CN105169474B - Polypeptide material capable of carrying out self-assembly to form hydrogel under neutral pH condition and applications thereof - Google Patents

Abstract

The invention provides a polypeptide material capable of carrying out self-assembly to form hydrogel under a neutral pH condition. The polypeptide material is prepared by the following steps: grafting a plurality of acidic amino acids and a plurality of alkaline amino acids to a RADA16-I sequence so as to form two short peptides with opposite charges, and mixing to carry out self-assembly to form the polypeptide material of hydrogel. The provided polypeptide material can carry out self-assembly in a neutral pH condition at a room temperature (similar to human physiological conditions) to form three-dimensional porous nano fiber hydrogel. The prepared material has a better biocompatibility, can be applied to in-situ cell/active cell loading and in-vivo in-situ injection, and has a wide application prospect and a high clinical application value in the fields such as three-dimensional cell cultivation, biological scaffold material used in tissue engineering, drug carriers, and the like.

Description

Polypeptide material self-assembled into hydrogel under neutral pH and application thereof

technical field

The invention belongs to the field of material preparation, and more specifically relates to a polypeptide material self-assembled into a hydrogel at a neutral pH and an application thereof.

Background technique

Three-dimensional porous nanoscaffolds can mimic the structure of natural extracellular matrix to provide support for cell growth, and are widely used in biomedicine, tissue engineering and other research fields. Almost all tissue cells grow in three-dimensional conditions in vivo, and the cells are wrapped in nanofibrous hydrogels composed of collagen fibers as the main component. In addition, the extracellular matrix also contains a large amount of insoluble matrix proteins and soluble growth factor. However, most of the cell culture techniques used in the current research are two-dimensional culture, that is, cells are cultured on culture plates or dishes, which is quite different from the growth environment in vivo, which affects cell growth and even affects cell genes or functions. Variety. How to construct artificial scaffold materials in vitro that mimic the structure and function of natural extracellular matrix and provide cells with a three-dimensional growth environment has always been a hot spot in basic research and product development in the fields of biology, medicine and materials engineering.

The self-assembled short peptide RADA16-I is a 4-repetition arginine-alanine-aspartic acid-alanine sequence, namely (arginine-alanine-aspartic acid-alanine) ₄ , when its aqueous solution is adjusted to pH = 7, it will form a hydrogel composed of nanofiber network, which is very similar to the structure of natural extracellular matrix. It has been widely used in the field of biomedical engineering as tissue engineering scaffold, drug carrier and hemostatic material , is a typical representative of self-assembled short peptide hydrogel materials. However, a significant disadvantage is that the aqueous solution of the material is obviously acidic (pH=3~4), and cannot be directly mixed with the cell suspension and active molecules to form a gel in situ, that is, it is difficult to realize cell embedding in RADA 16-I water. Three-dimensional growth in the gel.

The current reports on cell culture still use the two-dimensional culture mode of preparing hydrogel and then planting cells on its surface, which cannot realize true three-dimensional cell culture. However, when the material is directly injected into the body to repair damage and is used for hemostasis, the lower pH will cause damage to the host tissue. Since the material was first reported in 1993, people have done a lot of research on RADA16-I, but the shortcoming of its acidity still cannot be effectively solved.

Contents of the invention

According to the deficiency in current self-assembled hydrogel materials, the present invention provides a polypeptide material self-assembled into hydrogel at neutral pH.

Another object of the present invention is to provide the preparation method and application of the above polypeptide material.

Technical purpose of the present invention is achieved through the following technical solutions:

The present invention provides a polypeptide material that self-assembles into a hydrogel at neutral pH, including linking a plurality of acidic amino acids and a plurality of basic amino acids on the RADA16-I sequence to form two oppositely charged short The peptides, after mixing, self-assemble to form the polypeptide material of the hydrogel.

