CN111533926B - Chiral supramolecular nucleoside hydrogel based on boron ester bond and preparation method and application thereof - Google Patents

Abstract

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.

Description

Chiral supramolecular nucleoside hydrogel based on boron ester bond and preparation method and application thereof

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 the characteristics of good biodegradability, capability of promoting cell proliferation and differentiation and generating extracellular matrix, exchange channels of nutrient substances and metabolites, capability of adhering to and integrating with surrounding tissues and the like. The tissue scaffold can be divided into two types, namely a prefabricated porous scaffold and hydrogel according to the requirement of preparing the tissue scaffold 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 bonding and are mostly high molecular polymer hydrogels, which can be classified into synthetic high molecular polymers (e.g., polyvinyl alcohol, polyacrylate, polyamide, polyethylene, etc.) and natural high molecular polymers (e.g., 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 supramolecular hydrogel has unique properties in the aspects of stability, mechanical property, in-vivo degradation, metabolism and the like, the structure of the supramolecular hydrogel can generate important influence on the growth, proliferation, differentiation and the like of seed cells, and the supramolecular hydrogel 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, dynamic covalent bonds dominated by boron ester bonds (B-O), acylhydrazone bonds (-HC = N-NH-CO-) and reversible imine bonds (-C = N-) are one of the main means for synthesizing functional hydrogels.

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 a 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 guanosine in the solvent is 0.05 to 0.2M, 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 Na ₂ B ₄ O ₇ ·10H ₂ O。

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 samples ¹¹ B NMR (a) and infrared (B) characterization spectrograms; in the a graph, the test samples of 3 curves from top to bottom were 2.8% w/v L-G supramolecular hydrogel obtained in example 5, 1.4% w/v L-G supramolecular hydrogel obtained in example 4, sodium borate in order ¹¹ B-disester represents the chemical shift of the boric acid Diester bond, ¹¹ B-Monoester represents the chemical shift of the boric acid Monoester bond, ¹¹ b, chemical shift of free boric acid bond; in panel B, the B-O (-) curve represents raw material L-guanosine, and the B-O (+) curve represents 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. 5:2.8% 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) after injection into the subcutaneous tissue of the back of mice for different periods of time, and the "control group" was 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 injection subcutaneously in the back of mice for different times, "control" is the control result.

FIG. 7:2.8% w/v L-G supramolecular hydrogel 3D cell culture assay results: (a) Injecting LIVE/DEAD cell staining results of w/v L-G supramolecular hydrogel No. 6d at 2.8%, wherein green fluorescence indicates LIVE cells and red fluorescence indicates DEAD cells; (b) Percentage viable cells were counted, where 0d represents the results of the control group and 6d represents the results of the injection of 2.8% w/v L-G supramolecular hydrogel at 6 d.

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 used in the following examples and experimental examples was 7.35 to 7.45.

Example 1, 1.4% of the invention preparation of w/v D-G supramolecular hydrogels

mu.L of PBS buffer, 40. Mu.L of 0.25M sodium borate aqueous solution and D-guanosine (2.8 mg) were added to a glass vial, mixed, heated (90 ℃) until the solid powder was completely dissolved, and cooled at room temperature for 5 minutes to obtain 1.4% w/v D-G of the present invention.

Example 2, invention 2.8% w/v D-G preparation of supramolecular hydrogels

The mass of D-guanosine in example 1 was modified to 5.6mg, and 2.8% w/v D-G supramolecular hydrogel was prepared.

Example 3, preparation of 5.6% w/v D-G supramolecular hydrogel

The mass of D-guanosine in example 1 was modified to 11.2mg, and 5.6% w/v D-G supramolecular hydrogel was prepared.

Example 4, 1.4% of the invention preparation of w/v L-G supramolecular hydrogels

mu.L of PBS buffer, 40. Mu.L of 0.25M sodium borate aqueous solution and L-guanosine (2.8 mg) were added to a glass vial, mixed, heated (90 ℃) until the solid powder was completely dissolved, and cooled at room temperature for 5 minutes to obtain 1.4% w/v L-G of the present invention.

Example 5, 2.8% of the invention preparation of w/v L-G supramolecular hydrogels

The mass of L-guanosine was modified to 5.6mg in example 4 to obtain 2.8% w/v L-G supramolecular hydrogel.

Example 6, invention 5.6% w/v L-G preparation of supramolecular hydrogels

The mass of L-guanosine in example 4 was modified to 11.2mg, and 5.6% w/v L-G supramolecular hydrogel was prepared.

Table 1 raw materials and nomenclature of supramolecular hydrogels of examples 1 to 6

The following is a preparation method of a control hydrogel without introducing a boroester bond:

comparative example 1 preparation of comparative hydrogel 1

Control hydrogel 1 was prepared by replacing 160. Mu.L of PBS buffer and 40. Mu.L of 0.25M aqueous sodium borate solution from example 1 with 200. Mu.L of PBS buffer.

