CN113058076B

CN113058076B - Supermolecule nucleoside hydrogel and preparation method and application thereof

Info

Publication number: CN113058076B
Application number: CN202110333731.8A
Authority: CN
Inventors: 刘江; 赵行; 倪广成; 但红霞; 陈谦明
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2021-03-29
Filing date: 2021-03-29
Publication date: 2022-03-11
Anticipated expiration: 2041-03-29
Also published as: CN113058076A

Abstract

The invention provides a supramolecular nucleoside hydrogel and a preparation method and application thereof, belonging to the field of biomedical materials. In a powder X-ray diffraction pattern of the hydrogel, diffraction peaks exist at the diffraction angles of 26.7 degrees, 28.4 degrees and 40.5 degrees according to the 2 theta. The invention successfully constructs the supermolecule hydrogel based on D-configuration alpha-deoxyguanosine, the alpha-dG hydrogel has excellent stability, can not be disintegrated and damaged after being placed for 18 months, and the problem of poor stability of the supermolecule hydrogel formed by D-type guanosine in the prior art is solved. In addition, the alpha-dG hydrogel has good injectability, biocompatibility and drug delivery capability, can be used as a drug carrier, and is expected to be further applied to the field of biomedicine, such as local treatment of oral mucosa diseases.

Description

Supermolecule nucleoside hydrogel and preparation method and application thereof

Technical Field

The invention belongs to the field of biomedical materials, and particularly relates to a supramolecular nucleoside hydrogel and a preparation method and application thereof.

Background

The hydrogel is a high molecular polymer material which takes water as a dispersion medium, is hydrophilic and insoluble in water, and can absorb a large amount of water and has a cross-linked structure. It has good biocompatibility, and can reduce adverse reaction when used as a human body implant, so the material is widely applied as an excellent biomedical material.

Hydrogels are divided into two categories, chemical gels and physical gels. Gel molecules of the chemical gel are connected through covalent bonds without thermal reversibility; gel molecules of the physical gel are connected through intermolecular forces, hydrogen bonds, electrostatic interactions, pi-pi bonds, hydrophobic interactions, coordination bonds and the like, and have thermal reversibility.

The supermolecular hydrogel is a novel hydrogel, and the construction of the novel supermolecular system is based on the multi-level assembly of the synergy of multiple non-covalent interactions. Namely, the small molecule building units are firstly assembled into a supramolecular polymer instead of a non-covalent polymer, and then a three-dimensional cross-linked structure of gel is formed through multilayer assembly.

Supramolecular nucleoside hydrogels are a research hotspot in supramolecular hydrogels. There are 5 kinds of nucleosides mainly in human body, including 2 kinds of purine nucleosides (adenosine and guanosine), and 3 kinds of pyrimidine nucleosides (including cytidine, thymidine, uridine). Wherein, guanosine has 2 hydrogen bond donors and 2 hydrogen bond acceptors, which means that guanosine can not only be combined with other molecules, but also be combined with itself. Therefore, guanosine shows great advantages in the field of supramolecular hydrogels with its unique structure.

However, the research shows that the supermolecular gel formed by D-type guanosine is easy to crystallize in a short time, so that the stability is poor, and the application of the supermolecular gel is greatly limited. Although patent CN109180963A found that supramolecular hydrogel formed by self-assembly of L-type guanosine is more stable than supramolecular hydrogel formed by self-assembly of D-type guanosine. However, the improvement of the stability of D-type guanosine hydrogel is still a problem.

The D-type guanosine hydrogel cannot meet the application as a drug sustained-release preparation and a soft material of tissue engineering due to poor stability. Further improving the stability of the hydrogel is of great significance.

Disclosure of Invention

The invention aims to provide a supramolecular nucleoside hydrogel and a preparation method and application thereof.

The invention provides a supramolecular nucleoside hydrogel, wherein in a powder X-ray diffraction pattern of the hydrogel, diffraction peaks exist at the positions of 26.7 degrees, 28.4 degrees and 40.5 degrees of 2 theta diffraction angles.

Further, in the powder X-ray diffraction pattern of the hydrogel, the relative intensity values of diffraction peaks at 2 theta diffraction angles are as follows:

2 theta, degree	Strength%
		26.7	5.42
28.4	100
		40.5	71.84

。

Further, the powder X-ray diffraction pattern of the hydrogel is shown in FIG. 2B.

