

The present invention will be further described below in conjunction with accompanying drawing:

Fig. 1 is the NH ₂ -VdopaVKVKVK-VDPPT-KVKVKVKV-NH ₂ reversed-phase high performance liquid chromatogram synthesized by the solid-phase synthesis method in the present invention;

Fig. 2 is an atomic force microscope topography diagram of self-assembly of NH ₂ -VdopaVKVKVK-VDPPT-KVKVKVKV-NH ₂ in Tris-HCl (pH7.4) solution of the present invention;

Fig. 3 is a transmission electron microscope topography diagram of self-assembly of NH ₂ -VdopaVKVKVK-VDPPT-KVKVKVKV-NH ₂ in Tris-HCl (pH 7) solution of the present invention;

Fig. 4 is an atomic force microscopy image of self-assembly of NH ₂ -VdopaVKVKVK-VDPPT-KVKVKVKV-NH ₂ in Tris-HCl (pH 10) solution of the present invention;

Fig. 5 is a transmission electron microscope topography diagram of self-assembly of NH ₂ -VdopaVKVKVK-VDPPT-KVKVKVKV-NH ₂ in Tris-HCl (pH 10) solution of the present invention;

Fig. 6 is the relationship between the mechanical strength (storage modulus and loss modulus) and frequency of hydrogel formed by NH ₂ -VdopaVKVKVK-VDPPT-KVKVKVKV-NH ₂ of the present invention after 24 hours;

Fig. 7 is the topography of the hydrogel formed by NH ₂ -VdopaVKVKVK-VDPPT-KVKVKVKV-NH ₂ of the present invention after adding FeCl ₃ ;

Fig. 8 is a transmission electron micrograph of the hydrogel formed by NH ₂ -VdopaVKVKVK-VDPPT-KVKVKVKV-NH ₂ of the present invention after adding FeCl ₃ .

detailed description

The present invention will be described in further detail below in conjunction with specific embodiments.

At first the specifications and models of the selected main experimental instruments of the present invention are briefly described, and the following experimental instruments can be purchased through commercial channels:

Electric constant temperature incubator (DNP-9082 type, Shanghai Jinghong Experimental Equipment Co., Ltd.);

Drying and disinfection oven (DHG-9246A type, Shanghai Jinghong Test Equipment Co., Ltd.);

UV Spectrophotometer (American Biotech Instruments);

Desktop centrifuge (Eibendorf, Germany);

Atomic Force Microscope (AFM) (Nanoscope Iva Multimode AFM, Digital Instruments)

Transmission electron microscope (TEM) (JEOL JEM1400Plus type)

Rheometer (Mars III, Harker)

Ultra-clean bench (Airtech type, Jiangsu Antai)

Constant temperature cell incubator (HERACELL 150i, Thermoelectric Company, France)

Inverted microscope (TS100, Nikon, Japan)

Fluorescence inverted microscope (DMI3000B, Leica, Germany)

Confocal microscope (Nikon A1, Japan)

Disposable cell culture flask (25cm ² , Corning, costar type)

Disposable pipette (5mL, accuracy 0.1mL, Corning, costar type)

Disposable cell culture plate (Cat. No. 3599, Corning, costar type)

Disposable cell culture plate (Cat. No. 3548, Corning, costar type)

Liquid nitrogen container (YDS-30-125 type, Dongya liquid nitrogen container)

Microwave-assisted peptide synthesizer (Liberty1 microwave peptide automatic synthesizer from CEM Company).

Secondly, the polypeptide NH ₂ -VdopaVKVKVK-VDPPT-KVKVKVKV-NH ₂ selected for the preparation of the hydrogel in the present invention is self-made, and its specific preparation method is as follows:

Step 1. Distill N,N-dimethylformamide (DMF) and piperidine (Piperidine) solvents

Distill the purchased DMF solution under reduced pressure at 60°C to obtain pure DMF solvent; add a small amount of CaH2 to the purchased piperidine and heat to reflux for 1-2 hours, and receive the fraction with boiling point temperature (106°C) to obtain pure piperidine Pyridine solvent;

Step 2, preparation of amino acids, resins, activators, capping agents, deprotecting agents, etc.