The invention is a hydrogel based on low-molecular-weight self-assembled short peptides, which provides space for three-dimensional growth of cells and is used for constructing tissue engineering scaffolds. This material can form nanofibrous hydrogel under the condition of normal temperature and neutral pH, which imitates the structure of natural extracellular matrix, has good physical and chemical properties and biocompatibility, supports three-dimensional growth of cells, meets the requirements of tissue engineering scaffolds, and can Load active molecular drugs, such as growth factors, short peptide drugs. It can be widely used in the regeneration and damage repair of artificial organs such as cartilage, blood vessels, nerves, and skin.

Preferably, the two oppositely charged short peptide sequences are

Sequence 1: (arginine-alanine-aspartic acid-alanine) ₄ -arginine-isoleucine-lysine-valine-alanine-valine;

Sequence 2: (arginine-alanine-aspartic acid-alanine) ₄ -glutamic acid-glutamic acid-tyrosine-isoleucine-glycine-serine-arginine.

Preferably, the two oppositely charged short peptide sequences are

Sequence 3: (arginine-alanine-aspartic acid-alanine) _4- (lysine) _n ;

Sequence 4: (arginine-alanine-aspartic acid-alanine) ₄ -glutamic acid-glutamic acid-aspartic acid-aspartic acid-aspartic acid;

Wherein, n is any natural number within 1~10.

The applicant found that the number of lysine grafted on (arginine-alanine-aspartic acid-alanine) ₄ and mixed with sequence 4 can be 1~10 Any natural number in , the two pairs of short peptides formed in this way can realize the ability of self-assembly at neutral pH after mixing.

Preferably, the two oppositely charged short peptides are formulated into 5-15 mg/mL aqueous solutions, mixed according to a volume ratio of 1:1, and the pH is adjusted to neutral to obtain the polypeptide material.

The technical scheme adopted in the present invention is: on the short peptide (arginine-alanine-aspartic acid-alanine) ₄ sequence, a plurality of acidic amino acid-based sequences are connected through chemical covalent bonds, such as aspartic acid and glutamic acid, or linked to sequences dominated by multiple basic amino acids, such as lysine, arginine, and histidine. Finally, two short peptide sequences with a net positive charge and a net negative charge were obtained under neutral pH conditions. After being dissolved in water, the pH was adjusted to 7, and the two were mixed to form a three-dimensional nanoporous hydrogel. The hydrogel maintains a neutral pH value and has a stable three-dimensional structure.

The synthesis of the two pairs of short peptide sequences in the present invention is obtained by using the existing grafting direction. Since there is currently no established theory to support which modification (ie, grafting sequence and grafting position) can be obtained under neutral conditions. The synthesized self-assembled polypeptide hydrogels have different sequence arrangements and charge distributions. At the same time, the internal self-assembly mode of the structure will affect the final grafting effect. Therefore, the specific grafting sites and grafting properties of the above-mentioned grafts The sequence arrangement of branches has a great influence on the final effect.

Compared with the existing polypeptide self-assembled hydrogel, the present invention can form a three-dimensional porous hydrogel under physiological conditions (pH 7-7.4), which is composed of a nanofiber network with a diameter of about 20 nm, and the gel network structure is stable , can provide support for three-dimensional cell culture. The introduction of functional amino acid sequences can promote cell growth and adhesion, and can also be loaded with active molecular drugs, such as growth factors and short peptide drugs, to obtain controlled release. It can also be used for injection, and is easy to operate, and is an ideal biological material.

The polypeptide material self-assembled into hydrogel under neutral pH of the present invention is neutral during use and will not cause harm to the nucleus host tissue, and can be used for real three-dimensional culture cells. The suspension is mixed with the two oppositely charged short peptides respectively, and then the two are mixed to obtain a cell-embedded hydrogel/cell complex, which is added to a culture medium for culturing.

Furthermore, the polypeptide material self-assembled into hydrogel at neutral pH has great application prospects in the regeneration and repair of cartilage, blood vessels, nerves or skin.

Compared with the prior art, the present invention has the following beneficial effects:

The invention provides a three-dimensional porous nanofiber hydrogel that can be self-assembled under normal temperature and neutral pH conditions, that is, under human physiological conditions. The material prepared by using the technology of the present invention has better biocompatibility, can realize in situ loading of cells/active molecules and in situ injection in vivo, and has very broad applications in the fields of three-dimensional cell culture, tissue engineering bioscaffold materials and drug carriers Prospect and clinical application value.

