CN105363069B - A kind of FeCl3the intelligent polypeptide hydrogel of regulation and control, preparation method and applications - Google Patents

Abstract

The invention discloses a kind of FeCl₃Regulation and control intelligent polypeptide hydrogel, preparation method and applications, its be select structural formula be NH₂‑VdopaVKVKVK‑VDPPT‑KVKVKVKV‑NH₂Polypeptide be self-assembly of in the Tris HCl solution that pH is 9～10, gained hydrogel is at FeCl₃Effect under can cave in for liquid, the hydrogel that the present invention prepares can be as cell culturing bracket.The hydrogel that the Study of Self-assembling Peptides that the present invention proposes is formed, can pass through Fe³⁺Addition so that it is cave in for solution state, be a kind of Fe³⁺The progress in Intelligent Hydrogel of regulation and control, is the ideal nano-structure engineering material of a class, significant to human life's health.

Description

A kind of FeCl3The intelligent polypeptide hydrogel of regulation and control, preparation method and applications

Technical field

The present invention relates to the preparing technical field of hydrogel, be specifically related to a kind of FeCl₃The intelligent polypeptide water-setting of regulation and control Glue, preparation method and applications.

Background technology

Preferably tissue engineering material not only needs possess the grid structure supporting and connecting tissue, and needs in regulation Tissue occurs and the physiological activity aspect of cell has advantage.(shell gathers current most of timbering material many employings biomacromolecule Sugar, alginate, collagen protein) or synthesis macromolecule (Polyethylene Glycol) etc., in order to provide the microenvironment supporting cell growth.But Chemical residual, source of disease is propagated and is synthesized the problems such as cost and limits its deeply application.

Hydrogel of the prior art, its preparation method is usually little molecular self-assembling and high molecular crosslink two class.At present, Little molecule synthesis easy, with low cost but owing to its structure is engineer, it is difficult to realize degraded；High-molecular gel preparation letter Single, structure-controllable, but it is difficult to rationally embedding and degraded of biological response site.This research, by the most embedding for corresponding site Enter in this little molecule of the convenient polypeptide of synthesis, it is achieved that clear and definite degradation capability.In the organizational project cultivated for cell In material, when cell grows into some, it is not necessary to the when that support supporting, it is desirable to tissue engineering material can effectively drop Solve, thus allow cell preferably be dissolved in body tissue.

Summary of the invention

It is an object of the invention to provide a kind of intelligent polypeptide hydrogel and preparation method thereof, this hydrogel is at FeCl₃ Effect under can cave in for liquid.

One of task of the present invention is to provide the preparation method of hydrogel.

A kind of preparation method of hydrogel, it is that selection structural formula is:

NH₂-VdopaVKVKVK-VDPPT-KVKVKVKV-NH₂Polypeptide in the Tris-HCl solution that pH is 9～10 from Assembling formation, this hydrogel is at FeCl₃Effect under can cave in for liquid.

The Advantageous Effects that technique scheme is directly brought is: first, and selection can use solid phase synthesis technique side Just the small peptide synthesized so that synthesize with low cost, purification is convenient；Meanwhile, the polypeptide of design synthesis uses ripe self assembly Model, possesses good assembling, forms the ability of hydrogel；Second, use the physical method system of regulation pH at ambient temperature Standby hydrogel, it is to avoid the injury that tissue is caused by additional chemical agent, ultraviolet light etc., reduces compared to existing technology and causes tissue The risk of injury；3rd, by adding FeCl₃Degrade, it is achieved that coagulated by the physical action of simple inorganic ions The function of glue degraded, it is to avoid introduce potential source biomolecule toxicity and immunogenicity that chemical reaction causes.

As a preferred version of the present invention, hydrogel and FeCl₃Volume ratio be 3:1.

In Tris-HCl solution, the concentration of polypeptide is 4mM.

Another task of the present invention is to provide the hydrogel that above-mentioned preparation method prepares.

Above-mentioned hydrogel is nanofibrous structures.

The application in cell culturing bracket of the above-mentioned hydrogel.

The Advantageous Effects that the present invention is brought:

The invention provides the preparation method of a kind of hydrogel, the difference that the hydrogel prepared is maximum with prior art It is: can be at FeCl₃Effect under degrade；In terms of the choosing of raw material, the present invention selected polypeptide in Tris-HCl solution, control PH processed is 9 to be self-assembly of, self-assembling polypeptide the nanofiber formed is support, the hydrogel that preparation property is stable, thus Make it have corresponding biological function, enrich the kind that peptide hydrogel is formed.