The amount of amino acids and other reagents required for the preparation of 0.25mM NH2-VdopaVKVKVKVDPPTKVKVKVKV-NH2 was calculated on the peptide solid-phase synthesizer (in order to ensure the purity of the peptide during peptide synthesis, the amount of amino acid was doubled, and its final concentration was 0.2M):

Lys (lysine): 1.03g dissolved in 11mL DMF;

Val (isoleucine): 2.97g dissolved in 42mL DMF;

Resin (loaded at 0.6mmol/g): 0.417g;

Note: The a-amino groups of amino acids are protected by Fmoc, and the side chain amino groups of Lys are also protected;

Activator: benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU): 6.83g; 1-hydroxybenzotriazole (HOBT): 2.43g; dissolved in 40mL DMF;

Activation base: N,N-diisopropylethylamine (DIEA): 6.96mL; DMF: 13.04mL;

Capping agent: acetic anhydride: 4ml; N,N-diisopropylethylamine (DIEA): 0.44g; HOBT: 0.04g; dissolved in 16mL of DMF;

Cracking agent: trifluoroacetic acid (TFA): 23.5mL; triisopropylsilane (TIS): 0.25mL; H2O: 0.625mL; 1,2-ethanedithiol (EDT) 0.625mL;

Deprotecting agent: piperidine: 46 mL; DMF: 184 mL; 1-hydroxybenzotriazole (HOBT): 3.11 g.

Step 3. Solid-phase synthesis and purification of polypeptides

Add the prepared drug in step 2 into the designated container of the microwave solid-phase synthesizer, start to synthesize Ac-IIIIKK-NH2 from the C-terminal to the N-terminal, and the instrument will automatically synthesize. After the synthesis of the peptide is completed, pour the product in the product tube into a round bottom flask, add a cracking agent, stir at room temperature for 4 hours, collect the filtrate after vacuum filtration, wash the resin for 3 times with TFA, combine the filtrate and washing liquid, and pour it into a distillation flask Medium distillation (to remove residual TFA), the product after distillation was poured into a 10mL centrifuge tube, added cold diethyl ether, centrifuged for 15min at a speed of 9000rpm/min, repeated more than 10 times, purified by preparative reverse-phase high-performance liquid phase, and finally the product Put it into a high-pressure freeze dryer for freeze-drying, and store it in a refrigerator after freeze-drying. Matrix-assisted laser desorption time-of-flight mass spectrometry analysis shows that its molecular weight is the molecular weight of the polypeptide we synthesized. Reversed-phase high-performance liquid chromatography analysis, as shown in Figure 1, shows that the purity of the polypeptide synthesized by the present invention is above 98%.

Detection: Self-assembly morphology detection of NH ₂ -VdopaVKVKVKVDPPTKVKVKVKV-NH ₂ in Tris-HCl buffer (AFM, TEM)

The specific detection method is as follows:

AFM scanning: Take 10 μL of the prepared peptide sample and drop it on the surface of a clean mica sheet, adsorb for 30 seconds, then blow dry the sample with high-purity nitrogen, and complete the scanning under the AFM microscope in tapping mode with a scanning angle of 0°. The scan rate is 1-1.5 Hz, the probe is an RTESP silicon probe (Veeco, Santa Barbara, CA), the tip radius is about 10 nm, the vibration arm length is 125 μm, and the elastic coefficient is 42 N/m. The same sample is scanned 5 times at different positions, and the The results showed that the polypeptide NH ₂ -VdopaVKVKVKVDPPTKVKVKVKV-NH ₂ self-assembled into a very thin nanofiber structure in Tris-HCl buffer, as shown in Figure 2 and Figure 3 .

TEM: Draw a drop of polypeptide solution on the surface of the sealing film, cover the 400-mesh copper mesh with carbon film on the droplet, and let it absorb for 5 minutes. Remove the remaining liquid around the removed copper mesh with filter paper, and then use 2% acetic acid Stain with uranyl for 10 minutes. After the electron microscope examination, the results showed that the peptide sample NH ₂ -VdopaVKVKVKVDPPTKVKVKVKV-NH ₂ observed by the transmission electron microscope self-assembled into a fiber structure in Tris-HCl buffer, which was consistent with the observation by the atomic force microscope, both of which were very fine. The fibrous structure, as shown in Figure 4 and Figure 5.

Example 1:

The preparation method of the present embodiment hydrogel comprises the following steps:

Select the polypeptide NH ₂ -VdopaVKVKVK-VDPPT-KVKVKVKV-NH ₂ prepared above to be diluted to a concentration of 4 mM under the action of buffer Tris-HCl solution, add NaOH to adjust the pH to 9, and self-assemble into a hydrogel at room temperature for 2 hours .