Comparative example 2 preparation of comparative hydrogel 2

Control hydrogel 2 was prepared by replacing 160. Mu.L of PBS buffer and 40. Mu.L of 0.25M aqueous sodium borate solution from example 2 with 200. Mu.L of PBS buffer.

Comparative example 3 preparation of comparative hydrogel 3

Control hydrogel 3 was prepared by replacing 160. Mu.L of PBS buffer and 40. Mu.L of 0.25M aqueous sodium borate solution from example 3 with 200. Mu.L of PBS buffer.

Comparative example 4 preparation of comparative hydrogel 4

Control hydrogel 4 was prepared by replacing 160. Mu.L of PBS buffer and 40. Mu.L of 0.25M aqueous sodium borate solution from example 4 with 200. Mu.L of PBS buffer.

Comparative example 5 preparation of comparative hydrogel 5

Control hydrogel 5 was prepared by replacing 160. Mu.L of PBS buffer and 40. Mu.L of 0.25M aqueous sodium borate solution from example 5 with 200. Mu.L of PBS buffer.

Comparative example 6 preparation of comparative hydrogel 6

Control hydrogel 6 was prepared by replacing 160. Mu.L of PBS buffer and 40. Mu.L of 0.25M aqueous sodium borate solution from example 6 with 200. Mu.L of PBS buffer.

TABLE 1 COMPARATIVE EXAMPLES 1-6 materials and nomenclature of each control hydrogel

Comparative example	Sodium borate	Guanosine concentration	Guanosine	Control hydrogel nomenclature
					Comparative example 1	Is free of	0.05M	D-guanosine	Control hydrogel	1
Comparative example 2	Is free of	0.1M	D-guanosine	Control hydrogel							2
					Comparative example 3	Is free of	0.2M	D-guanosine	Control hydrogel	3
Comparative example 4	Is composed of	0.05M	L-guanosine	Control hydrogel							4
					Comparative example 5	Is free of	0.1M	L-guanosine	Control hydrogel	5
Comparative example 6	Is free of	0.2M	L-guanosine	Control hydrogel							6

The beneficial effects of the present invention are demonstrated by the following experimental examples.

Experimental example 1 structural characterization of supramolecular hydrogel

1. Experimental methods

Taking the supramolecular hydrogel obtained in examples 4 and 5, and performing nuclear magnetism ( ¹¹ B NMR) characterization, and taking the raw material sodium borate as a reference;

the supramolecular hydrogel prepared in example 5 was taken for infrared characterization, and raw material L-guanosine was used as a control.

2. Results of the experiment

As shown in fig. 1a, it can be seen that the supramolecular hydrogels prepared in example 4 and example 5 of the present invention both successfully incorporate boroester bonds. Further, as shown in FIG. 1b, the infrared spectrum analysis revealed that the peak signal of the nucleotide hydroxyl group in the raw material guanosine (v (-OH, 3300 cm) ^-1 ) The boron ester bond is obviously weakened after the introduction, which indicates that nucleoside glycosyl 2'-OH and nucleoside glycosyl 3' -OH participate in the formation of the boron ester bond.

Experimental example 2 evaluation of stability of supramolecular hydrogel

1. Experimental methods

(1) Evaluation object

The supramolecular hydrogels obtained in examples 1 to 6 and the control hydrogels obtained in comparative examples 1 to 6.

(2) Stability evaluation method

And (3) after the glass bottle for preparing the supermolecule hydrogel is heated, the glass bottle is cooled for 5 minutes at room temperature, and then the glass bottle is inverted. The glass bottle was kept in an inverted state for 5 minutes (5 min), 24 hours (24 h) and 8 days (8 d), and then the gel state was observed.

2. Results of the experiment

The results are shown in FIG. 2. It can be seen that, at each guanosine concentration, the control hydrogel formed from guanosine without sodium borate formed a large amount of crystals within 24 hours, and had poor stability; after the sodium borate is added, no obvious crystallization is found in the supramolecular hydrogel formed by the action of the guanosine and the sodium borate within 24 hours, and particularly, the supramolecular hydrogels shown as d1 (prepared by example 2) and d2 (prepared by example 5) in figure 2 can be gelled in a short time and still have no obvious crystallization on the 8 th day of inversion.

Therefore, the supramolecular hydrogel prepared by adding the sodium borate aqueous solution is better in stability than the hydrogel prepared without adding the sodium borate aqueous solution by taking the D-guanosine or the L-guanosine as a raw material; the stability of the obtained guanosine supramolecular hydrogel is improved after the introduction of the boron ester bond.