Further, the supramolecular nucleoside hydrogel is formed by crosslinking deoxyguanosine in the presence of alkali metal ions.

Further, the molar ratio of the deoxyguanosine to the alkali metal ions is (1-10): (1-10);

preferably, the molar ratio of the deoxyguanosine to the alkali metal ion is 1: 4.

Further, the alkali metal ion is K⁺。

Further, the deoxyguanosine is alpha-deoxyguanosine;

preferably, the alpha-deoxyguanosine is alpha-deoxyguanosine with a D configuration, and the structure of the alpha-deoxyguanosine is shown as a formula I:

the invention also provides a preparation method of the supramolecular nucleoside hydrogel, which comprises the following steps:

(1) dissolving deoxyguanosine in a solution containing alkali metal ions;

(2) and (5) cooling to obtain the product.

Further, in the step (1), the concentration of deoxyguanosine in the solution containing alkali metal ions is 0.5-2.8% w/v; and/or in the step (1), the concentration of alkali metal ions in the solution containing alkali metal ions is 0.05-0.2 mol/L; and/or in the step (1), the dissolving is heating dissolving, and the heating temperature is 80-100 ℃; and/or, in the step (2), the cooling is cooling at room temperature;

preferably, the first and second electrodes are formed of a metal,

in the step (1), the concentration of the deoxyguanosine in the solution containing the alkali metal ions is 1.4% w/v; and/or in the step (1), the concentration of alkali metal ions in the solution containing alkali metal ions is 0.2 mol/L; and/or, in the step (1), the heating temperature is 90 ℃.

The invention also provides application of the supramolecular nucleoside hydrogel in preparation of self-repairing materials, coating materials, drug sustained-release preparations and soft materials for tissue engineering.

The invention successfully constructs the supermolecule hydrogel based on D-configuration alpha-deoxyguanosine, the alpha-dG hydrogel has excellent stability, can not be disintegrated and damaged after being placed for 18 months, and the problem of poor stability of the supermolecule hydrogel formed by D-type guanosine in the prior art is solved. In addition, the alpha-dG hydrogel has good injectability, biocompatibility and drug delivery capability, can be used as a drug carrier, and is expected to be further applied to the field of biomedicine, such as local treatment of oral mucosa diseases.

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 shows the stability results of the constructed α -dG hydrogel of the present invention after 18 months of storage.

FIG. 2 shows the resolution of the molecular configuration of the α -dG hydrogel of the present invention: a is a gel cross-linked network formed by self-assembly of guanosine and derivatives thereof through G-tetramer and pi-pi accumulation in the prior art; b is a PXRD pattern of the alpha-dG hydrogel; c is the VT-NMR result of the alpha-dG hydrogel; d is the NOE pattern of the alpha-dG hydrogel.

FIG. 3 shows the results of the biocompatibility studies of the α -dG hydrogel of the present invention: a is the in vitro cytotoxicity test result of each hydrogel; b is the in vitro cytotoxicity test result of the alpha-dG hydrogel with different concentrations; c is the result of the in vivo acute toxicity test of each hydrogel.

FIG. 4 shows the results of the study on the drug delivery capacity of the α -dG hydrogel of the present invention: a is a schematic diagram of an in vitro drug delivery device; b is a dexamethasone sodium phosphate standard curve; c is the in vitro slow release result of dexamethasone sodium phosphate under different delivery systems.

Detailed Description

The raw materials and equipment used in the embodiment of the present invention are known products and obtained by purchasing commercially available products.

The alpha-deoxyguanosine used in the application is alpha-deoxyguanosine (alpha-dG) with a D configuration, and the structural formula of the alpha-deoxyguanosine is as follows:

the beta-deoxyguanosine used in the application is beta-deoxyguanosine (beta-dG) with a D-configuration, and the structural formula of the beta-deoxyguanosine is as follows:

example 1 preparation of supramolecular nucleoside hydrogels of the invention

Alpha-deoxyguanosine (alpha-dG) is dissolved in a KCl water solution with the concentration of 0.2mol/L, the solution is heated (80-100 ℃) until the alpha-dG is fully dissolved, and the concentration of the alpha-dG in the obtained solution is 1.4% w/v (namely 1.4mg/100 mu L). And cooling the solution at room temperature (25 ℃) for 5-10 minutes to obtain semitransparent alpha-dG hydrogel (the supermolecular nucleoside hydrogel provided by the invention).

