阅读说明：本技术 一种基于硼酯键的手性超分子核苷水凝胶及其制备方法和用途 (Chiral supramolecular nucleoside hydrogel based on boron ester bond and preparation method and application thereof ) 是由 刘江 杜玉琦 刘天楠 丁婷婷 曾昕 于 2020-05-18 设计创作，主要内容包括：本发明涉及一种基于硼酯键的手性超分子核苷水凝胶及其制备方法和用途。具体提供了一种超分子水凝胶,所述超分子水凝胶是以由鸟嘌呤核苷和硼酸盐为原料,在溶剂中混合得到的；所述鸟嘌呤核苷为D-鸟嘌呤核苷和/或L-鸟嘌呤核苷。该超分子水凝胶具有优异的稳定性、可注射性和自修复性,同时具有良好的生物相容性,在动物体内未表现明显急性毒性,能够在体内降解,可作为细胞外基质用于细胞的3D培养。本发明提供的超分子水凝胶作为支架材料在组织工程领域具有非常好的应用前景。(The invention relates to a chiral supramolecular nucleoside hydrogel based on a boron ester bond, a preparation method and application thereof. The supermolecule hydrogel is prepared by mixing guanosine and borate serving as raw materials in a solvent; the guanosine is D-guanosine and/or L-guanosine. The supramolecular hydrogel has excellent stability, injectability and self-repairing property, has good biocompatibility, does not show obvious acute toxicity in animal bodies, can be degraded in the bodies, and can be used as extracellular matrix for 3D culture of cells. The supermolecule hydrogel provided by the invention has a very good application prospect in the field of tissue engineering as a scaffold material.)

1. A supramolecular hydrogel, comprising: the supermolecule hydrogel is prepared by mixing guanosine and borate serving as raw materials in a solvent; the guanosine is D-guanosine and/or L-guanosine, the structure of the D-guanosine is shown as a formula (I), and the structure of the L-guanosine is shown as a formula (II):

2. the supramolecular hydrogel of claim 1, wherein: the guanosine is D-guanosine or L-guanosine, preferably L-guanosine.

3. The supramolecular hydrogel of claim 2, wherein: the borate is sodium borate, potassium borate, lithium borate, magnesium borate or calcium borate, and preferably sodium borate.

4. The supramolecular hydrogel of claim 3, wherein: the solvent is water or an aqueous solution, the aqueous solution is preferably PBS buffer solution, and the pH range of the PBS buffer solution is preferably 7.35-7.45.

5. The supramolecular hydrogel of any one of claims 1 to 4, wherein: the ratio of borate ions to the amount of guanosine in the borate is 1: (1-4), preferably 1: 2.

6. The supramolecular hydrogel of claim 5, wherein: the concentration of the guanosine in the solvent is 0.05-0.2M, and preferably 0.1M.

7. The supramolecular hydrogel of claim 1, wherein: the supramolecular hydrogel comprises a boron ester bond.

8. A method of preparing the supramolecular hydrogel as claimed in any one of claims 1 to 7, wherein: the method comprises the steps of uniformly mixing guanosine, borate and a solvent, and heating to obtain the compound.

9. The method of claim 8, wherein: the heating temperature is 80-100 ℃, and preferably 90 ℃;

and/or the heating time is the time until the raw materials are completely dissolved.

10. Use of the supramolecular hydrogel according to any one of claims 1 to 7 for the preparation of extracellular matrix or scaffold materials.

Technical Field

The invention belongs to the field of hydrogel, and particularly relates to chiral supramolecular nucleoside hydrogel based on a boron ester bond, and a preparation method and application thereof.

Background

Oral mucosal disease refers to a general term for various diseases of different types and kinds occurring in oral mucosa and soft tissues. In clinical work, certain oral mucosa diseases are often manifested as persistent, extensive and serious erosive or ulcerative surfaces, such as oral lichen planus, severe recurrent aphthous ulcer and the like; such diseases are histopathologically manifested as a tissue defect of a portion of epithelium or a full layer of epithelium. Current treatment modalities include removal of primary factors, medication, surgical treatment, laser treatment, and the like. However, the traditional treatment method has the defects of unclear or difficult removal of some primary factors, poor curative effect of the medicine, large side effect and the like, so that the clinical needs are difficult to meet.

In recent years, tissue engineering techniques have been advanced significantly in the regeneration of bone and cartilage tissues, and are probably a new concept for the treatment of oral mucosal diseases. Tissue engineering is generally composed of three elements, namely a scaffold material, seed cells and growth factors. Common tissue engineering strategies are: the seed cells grow and expand in the three-dimensional porous tissue scaffold under the precisely controlled condition to form a structure, then the cell/scaffold structure is implanted into a required part in a body to guide new tissues to form in the scaffold, the scaffold is gradually degraded and disappeared along with the formation of the tissues, and damaged tissues and organs are reconstructed. The scaffold material not only plays a supporting role and provides a foundation for differentiation and migration of seed cells, but also can be used as a slow release platform of medicines and cytokines to promote repair and reconstruction of damaged tissues and organs.