The hydrogel that the Study of Self-assembling Peptides that the present invention proposes is formed, can pass through Fe³⁺Addition so that it is cave in for solution State, is a kind of Fe³⁺The progress in Intelligent Hydrogel of regulation and control, is the ideal nano-structure engineering material of a class, to human life Healthy significant.

Accompanying drawing explanation

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

Fig. 1 is the NH after the present invention is synthesized by solid-phase synthesis₂-VdopaVKVKVK-VDPPT-KVKVKVKV-NH₂Instead Phase high-efficient liquid phase chromatogram；

Fig. 2 is NH of the present invention₂-VdopaVKVKVK-VDPPT-KVKVKVKV-NH₂In Tris-HCl (pH7.4) solution certainly Assemble atomic force microscope shape appearance figure；

Fig. 3 is NH of the present invention₂-VdopaVKVKVK-VDPPT-KVKVKVKV-NH₂From group in Tris-HCl (pH7) solution Dress transmission electron microscope shape appearance figure；

Fig. 4 is NH of the present invention₂-VdopaVKVKVK-VDPPT-KVKVKVKV-NH₂In Tris-HCl (pH10) solution certainly Assemble atomic force microscope shape appearance figure；

Fig. 5 is NH of the present invention₂-VdopaVKVKVK-VDPPT-KVKVKVKV-NH₂In Tris-HCl (pH10) solution certainly Assemble transmission electron microscope shape appearance figure；

Fig. 6 is NH of the present invention₂-VdopaVKVKVK-VDPPT-KVKVKVKV-NH₂The machinery of hydrogel is formed after 24 hours Relation between intensity (storage modulus and loss modulus) and frequency；

Fig. 7 is NH of the present invention₂-VdopaVKVKVK-VDPPT-KVKVKVKV-NH₂The hydrogel formed adds FeCl₃The most former Sub-force microscope shape appearance figure；

Fig. 8 is NH of the present invention₂-VdopaVKVKVK-VDPPT-KVKVKVKV-NH₂The hydrogel formed adds FeCl₃Rear saturating Penetrate ultramicroscope shape appearance figure.

Detailed description of the invention

Below in conjunction with specific embodiment, the present invention is described in further details.

First being described briefly the specification of major experimental instrument selected by the present invention, model, following experimental apparatus is equal Can be bought by commercial channel and obtain:

Electric heating constant temperature incubator (DNP-9082 type, upper Nereid grand testing equipment company limited)；

Dry disinfection baking box (DHG-9246A type, upper Nereid grand testing equipment company limited)；

Ultraviolet spectrophotometer (biotechnology instrument company of the U.S.)；

Desk centrifuge (Ai Bendefu company of Germany)；

Atomic force microscope (AFM) (Nanoscope Iva type multiple-pattern atomic force microscope, Digital Instruments)

Transmission electron microscope (TEM) (NEC company JEM1400Plus type)

Flow graph (Mars type III, Haake company)

Superclean bench (Airtech type, Jiangsu is safe and sound)

Constant temperature cell culture incubator (HERACELL 150i, power & light company, France)

Inverted microscope (TS100 type, NIKON, Japan)

Fluorescence inverted microscope (DMI3000B type, Lycra company, Germany)

Laser Scanning Confocal Microscope (NIKON's A1 type, Japan)

Disposable Tissue Culture Flask (25cm², Corning Incorporated, costar type)

Disposable pipet (5mL, accuracy 0.1mL, Corning Incorporated, costar type)

Disposable Tissue Culture Plate (article No. 3599, Corning Incorporated, costar type)

Disposable Tissue Culture Plate (article No. 3548, Corning Incorporated, costar type)

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

Microwave-assisted Peptide synthesizer (the Liberty1 many automatic peptide synthesizers of type microwave of CEM company).