In addition, in the supramolecular hydrogel prepared by adding the sodium borate aqueous solution, when the ratio of borate ions in the raw material sodium borate to the amount of the substance of the raw material guanosine is 1:2, the stability of the obtained supramolecular hydrogel is optimal.

Experimental example 3 rheological test of supramolecular hydrogel

1. Experimental methods

(1) Characterizing an object

The supramolecular hydrogels prepared in examples 2 and 5;

control hydrogel 2' was prepared by replacing 160. Mu.L of PBS buffer and 40. Mu.L of 0.25M aqueous sodium borate solution from example 2 with 200. Mu.L of 0.2M aqueous KCl solution;

a control hydrogel 5' was prepared by replacing 160. Mu.L of PBS buffer and 40. Mu.L of 0.25M aqueous sodium borate in example 5 with 200. Mu.L of 0.2M aqueous KCl.

(2) Test method

(2.1) rheological measurements at different shear frequencies: heating each hydrogel until the hydrogel is completely dissolved to form a solution, sucking 1.2mL of the solution on a parallel plate of a rheometer preheated to 80 ℃, descending a conical plate PC50 until the gap distance is 0.5mm, scraping redundant samples, dripping a proper amount of standard oil along the periphery of the parallel plate to prevent the samples from volatilizing, and cooling to 20 ℃; the temperature is reduced, the test is started after 5min or 10min, the shearing strain is set to be 1 percent, and the shearing frequency is reduced from 100rad/s to 0.1rad/s.

(2.2) rheology test at different shear strains: heating each hydrogel until the hydrogel is completely dissolved to form a solution, sucking 1.2mL of the solution on a parallel plate of a rheometer preheated to 80 ℃, descending a conical plate PC50 until the gap distance is 0.5mm, scraping redundant samples, dripping a proper amount of standard oil along the periphery of the parallel plate to prevent the samples from volatilizing, and cooling to 20 ℃; calculating from the beginning of cooling, starting the test after 5min or 10min, and setting the strain at low shear to be 1 percent and the frequency to be 1rad/s; the strain at high shear is 300%, the frequency 1rad/s.

2. Results of the experiment

2.1 Rheological measurements at different shear frequencies

The results are shown in FIG. 3. The test results show that the shear frequency is gradually reduced from 100rad/s to 0.1rad/s, the elastic modulus G' of the supramolecular hydrogel prepared in the embodiment of the invention is always larger than the loss modulus G "(fig. 3 c-d), and the supramolecular hydrogel prepared in the embodiment of the invention is always kept in a gel state. Moreover, the supramolecular hydrogel G' produced by the example of the invention was consistently larger than the control hydrogel without incorporation of boroester bonds in the measured frequency range (fig. 3).

Furthermore, the crosslink density of different samples can be compared based on the G 'value at low shear frequency, the higher the G' value, the harder the material. As seen from FIG. 3, the supramolecular hydrogel produced in example of the present invention increased in degree of crosslinking and increased in hardness after introduction of boroester bonds, and 2.8% w/v D-G supramolecular hydrogel produced in example 2 was more in degree of crosslinking and hardness than 2.8% w/v L-G supramolecular hydrogel produced in example 5.

2.2 Rheology test at different shear strains

The results are shown in FIG. 4. The test results show that the supramolecular hydrogel prepared in the example of the invention exhibits gel properties at 1% shear strain G' > G "(fig. 4 c-d). After a high shear strain of 300%, G 'and G "drop rapidly, and G' < G", indicating that the gel is broken and turned into a liquid; when the shear strain is restored to 1%, G 'and G "rise rapidly, and G" < G', indicating that the liquid is again transformed into a gel. After the three high shear strains are finished, the G 'value of the hydrogel is equivalent to the initial value and is higher than the G' value.

Therefore, the supramolecular hydrogel prepared by the embodiment of the invention can be converted into liquid from gel after being subjected to high shear stress, and the supramolecular hydrogel has injectability; when the shear stress is relieved, the supramolecular hydrogel is restored to a gel state from liquid, and the supramolecular hydrogel is proved to have self-repairing property.

Experimental example 4 biocompatibility characterization of supramolecular hydrogel

(1) Experimental methods

The supramolecular hydrogel was prepared according to the method of example 2 or 5, cooled for 10min, and then 1mL was injected subcutaneously into the back of 6-8 week-old Babl/c female mice, and then the heart, liver, spleen, lung, and kidney were removed at 0h, 3h, 6h, 12h, and 24h for HE staining, and the tissue change was observed under a microscope.

NaBO injected in the same volume ₃ A control group was prepared by mixing an aqueous solution (0.25M) with PBS buffer, in which NaBO was contained ₃ Volume ratio of aqueous solution to PBS buffer v: v =2:8.