Gel formation was confirmed using a vial inversion test at room temperature, the hydrogel formation was confirmed by inverting the hydrogel-loaded vial and observing hydrogel stability if no flow of sample was observed. The stability of the hydrogel prepared in example 1 is shown in FIG. 1. As can be seen from fig. 1: the alpha-dG hydrogel shows good stability, and the hydrogel does not have obvious disintegration damage after being kept stand for more than 18 months at room temperature.

Comparative example 1 preparation of beta-deoxyguanosine hydrogel

Dissolving beta-deoxyguanosine (beta-dG) in a KCl aqueous solution with the concentration of 0.2mol/L, heating the solution (80-100 ℃) until the beta-dG is fully dissolved, and obtaining the solution with the concentration of the beta-dG of 1.4% w/v (namely 1.4mg/100 mu L). And cooling the solution at room temperature (25 ℃) for 5-10 minutes to obtain the beta-dG hydrogel.

The advantageous effects of the present invention are demonstrated by specific test examples below.

Test example 1 stability test of supramolecular nucleoside hydrogel according to the present invention

1. Test method

After the formation of gel was confirmed by subjecting the α -dG hydrogel prepared in example 1 and the β -dG hydrogel prepared in comparative example 1 to a vial inversion test at room temperature, respectively, the two hydrogels were left at room temperature (25 ℃), and the states of the two hydrogels, including the presence or absence of crystal precipitation, liquid exudation, and collapse of the gel structure, were periodically observed and recorded. The lifetime stability of both gels was evaluated as the length of time from gel formation to gel disintegration.

2. Test results

The research finds that: the life of the beta-dG hydrogel which is kept standing at room temperature can only be maintained for 1-2 hours, and then the hydrogel is disintegrated and destroyed. In contrast, the alpha-dG hydrogel is observed to have no obvious crystal precipitation or gel disintegration phenomenon for more than 18 months under the same environment. This indicates that the α -dG hydrogels have excellent lifetime stability far exceeding that of the β -dG hydrogels.

Test example 2 verification of molecular configuration of supramolecular nucleoside hydrogel according to the present invention

In the prior art, guanosine and derivatives thereof generally form a gel cross-linked network by means of G-tetramer (G-quartz) and pi-pi stacking (as shown in FIG. 2A). To verify the formation method and structure of the supramolecular nucleoside hydrogel of the present invention, the following experiment was performed:

the molecular configuration of the α -dG hydrogel prepared in example 1 was explored and subjected to powder X-ray diffraction (PXRD) analysis, which showed a powder X-ray diffraction pattern as shown in FIG. 2B: the 2 θ diffraction angles had diffraction peaks at 26.7 °, 28.4 ° and 40.5 °, and the relative intensity values of the diffraction peaks at the 2 θ diffraction angles are shown in table 1. The alpha-dG hydrogel sample can see a characteristic peak of pi-pi stacking at 2 theta ≈ 26.7 degrees, and the pi-pi stacking phenomenon in the alpha-dG hydrogel is confirmed.

TABLE 1 relative intensity values of characteristic peaks at 2 theta diffraction angles of powder X-ray diffraction patterns

2 theta, degree	Strength%
		26.7	5.42
28.4	100
		40.5	71.84

To verify the presence of G-tetramer in the hydrogel system, samples of α -dG hydrogel prepared in example 1 were subjected to temperature-variable nuclear magnetic resonance (VT-NMR) and Nuclear Overhauser Effect (NOE) signal detection, and the results are shown in FIGS. 2C and 2D. As can be seen from fig. 2C and 2D: N1H (delta 10.78 → delta 10.67ppm), C8H (delta 7.98 → delta 7.94ppm), and 2-NH in the alpha-dG hydrogel as the temperature is increased from 25 ℃ to 65 ℃₂(delta 6.62 → delta 6.47ppm) all show chemical shift changes, indicating that during self-assembly, all of these hydrogen atoms participate in the formation of intermolecular hydrogen bonds, whereas in the G-tetramer, only N1H and 2-NH are usually present₂Participate in the formation of intermolecular hydrogen bonds. Further, for the G-tetramer, due to the close spatial proximity, C8H is present with 2-NH₂N1H gave a corresponding signal in the NOE pattern, but in none of the α -dG hydrogel samples, this indicates that in the α -dG hydrogel system, the conventional G-tetramer may not be in its configuration.