Description of drawings

Figure 1 shows the hydrogel material (P1+P2) formed after mixing two modified short peptides (P1, P2). The color of the cell culture medium diffused into the hydrogel did not change, indicating the formation of hydrogel The gel has a neutral pH; the atomic force microscope shows the morphology of nanofibers, indicating that the formed hydrogel is a network composed of nanofibers.

Figure 2 is a comparison chart of the rheological properties of the three materials, P1 and P2 are modified short peptides, and P1+P2 is a mixture of the two. G' is the storage modulus, and G" is the loss modulus. After mixing P1 and P2, G' increases greatly, indicating that a stable hydrogel is formed.

Figure 3 is the three-dimensional growth morphology of neural stem cell spheres in different nanofibrous hydrogels. A is the nanofiber hydrogel prepared by the patent of the present invention; B is the RADA 16-I nanofiber hydrogel; it can be seen from the figure that neural stem cell balls can grow three-dimensionally in the nanofiber hydrogel prepared by the patent of the present invention, And grow longer axons, while in RADA 16-I hydrogel, no axons grow.

Figure 4 shows the detection of the survival of neural stem cells in different three-dimensional scaffolds using the living and dead cell detection kit, green represents living cells, and red represents dead cells. A is the nanofiber hydrogel prepared by the patent of the present invention; B is the RADA 16-I nanofiber hydrogel; it can be seen from the figure that neural stem cells survive in large quantities in the nanofiber hydrogel prepared by the patent of the present invention, and the survival rate It reaches 100%, and can grow three-dimensionally, and grow longer axons, while in RADA 16-I hydrogel, a large number of dead, layered spherical, without any axon growth.

detailed description

The present invention is described in detail by the following examples, it is necessary to point out that this example is only used to further illustrate the present invention, but can not be interpreted as limiting the protection scope of the present invention, those skilled in the art can according to above-mentioned this invention The content of the invention makes some non-essential improvements and adjustments.

Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Embodiment 1: Preparation of polypeptide material of hydrogel:

Sequence 1 (P1): (arginine-alanine-aspartic acid-alanine) ₄ -arginine-isoleucine-lysine-valine-alanine-valine After being dissolved in ultrapure water, it is configured into a solution with a concentration of 5-15ml/mL, and its pH value is adjusted to 7 with NaOH to obtain a positively charged short peptide aqueous solution.

Sequence 2 (P2): (arginine-alanine-aspartic acid-alanine) ₄ -glutamic acid-glutamic acid-tyrosine-isoleucine-glycine-serine-arginine After dissolving in ultrapure water, configure it into a solution with a concentration of 5-15ml/mL, adjust its pH value to 7 with NaOH, and obtain a negatively charged short peptide aqueous solution.

Then mix the two solutions at a volume ratio of 1:1, and quickly form a gel after standing still, and put it into use after the shape is stable (see Figure 1).

As shown in Figure 1, after the two are mixed, a hydrogel is formed and fluidity is lost.

The atomic force microscope showed the morphology of nanofibers, which consisted of a network of nanofibers with a diameter of about 20 nm, indicating that the formed hydrogel was a network of nanofibers, and the structure of the gel network was stable, which could provide support for three-dimensional cell culture.

Embodiment 2: preparation of the polypeptide material of hydrogel:

Preparation method is with embodiment 1,

Sequence 3 (P1'): (arginine-alanine-aspartic acid-alanine) ₄ -(lysine) _1-10 ; n is any natural number within 1-10.

Sequence 4 (P2'): (arginine-alanine-aspartic acid-alanine) ₄ -glutamic acid-glutamic acid-aspartic acid-aspartic acid-aspartic acid.