(2) Results of the experiment

The results are shown in FIG. 5. The results showed that 2.8% w/v D-G and 2.8% w/v L-G supramolecular hydrogel group did not show significant tissue edema, inflammation, and defect, clear tissue structure and cell contour, alignment, significant cell atrophy or necrosis, and significant inflammatory exudation and hemorrhage between cells, compared to the control group. Therefore, the supramolecular hydrogel prepared by the embodiment of the invention does not show obvious acute toxicity in animals.

Experimental example 5 characterization of in vivo degradation Rate of supramolecular hydrogels

(1) Experimental methods

Supramolecular hydrogel was prepared according to the method of example 2 or 5, cooled for 10min, 1mL was injected subcutaneously into the back of 6-8 week old Babl/c female mice, and the subcutaneous hydrogel residual amount was observed at 0h, 3h, 6h, 12h, 24 h.

NaBO injected in the same volume ₃ A control group was prepared by mixing an aqueous solution (0.25M) with PBS buffer, in which NaBO was present ₃ Volume ratio of aqueous solution to PBS buffer v: v =2:8.

(2) Results of the experiment

The results are shown in FIG. 6. The results indicated that 2.8% w/v D-G supramolecular hydrogel was completely degraded at 24 h; 2.8% w/v L-G supramolecular hydrogel still had a small residue at 24 h. Thus, the supramolecular hydrogels produced by the examples of the invention were all degradable in vivo, with 2.8% w/v L-G supramolecular hydrogel being more slowly degraded in vivo than 2.8% w/v D-G supramolecular hydrogel.

Experimental example 6 application of supramolecular hydrogel as scaffold material

(1) Experimental methods

The effect of the 2.8% w/v L-G supramolecular hydrogel of the invention as extracellular matrix for 3D culture of cells was verified by LIVE/DEAD cell viatility experiments.

2.8% w/v L-G supramolecular hydrogel was prepared according to the method of example 5, heated until the gel was completely dissolved; simultaneously collecting well-grown NOK cells, preparing a cell suspension, and adjusting the cell density to 1-2 x 10 ⁵ one/mL. Mixing 50 μ L of cell suspension and 100 μ L of preheated gel liquid, cooling at room temperature for 10min, dripping 50 μ L of culture medium along the periphery of hydrogel, changing the solution 1 time every day, staining LIVE/DEAD cells at 6d, observing and photographing under a fluorescence microscope, and counting by using Bitplane Imaris 7.4.2 software.

50 μ L of cell suspension and 100 μ L of pre-heated gel liquid were mixed, cooled at room temperature for 10min, and then LIVE/DEAD cell staining was immediately performed as a control.

(2) Results of the experiment

The results are shown in FIG. 7. The results showed that the cells grew scattered or aggregated in the gel after 6D of culture, and no significant dead cells were observed (FIG. 7 a), and no significant difference in the percentage of viable cells counted compared to the control group (FIG. 7 b), indicating that the 2.8% w/v L-G supramolecular hydrogel produced by the present invention can be used as extracellular matrix for 3D culture of cells.

The experiments show that the 2.8% w/v L-G supramolecular hydrogel prepared by the invention has good biocompatibility, does not show obvious acute toxicity, and can be used for 3D culture of cells as an extracellular matrix.

In conclusion, the invention provides the chiral supramolecular hydrogel based on the boron ester bond, the supramolecular hydrogel 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.

Claims (10)

1. Use of a supramolecular hydrogel in the preparation of an extracellular matrix or a scaffold material, characterized in that: the supermolecule hydrogel is prepared by mixing L-guanosine and borate serving as raw materials in a solvent; the ratio of borate ions in the borate to the amount of L-guanosine in the borate is 1, the concentration of L-guanosine in a solvent is 0.1M, and the structure of L-guanosine is shown as a formula (II):

。

2. use according to claim 1, characterized in that: the borate is sodium borate, potassium borate, lithium borate, magnesium borate or calcium borate.

3. Use according to claim 2, characterized in that: the borate is sodium borate.

4. Use according to claim 1, characterized in that: the solvent is water or an aqueous solution.

5. Use according to claim 4, characterized in that: the aqueous solution was PBS buffer.

6. Use according to claim 5, characterized in that: the pH range of the PBS buffer solution is 7.35 to 7.45.

7. Use according to claim 1, characterized in that: the supramolecular hydrogel comprises a boron ester bond.

8. The use of any one of claims 1~7, wherein: the preparation method of the supramolecular hydrogel comprises the steps of uniformly mixing L-guanosine, borate and a solvent, and heating to obtain the supramolecular hydrogel.

9. Use according to claim 8, characterized in that: the heating temperature is 80 to 100 ℃;

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

10. Use according to claim 9, characterized in that: the heating temperature was 90 ℃.

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