Test example 3 biocompatibility test of supramolecular nucleoside hydrogel of the present invention

The biocompatibility of the α -dG hydrogel prepared in example 1 of the present application was investigated in comparison with a hydrogel constructed from β -dG endogenous to the organism (i.e., β -deoxyguanosine in the D configuration).

The preparation method of the hydrogel constructed by the beta-dG is the same as that of the hydrogel constructed by the comparative example 1: adding the beta-dG into a KCl aqueous solution with the concentration of 0.2mol/L, and heating the solution (80-100 ℃) until the beta-dG is fully dissolved to obtain a solution with the concentration of the beta-dG of 1.4% w/v (namely 1.4mg/100 mu L). And cooling the solution at room temperature (25 ℃) for 5-10 minutes to obtain the beta-dG hydrogel.

In vitro toxicity test of the supramolecular nucleoside hydrogel

The in vitro toxicity of the α -dG hydrogels of the present application was first explored and the test methods were as follows:

preparing Normal Oral Keratinocyte (NOK) with good growth state into cell suspension, and adjusting the density to about 2-4 × 10⁴After/ml, cells were seeded in 96-well plates with 90. mu. L K-SFM medium and 10. mu.l α -dG hydrogel or β -dG hydrogel (hydrogel injectable) or KCl aqueous solution (0.2mol/L) in a 9:1 ratio for a total of 100. mu.l per well. The control group was added with a mixture of equal amounts of medium and Phosphate Buffered Saline (PBS) (90. mu.l medium and 10. mu.l PBS) at the same ratio. 5% CO at 37 deg.C₂After 24h of incubator culture, adding 10% CCK8 for 1h of incubation, and after the color change in the pore plate is obvious, detecting the absorbance value (OD) of each pore at the wavelength of 450nm by using a multifunctional microplate reader. In addition, the effect of different concentrations of α -dG hydrogel on NOK cell activity after incubation for different periods of time was also explored. Percent (%) cell activity (experimental OD-blank OD)/(control OD-blank OD) × 100%.

The results are shown in FIG. 3A: the KCl-treated group showed almost no significant (> 99%) decrease in cellular activity compared to the PBS control group; after 24 hours of treatment by the alpha-dG hydrogel, the percentage of NOK cell activity is about 81 percent; in contrast, the percent cell viability after β -dG hydrogel treatment was only about 42%, and the data were statistically different. It is shown that the α -dG hydrogels of the present invention exhibit better biocompatibility than the β -dG hydrogels in vitro cytotoxicity studies.

Furthermore, after the alpha-dG hydrogel with different concentrations is treated for 24 hours or 48 hours, the NOK cell activity percentage is maintained to be more than 70% (as shown in figure 3B), and the good biocompatibility of the alpha-dG hydrogel in an in vitro environment is further proved.

Second, the in vivo acute toxicity test of the supramolecular nucleoside hydrogel

The in vivo acute toxicity of the α -dG hydrogels of the present application was then explored and the test methods were as follows:

1.4% w/v of α -dG and β -dG hydrogels were prepared according to the method described in example 1 and test example 1, and after cooling both hydrogels in a 1ml syringe for 10min, the backs of 7-week-old female BALB/c mice were subcutaneously injected with 200 μ L of hydrogel or an equivalent amount of 0.2mol/L KCl aqueous solution, respectively, and were bred. After each hydrogel was completely degraded by visual observation, the mice were sacrificed (approximately 1 day of feeding), and important organs (heart, liver, spleen, lung, and kidney) were removed and stained with Hematoxylin-Eosin (H & E), and the state of each tissue was observed under a microscope.

The results are shown in FIG. 3C: after KCl and the two hydrogels are injected to the back of a mouse subcutaneously, the tissue structure and the cell morphology of each important organ of the mouse are normal, abnormal mitotic picture, cell necrosis or inflammatory infiltration are not found, the two hydrogels do not show obvious in-vivo acute toxicity, and the alpha-dG hydrogel has good biocompatibility in vivo.