Secondly, the present invention prepares the polypeptide NH selected by hydrogel₂-VdopaVKVKVK-VDPPT-KVKVKVKV-NH₂It is Homemade, its concrete preparation method is:

Step 1, distillation N,N-dimethylformamide (DMF) and piperidines (Piperidine) solvent

The DMF solution bought is reduced pressure distillation under the conditions of 60 DEG C, obtains pure DMF solvent；The piperidines of purchase will be added Enter a small amount of CaH2 to be heated to reflux 1-2 hour, receive the fraction of boiling temperature (106 DEG C), obtain pure piperidines solvent；

The preparation of step 2, aminoacid, resin, activator, block agent, deprotection agent etc.

Needed for calculating preparation 0.25mM NH2-VdopaVKVKVKVDPPTKVKVKVKV-NH2 on Solid-phase synthesis peptides instrument (for the purity of polypeptide during guarantee Peptide systhesis, amino acid whose consumption doubles the consumption of aminoacid and other reagent, and it is final concentration of 0.2M):

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

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

Resin (carrying capacity is 0.6mmol/g): 0.417g；

Note: amino acid whose a-amino is Fmoc protection, and the side-chain amino group of Lys is also protected；

Activator: BTA-N, N, N', N'-tetramethylurea hexafluorophosphoric acid ester (HBTU): 6.83g；1-hydroxy benzo Triazole (HOBT): 2.43g；It is dissolved in 40mL DMF；

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

Block agent: acetic anhydride: 4ml；N, N-diisopropylethylamine (DIEA): 0.44g；HOBT:0.04g；It is dissolved in 16mL In DMF；

Decomposition agent: trifluoroacetic acid (TFA): 23.5mL；Tri isopropyl silane (TIS): 0.25mL；H2O:0.625mL；1,2- Dithioglycol (EDT) 0.625mL；

Deprotection agent: piperidines: 46mL；DMF:184mL；I-hydroxybenzotriazole (HOBT): 3.11g.

Step 3, the solid phase synthesis of polypeptide and purification

Ready medicine in step 2 is joined in solid-state reaction under microwave instrument specified containers, start from C end to N end Synthesis Ac-IIIIKK-NH2, instrument is automatically synthesized.After Peptide systhesis, the product in product pipe is poured in round-bottomed flask, Adding decomposition agent, 4h is stirred at room temperature, collect filtrate after vacuum filtration, TFA washing resin 3 times, merging filtrate and cleaning mixture, by it Pouring in alembic and distill (TFA removing remaining), the product after distillation is poured in 10mL centrifuge tube, adds cold diethyl ether, centrifugal 15min, rotating speed is 9000rpm/min, is repeated 10 times above, preparative RP-HPLC purification, finally product is put into height Lyophilizing in pressure freeze dryer, puts into refrigerator and preserves after lyophilizing, substance assistant laser desorpted flying time mass spectrum analysis shows its point The molecular weight that son amount is the polypeptide of our synthesis.Rp-hplc analysis, as it is shown in figure 1, the polypeptide of present invention synthesis Purity be more than 98%.

Detection: NH₂-VdopaVKVKVKVDPPTKVKVKVKV-NH₂Self assembly pattern inspection in Tris-HCl buffer Survey (AFM, TEM)

Concrete detection method is as follows:

AFM scan: take polypeptide sample dropping that 10 μ L prepare in clean mica sheet surface, adsorb 30s, then High Purity Nitrogen Air-blowing dry-eye disease, completes scanning with tapping-mode (tapping mode) under AFM microscope, and scanning angle is 0 °, sweep speed 1 ～1.5Hz, probe is RTESP type silicon probe (Veeco, Santa Barbara, CA), and needle type radius is about 10nm, length of raising one's arm 125 μm, coefficient of elasticity 42N/m, same sample scans 5 times at diverse location, and its result shows, polypeptide NH₂- VdopaVKVKVKVDPPTKVKVKVKV-NH₂The thinnest nanofibrous structures it is self-assembled into, such as figure in Tris-HCl buffer Shown in 2 and Fig. 3.

The 400 mesh copper mesh with carbon film are covered above drop by TEM: draw a polypeptide solution on sealed membrane surface, Staticaccelerator adsorption 5min, periphery residual liquid is absorbed by the copper mesh filter paper taken off, then with 2% uranium acetate dyeing 10min. Rear Electron microscopy, its result shows, the polypeptide sample NH arrived by transmission electron microscope observation₂- VdopaVKVKVKVDPPTKVKVKVKV-NH₂It is self-assembled into fibre structure, with atomic force microscopy in Tris-HCl buffer It is consistent that sem observation arrives, and is the thinnest filamentary structure, as shown in Figure 4 and Figure 5.