The ideal scaffold material has good biodegradability, can promote cell proliferation and differentiation and generate extracellular matrix, has exchange channels of nutrient substances and metabolites, and can be adhered and integrated with surrounding tissues. The tissue scaffold can be divided into two types, namely a prefabricated porous scaffold and hydrogel, according to whether the tissue scaffold needs to be prepared in advance. Among them, the hydrogel material is a polymer having a three-dimensional network structure, which can absorb a large amount of water in water to swell, and can continuously maintain its original structure after swelling without being dissolved. The three-dimensional network structure of the material is similar to that of a natural extracellular matrix, and meanwhile, the material is rich in water, is beneficial to the survival of seed cells, and is a bracket material widely applied to various tissue engineering researches.

Hydrogels can be classified as chemical hydrogels and physical hydrogels, depending on the bonding network. Chemical hydrogels are formed by covalent bond crosslinking, and are mostly high molecular polymer hydrogels, which can be artificially synthesized high molecular polymers (such as polyvinyl alcohol, polyacrylate, polyamide, polyethylene, etc.) and natural high molecular polymers (such as gelatin, collagen, agar, starch, etc.) according to their sources. Although the high molecular polymer hydrogel has excellent stability and mechanical properties, the hydrogel formed by the artificially synthesized polymer lacks a signal for cell recognition, and cannot be degraded under physiological conditions, or degradation products are toxic, so that the biological safety of the material is seriously influenced; the natural high molecular polymer has different structure sources, the difference between structures and properties, poor material property repeatability, insufficient mechanical strength and narrow adjustable range of the structure and the properties, and limits the application of the chemical hydrogel in the bracket material.

The physical hydrogel is formed by weak non-covalent bond interaction (such as hydrogen bond, ionic bond, pi-pi accumulation, van der waals force, electrostatic interaction and the like) among molecules, can easily generate reversible sol-gel behavior under the action of external force, is simple to prepare, does not need chemical reaction, and is more beneficial to the application of the hydrogel in the field of biomedicine. In recent years, low molecular weight gel-forming molecules (LMWG) have attracted increasing attention from researchers. However, physical hydrogels are less stable than chemical hydrogels, which greatly limits the use of physical hydrogels as scaffold materials.

Certain low molecular weight compounds such as amino acids, nucleic acids, etc. can form Self-Assembled fiber Networks (SAFINs) through non-covalent interactions in a specific solvent, and the network structures limit the free movement of solvent molecules to a certain extent, so that viscoelastic solid-like substances, namely supramolecular hydrogels, are formed. Compared with the traditional polymer hydrogel, the supermolecule hydrogel has unique properties in the aspects of stability, mechanical property, in-vivo degradation, metabolism and the like, has a structure which can generate important influence on the growth, proliferation, differentiation and the like of seed cells, and has wide application prospect in the aspects of drug-loaded systems, tissue engineering and the like.

It has been found that guanosine and its derivatives form tetramers (G-quatets) in salt solutions of certain metal ions, and these tetramers are stacked layer by layer to form fiber-like structures, which are cross-linked to each other to form a network. Guanosine and derivatives thereof have better potential in the preparation of supramolecular hydrogel. However, the guanosine molecules have the tendency of escaping from the network structure, and as time goes on, more and more molecules escape from the network, aggregate with each other, gradually form crystals or precipitates, so that the gel collapses, and the service life and stability of the gel are seriously influenced.

In addition, the appearance of novel dynamic covalent bond hydrogels (DCB gels) opens up a new development path for the application of functional hydrogels in the biomedical field. The dynamic covalent bond hydrogel is a three-dimensional network structure which is constructed by taking dynamic covalent bonds as crosslinking points, integrates the stability of chemical gel and the reversibility of physical gel, and generally has good self-repairing property (or self-healing property) and injection molding property. The unique adjustability enables the drug delivery system to have potential application value and development prospect in a plurality of fields such as drug delivery, sensors, tissue engineering, biomedical engineering and the like. Currently, one of the main means for synthesizing functional hydrogels is a dynamic covalent bond that is dominated by a boroester bond (B-O), an acylhydrazone bond (-HC ═ N-NH-CO-) and a reversible imine bond (-C ═ N-).

Therefore, the preparation of the functional supramolecular hydrogel integrating excellent stability, self-repairing property (or self-healing property) and injection molding has very important significance in the fields of tissue engineering scaffold materials and the like.

Disclosure of Invention

The invention aims to provide a chiral supramolecular nucleoside hydrogel based on a boron ester bond, and a preparation method and application thereof.

The invention provides a supermolecule hydrogel which is prepared by mixing guanosine and borate serving as raw materials in a solvent; the guanosine is D-guanosine and/or L-guanosine, the structure of the D-guanosine is shown as a formula (I), and the structure of the L-guanosine is shown as a formula (II):

further, the guanosine is D-guanosine or L-guanosine, and preferably the L-guanosine.