Hydrogel degradation test: at pH = 9, after the peptide NH ₂ -VdopaVKVKVK-VDPPT-KVKVKVKV-NH ₂ forms a hydrogel for 24 hours, the self-assembled morphology is a nanofiber structure, and the storage modulus and energy dissipation modulus The ratio of the amounts was around 10 and the rheological data indicated hydrogel formation. Then add a 1/3 molar concentration of FeCl ₃ solution on top of the hydrogel, and after standing for 1 hour, the nanofiber structure disappears without self-assembled morphology, and the hydrogel degrades with the change of macrostructure A solution is formed; the rheological experimental data show that the ratio of the storage modulus to the loss modulus is about 1, and the storage modulus is lower than 10Pa.

Example 2:

The preparation method of the present embodiment hydrogel comprises the following steps:

Select the polypeptide NH ₂ -VdopaVKVKVK-VDPPT-KVKVKVKV-NH ₂ prepared above and dilute it to a concentration of 4mM under the action of the buffer Tris-HCl solution, add NaOH to adjust the pH to 10, and self-assemble into a hydrogel at room temperature for 2 hours .

The hydrogel prepared in embodiment 2 is measured:

Draw 10 μL of hydrogel sample and drop it on the surface of new mica sheet. After static adsorption for 5 seconds, high-purity nitrogen gas blows dry the sample. AFM scanning and atomic force microscope observation show that after the polypeptide forms a hydrogel, the nanofibers become longer and thicker, as shown in the figure 6; during electron microscope observation, take a complete piece of hydrogel and place it on the surface of the sealing film, cover the 400-mesh copper mesh with carbon film on the top of the gel, let it absorb for 3 minutes, remove the copper mesh and filter it with filter paper. The residual liquid around the copper grid was blotted and stained with 2% uranyl acetate for 1 min. After absorbing the residual liquid on the copper grid, the electron microscope was used to detect the structure. The result was consistent with the structure shown by the atomic force microscope.

After forming a hydrogel with 4mmol peptide NH ₂ -VdopaVKVKVK-VDPPT-KVKVKVKV-NH ₂ at pH=9 for 24 hours, add NIH 3T3 cells on top of the hydrogel and culture in a cell culture incubator, 5% CO ₂ , 37°C After 72 hours, 1/3 molar concentration of FeCl ₃ solution was added. After 1 hour, the hydrogel was destroyed to form a solution, and NIH 3T3 cells grew to form a complete membrane-like structure, which was suspended in the peptide solution, providing an experiment for the development of artificial epithelial tissue. train of thought.

Example 3:

The mechanical properties (viscoelasticity) of NH2-VdopaVKVKVKV ^D PPTKVKVKVKV-NH2 hydrogel samples were characterized by a Haake rheometer, and the measurement module used was a cone-plate with a diameter of 35mm and a taper of 2° and a corresponding sample loading platform, and the sample volume was measured each time 500μL, the temperature of the rheological experiment is 25°C, and the frequency is 1Hz for stress scanning, the scanning range is 0.01%-100%, the linear viscoelastic region of the gel is measured, and the appropriate stress is selected from the linear viscoelastic region for dynamic analysis. Frequency scanning, the scanning range is 0.01Hz-100Hz, to study the relationship between storage modulus G' and loss modulus G".

Dilute the polypeptide NH ₂ -VdopaVKVKVK-VDPPT-KVKVKVKV-NH ₂ to a concentration of 4mM under the action of the buffer Tris-HCl solution, add NaOH to adjust the pH to 10 and mix well, then place it in a water bath at 37°C After 2 hours, 500 μL was taken to perform a frequency sweep of 0.01-80 Hz under the action of a stress of 0.1%. The experimental results are shown in FIGS. 7 and 8 . The mechanical strength G' of the polypeptide hydrogel is about 800Pa.

It should be noted that any equivalent or obvious modification made by those skilled in the art under the teaching of this specification shall fall within the protection scope of the present invention.

Claims (5)

1. the preparation method of a hydrogel, it is characterised in that: it is that selection structural formula is

NH₂-VdopaVKVKVK-VDPPT-KVKVKVKV-NH₂Polypeptide self assembly in the Tris-HCl solution that pH is 9～10 Being formed, described hydrogel is at FeCl₃Effect under can cave in for liquid.

The preparation method of hydrogel the most according to claim 1, it is characterised in that: described hydrogel and FeCl₃Volume ratio For 3:1.

The preparation method of hydrogel the most according to claim 1, it is characterised in that: in Tris-HCl solution, polypeptide is dense Degree is 4mM.

4. the hydrogel prepared according to the preparation method of claim 1 or 2 or 3, it is characterised in that: described hydrogel is for receiving Rice fibre structure.

The hydrogel the most according to claim 4 application in cell culturing bracket.