Test example 4 test of drug delivery ability of supramolecular nucleoside hydrogel according to the present invention

1. Test method

After successfully formulating the α -dG hydrogel as described in example 1, the temperature was maintained at about 60 ℃ to ensure that the hydrogel was in a viscoelastic liquid state, dexamethasone sodium phosphate (0.5% w/v) was added to the hydrogel and the two were mixed thoroughly with shaking for 5 min. As shown in fig. 4A, the drug delivery device was set up with the drug-loaded hydrogel placed in the donor chamber, the receptor chamber containing an equal volume of PBS solution, and the donor chamber and receptor chamber separated by a semi-permeable membrane. Control group an equivalent amount of KCl solution containing 0.5% w/v dexamethasone sodium phosphate was added to the donor chamber. The device was placed in a 37 ℃ incubator for incubation, and 20. mu.l of the solution was taken from the receptor chamber at 0h, 1h, 2h, 4h, 6h, 9h and 12h, respectively, diluted 50-fold while the receptor chamber was supplemented with 20. mu.l of PBS solution. The method comprises the following steps of mixing methanol: 0.05mol/L KH₂PO₄The content of the drug in the solution taken out was quantitatively analyzed by drawing a standard curve of dexamethasone sodium phosphate (as shown in fig. 4B) by using a High Performance Liquid Chromatography (HPLC) at a column temperature of 30 ℃ and a wavelength of 240nm, with 70:30(v: v) as a mobile phase.

2. Test results

The results are shown in FIG. 4C: within 1h from the beginning of the experiment, dexamethasone sodium phosphate with the drug loading of over 80 percent is released into a receptor chamber in a solution system (a control group); at the same time point, only about 40% of dexamethasone sodium phosphate is released from the alpha-dG hydrogel system. After that, the drug release in the hydrogel system of the invention is gradually slowed down, and the release reaches about 90 percent of the total drug loading amount in about 12 hours, which is similar to the total drug release amount in the solution system at the moment, and the alpha-dG hydrogel of the invention shows good drug delivery effect.

In conclusion, the invention successfully constructs the supermolecule hydrogel based on D-configuration alpha-deoxyguanosine, the alpha-dG hydrogel has excellent stability, can not be disintegrated and damaged after being placed for 18 months, and the problem of poor stability of the supermolecule hydrogel formed by D-type guanosine in the prior art is solved. In addition, the alpha-dG hydrogel has good injectability, biocompatibility and drug delivery capability, can be used as a drug carrier, and is expected to be further applied to the field of biomedicine, such as local treatment of oral mucosa diseases.

Claims

1. A supramolecular nucleoside hydrogel, comprising: it is formed by the cross-linking of deoxyguanosine in the presence of alkali metal ions;

the deoxyguanosine is alpha-deoxyguanosine

The alpha-deoxyguanosine is D-configuration alpha-deoxyguanosine, and the structure of the alpha-deoxyguanosine is shown as a formula I:

2. the supramolecular nucleoside hydrogel of claim 1, wherein: the mole ratio of the deoxyguanosine to the alkali metal ions is (1-10): (1-10).

3. The supramolecular nucleoside hydrogel of claim 2, wherein: the molar ratio of the deoxyguanosine to the alkali metal ions is 1: 4.

4. The supramolecular nucleoside hydrogel of claim 1, wherein: the alkali metal ion is K⁺。

5. The method for preparing the supramolecular nucleoside hydrogel according to any one of claims 1 to 4, wherein: it comprises the following steps:

(1) dissolving deoxyguanosine in a solution containing alkali metal ions;

(2) and (5) cooling to obtain the product.

6. The method of claim 5, wherein: in the step (1), the concentration of deoxyguanosine in the solution containing the alkali metal ions is 0.5-2.8% w/v; and/or in the step (1), the concentration of alkali metal ions in the solution containing alkali metal ions is 0.05-0.2 mol/L; and/or in the step (1), the dissolving is heating dissolving, and the heating temperature is 80-100 ℃; and/or, in the step (2), the cooling is cooling at room temperature.

7. The method of claim 6, wherein: in the step (1), the concentration of the deoxyguanosine in the solution containing the alkali metal ions is 1.4% w/v; and/or in the step (1), the concentration of alkali metal ions in the solution containing alkali metal ions is 0.2 mol/L; and/or, in the step (1), the heating temperature is 90 ℃.

8. Use of the supramolecular nucleoside hydrogel as claimed in any one of claims 1 to 4 in preparation of self-repairing materials, coating materials, drug sustained release preparations, soft materials for tissue engineering.