Further, the borate is sodium borate, potassium borate, lithium borate, magnesium borate, calcium borate, preferably sodium borate.

Further, the solvent is water or an aqueous solution, the aqueous solution is preferably PBS buffer solution, and the pH range of the PBS buffer solution is preferably 7.35-7.45.

Further, the ratio of the borate ion to the amount of the substance of guanosine in the borate is 1: (1-4), preferably 1: 2.

Further, the concentration of the guanosine in the solvent is 0.05-0.2M, and preferably 0.1M.

Further, the supramolecular hydrogel includes a boron ester bond.

The invention also provides a method for preparing the supermolecule hydrogel, which comprises the steps of uniformly mixing guanosine, borate and a solvent, and heating.

Further, the heating temperature is 80-100 ℃, and preferably 90 ℃;

and/or the heating time is the time until the raw materials are completely dissolved.

The invention also provides application of the supramolecular hydrogel in preparation of extracellular matrix or scaffold materials.

Sodium borate, also known as sodium tetraborate, commonly known as borax, of formula Na₂B₄O₇·10H₂O。

The structure of the boroester bond is shown in the following formula (III):

experimental results show that the chiral supramolecular hydrogel based on the boron ester bond has excellent stability, injectability and self-repairability, has good biocompatibility, does not show obvious acute toxicity in animal bodies, can be degraded in the bodies, and can be used as an extracellular matrix for 3D culture of cells. The supermolecule hydrogel provided by the invention has a very good application prospect in the field of tissue engineering as a scaffold material.

The method for preparing the supermolecule hydrogel provided by the invention is simple, safe and nontoxic, and is suitable for expanded production.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1: of samples¹¹B NMR (a) and infrared (B) characterization spectrograms; in the graph a, the test samples of the top-to-bottom 3 curves are 2.8% w/v L-G supramolecular hydrogel prepared in example 5, 1.4% w/v L-G supramolecular hydrogel prepared in example 4, and sodium borate in this order¹¹B-disester represents the chemical shift of the boric acid Diester bond,¹¹B-Monoester represents the chemical shift of the boric acid Monoester bond,¹¹b, chemical shift of free boric acid bond; in the B diagram, the B-O (-) curve represents the raw material L-guanosine, and the B-O (+) curve represents the 2.8% w/v L-G supramolecular hydrogel.

FIG. 2: each hydrogel was inverted for 5min, 24h, and 8 d. Wherein a1 represents the hydrogel obtained in comparative example 1, c1 represents the hydrogel obtained in comparative example 2, e1 represents the hydrogel obtained in comparative example 3, a2 represents the hydrogel obtained in comparative example 4, c2 represents the hydrogel obtained in comparative example 5, and e2 represents the hydrogel obtained in comparative example 6; b1 represents the hydrogel obtained in example 1, d1 represents the hydrogel obtained in example 2, f1 represents the hydrogel obtained in example 3, b2 represents the hydrogel obtained in example 4, d2 represents the hydrogel obtained in example 5, and f2 represents the hydrogel obtained in example 6.

FIG. 3: results of rheological testing of each hydrogel at different shear frequencies. Wherein a represents a control hydrogel 2 ', b represents a control hydrogel 5', c represents the supramolecular hydrogel prepared in example 2, and d represents the supramolecular hydrogel prepared in example 5.

FIG. 4: results of rheological testing of each hydrogel at different shear strains. Wherein a represents a control hydrogel 2 ', b represents a control hydrogel 5', c represents the supramolecular hydrogel prepared in example 2, and d represents the supramolecular hydrogel prepared in example 5.

FIG. 5: the results of HE staining of important organs after injecting 2.8% w/v D-G supramolecular hydrogel (i.e. D-G in the figure) and 2.8% w/v L-G supramolecular hydrogel (i.e. L-G in the figure) into the dorsal subcutaneous of mice for different times, and the "control group" is the result of the control group.

FIG. 6: 2.8% w/v D-G supramolecular hydrogel (i.e., D-G in the figure) and 2.8% w/v L-G supramolecular hydrogel (i.e., L-G in the figure) were degraded in mice after being injected subcutaneously in the back of the mice for various times, "control" is the control result.

FIG. 7: 2.8% w/v L-G supramolecular hydrogel 3D cell culture experimental results: (a) LIVE/DEAD cell staining results of 6d injected with 2.8% w/v L-G supramolecular hydrogel, where green fluorescence indicates LIVE cells and red fluorescence indicates DEAD cells; (b) percentage of viable cells was counted, where 0d represents the results for the control group and 6d represents the results for the 6 th d injection of 2.8% w/v L-G supramolecular hydrogel.

Detailed Description

The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.

The pH range of the PBS buffer solution adopted in the following examples and experimental examples is 7.35-7.45.