Example 3: Three-dimensional cell culture

Using ultrapure water as solvent, prepare P1 and P2 solutions with a concentration of 5-15 mg/mL, mix the cell suspension with P1 and P2 adjusted to neutral pH respectively, to obtain a uniform cell/short peptide mixture, use The NaOH solution adjusted its pH to neutral, and then mixed it at a ratio of 1:1 to obtain a three-dimensional hydrogel embedded with cells. After standing still, the gel was formed rapidly, and the cell culture medium was added for cultivation.

In Example 3, the embedded cells are neural stem cell spheres, as shown in Figures 3 and 4, it can be seen that the neural stem cell spheres can grow three-dimensionally in the nanofiber hydrogel prepared by the patent of the present invention, reaching a 100% survival rate. And grow longer axons, while in RADA 16-I hydrogel, no axons grow.

Example 4: Three-dimensional cell culture

Use ultrapure water as solvent to prepare P1' and P2' solutions with a concentration of 5-15 mg/mL, and mix the cell suspension with P1 and P2 adjusted to neutral pH to obtain a uniform cell/short peptide mixture , the pH was adjusted to neutral with NaOH solution, and then mixed at 1:1 to obtain a three-dimensional hydrogel embedded with cells, which quickly formed a gel after standing still, and was added to cell culture medium for cultivation.

The embedded cells in Example 4 are neural stem cell spheres, and the detected phenomenon is the same as the result of Example 3. Compared with the RADA 16-I hydrogel, the nanofiber hydrogel provided by the present invention can grow three-dimensionally, A 100% survival rate was achieved with longer neurite outgrowth, whereas in RADA 16-I hydrogel there was no axon outgrowth.

Example: 5: In vivo in situ injection into a gel

Inject P1 and P2 sequentially at the defect site in the body to form a gel. The gel is neutral and will not cause damage to surrounding tissues.

Inject P1' and P2' sequentially at the defect site in the body to form a gel. The gel is neutral and will not cause damage to surrounding tissues.

Claims (6)

1. the polypeptide material of hydrogel is self-assembled under a kind of neutral pH, it is characterised in that is included in RADA16-I sequences and is divided Do not connect multiple acidic amino acids and multiple basic amino acids, the small peptide of two kinds of oppositely chargeds is formed with this, from group after mixing Dress forms the polypeptide material of the hydrogel；The short peptide sequence of described two oppositely chargeds is respectively

Sequence 1：（Arg-Ala-Asp-alanine）₄- arginine-Isoleucine-lysine-the ammonia of valine-the third Acid-valine；

Sequence 2：（Arg-Ala-Asp-alanine）₄- Glu-Glu-Tyrosine-Isoleucine-sweet ammonia Acid-Vitro By Serine/arginine；

Or,

The short peptide sequence of described two oppositely chargeds is respectively

Sequence 3：（Arg-Ala-Asp-alanine）₄-（Lysine）_n；

Sequence 4：（Arg-Ala-Asp-alanine）₄- Glu-Glu-Asp-Asp-day Winter propylhomoserin；

Wherein, n is any one natural number in 1 ~ 10.

2. the polypeptide material of hydrogel is self-assembled under neutral pH according to claim 1, it is characterised in that by described two The small peptide for planting oppositely charged is each configured to the aqueous solution of 5 ~ 15mg/mL, according to 1：1 volume ratio mixing, pH is into for regulation Property, obtain final product the polypeptide material.

3. the polypeptide material of hydrogel is self-assembled under the neutral pH described in a kind of claim 1 or 2 in organizational project biology Application in frame material preparation.

4. the polypeptide material of hydrogel is self-assembled under the neutral pH described in a kind of claim 1 or 2 in three-dimensional cell cultivation Application.

5. application according to claim 4, it is characterised in that by cell suspension respectively with described two oppositely chargeds Small peptide is mixed, and then mixes both, obtains the hydrogel/cell conjugate of embedding cell, and adds culture medium to carry out Culture.

6. be self-assembled under the neutral pH described in a kind of claim 1 or 2 polypeptide material of hydrogel prepare cartilage, blood vessel, Application in the regeneration and injury repair material of nerve or skin.