Embodiment 1:

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

Choose the above-mentioned polypeptide NH prepared₂-VdopaVKVKVK-VDPPT-KVKVKVKV-NH₂At buffer Tris- Under the effect of HCl solution, being diluted to concentration is 4mM, adds NaOH and regulates pH to 9, and room temperature 2h is i.e. self-assembled into hydrogel.

Hydrogel degraded test: when pH=9, peptide NH₂-VdopaVKVKVK-VDPPT-KVKVKVKV-NH₂Form water-setting After glue 24h, now self assembly pattern is nanofibrous structures, and storage modulus is about 10 with the ratio of Loss modulus, rheology The formation of data explanation hydrogel.Then above hydrogel, add the FeCl of 1/3 molar concentration₃Solution, after placing 1h, transmission Electronic Speculum and atomic force microscope observation, nanofibrous structures disappears, without self assembly pattern, simultaneously with the change of macrostructure, Hydrogel degraded forms solution；Rheology data show, storage modulus is about 1 with the ratio of Loss modulus, and energy storage Modulus is less than 10Pa.

Embodiment 2:

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

Choose the above-mentioned polypeptide NH prepared₂-VdopaVKVKVK-VDPPT-KVKVKVKV-NH₂At buffer Tris- Under the effect of HCl solution, being diluted to concentration is 4mM, adds NaOH and regulates pH to 10, and room temperature 2h is i.e. self-assembled into hydrogel.

The hydrogel preparing embodiment 2 is measured:

Drawing 10 μ L hydrogel sample to drip after new mica sheet surface, staticaccelerator adsorption 5s, high pure nitrogen dries up sample, AFM scan, after atomic force microscope observation finds that polypeptide forms hydrogel, nanofiber is elongated thicker, as shown in Figure 6；Electronics When microscope is observed, take one block of complete hydrogel and be placed in sealed membrane surface, the 400 mesh copper mesh with carbon film are covered at gel Top, staticaccelerator adsorption 3min, absorbs copper mesh periphery residual liquid with filter paper after taking off copper mesh, 2% uranium acetate dyeing 1min, after drawing the residual liquid on copper mesh, Electron microscopy, the structure one that its result and atomic force microscope show Cause.

At pH=9,4mmol peptide NH₂-VdopaVKVKVK-VDPPT-KVKVKVKV-NH₂After forming hydrogel 24h, at water NIH 3T3 cell is added, at cell culture incubator, 5%CO above gel₂, 37 DEG C of environment are cultivated after 72h, add 1/3 mole dense The FeCl of degree₃Solution, the destroyed solution that formed of hydrogel after 1h, the growth of NIH 3T3 cell forms complete membrane structure, suspends In peptide solution, the research and development for artificial epithelial tissue provide experimental considerations.

Embodiment 3:

Haake torque rheometer is used to characterize NH2-VdopaVKVKVKV^DThe mechanicalness of PPTKVKVKVKV-NH2 hydrogel sample Energy (viscoelasticity), the measurement module of employing is cone-plate and corresponding load sample platform, each measuring samples volume of diameter 35mm tapering 2 ° Being 500 μ L, rheological experiment temperature is 25 DEG C, carries out stress scans with frequency for 1Hz, and sweep limits is 0.01%-100%, surveys Determining the linear viscoelastic region of gel, choose suitable stress to carry out dynamic frequency scanning from linear viscoelastic region, sweep limits is 0.01Hz-100Hz, study storage modulus G ' and loss modulus G " between relation.

By polypeptide NH₂-VdopaVKVKVK-VDPPT-KVKVKVKV-NH₂Under the effect of buffer Tris-HCl solution, Being diluted to concentration is 4mM, after adding NaOH regulation pH to 10 fully mixing, is statically placed in water-bath 2 hours in 37 DEG C of water-baths, then Taking 500 μ L and carry out the frequency scanning of 0.01-80Hz under stress is 0.1% effect, experimental result is as shown in Figure 7 and Figure 8.Polypeptide Mechanical strength G of hydrogel ' it is about 800Pa.

It should be noted that any equivalent way that those skilled in the art are made under the teaching of this specification, or Substantially variant all should be within the 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.