阅读说明：本技术 一种防止和/或延缓口腔白斑病癌变的核苷水凝胶及其制备方法及用途 (Nucleoside hydrogel for preventing and/or delaying oral leukoplakia canceration and preparation method and application thereof ) 是由 赵行 丁婷婷 但红霞 曾昕 陈谦明 于 2021-10-19 设计创作，主要内容包括：本发明提供了一种防止和/或延缓口腔白斑病癌变的核苷水凝胶及其制备方法及用途,属于医药技术领域。该核苷水凝胶是由原料鸟嘌呤核苷、硼酸盐、碱金属离子和单宁酸在溶剂中交联而成。本发明基于DG和TA,通过硼酸二酯键成功构建了集载体与药效为一体的DG-TA水凝胶。该水凝胶具有良好的氧化特性,能够有效防止和/或延缓口腔白斑病癌变,效果优异。同时,该水凝胶具有优异的体内降解性能、良好的可注射性能,以及优异的皮肤黏附性能；不仅可以注射给药,同时还可以通过涂抹的方式给药,用药更加安全,效果更加优异。此外,该水凝胶制备简单,具有良好的应用前景。(The invention provides a nucleoside hydrogel for preventing and/or delaying oral leukoplakia canceration and a preparation method and application thereof, belonging to the technical field of medicines. The nucleoside hydrogel is formed by cross-linking raw materials of guanosine, borate, alkali metal ions and tannic acid in a solvent. The DG-TA hydrogel integrating the carrier and the drug effect is successfully constructed on the basis of DG and TA through a boric acid diester bond. The hydrogel has good oxidation characteristic, can effectively prevent and/or delay oral leukoplakia canceration, and has excellent effect. Meanwhile, the hydrogel has excellent in-vivo degradation performance, good injectability and excellent skin adhesion performance; not only can inject and administer, but also can administer by smearing mode simultaneously, and the medicine is safer to use, and the effect is more excellent. In addition, the hydrogel is simple to prepare and has a good application prospect.)

1. A nucleoside hydrogel, characterized by: it is prepared by cross-linking raw materials of guanosine, borate, alkali metal ions and tannic acid in a solvent.

2. The nucleoside hydrogel according to claim 1, characterized in that: the mole ratio of the guanosine, the borate, the alkali metal ions and the tannic acid is (5-20): (5-20): (5-20): 1.

3. the nucleoside hydrogel according to claim 2, characterized in that: the mole ratio of the guanosine, the borate, the alkali metal ions and the tannic acid is 10: 10: 10: 1.

4. the nucleoside hydrogel according to claim 1, characterized in that: the concentration of the guanosine is 1-2% w/v.

5. The nucleoside hydrogel according to claim 4, characterized in that: the concentration of guanosine was 1.4% w/v.

6. The nucleoside hydrogel according to any one of claims 1 to 5, characterized in that: the guanosine is D-configuration guanosine, and the structural formula is as follows:

7. the nucleoside hydrogel according to any one of claims 1 to 5, characterized in that: the borate is p-phenylboronic acid or boric acid;

and/or, the alkali metal ion is selected from KOH, LiOH, NaOH, RbOH or CsOH;

and/or the solvent is deionized water.

8. The method for producing a nucleoside hydrogel according to any one of claims 1 to 7, characterized in that: it comprises the following steps:

heating and dissolving guanosine, borate, alkali metal ions and tannic acid in deionized water, and cooling to obtain the product.

9. The method of claim 8, wherein: it comprises the following steps:

(1) preparing a guanosine aqueous solution;

(2) adding borate and alkali metal ions into the guanosine aqueous solution, and heating for dissolving;

(3) adding tannic acid into the solution obtained in the step (2), and heating for dissolving;

(4) and cooling the solution to obtain the hydrogel.

10. Use of the nucleoside hydrogel of any one of claims 1 to 7 in the preparation of a medicament for preventing and/or delaying oral leukoplakia canceration.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a nucleoside hydrogel for preventing and/or delaying oral leukoplakia canceration, a preparation method and application thereof.

Background

Oral Squamous Cell Carcinoma (OSCC) is the most common malignancy of the oral maxillofacial area. Despite the significant advances in the diagnosis and treatment of malignancies in recent years, the prognosis of OSCC patients has not improved significantly. OSCC usually begins with one or more pre-existing diseases, so-called oral potential malignant conditions (OPMDs). Therefore, shifting the focus of OSCC therapy to the OPMD stage should greatly improve the quality of life of the patient. Leukoplakia stomatitis (OLK) is the most common and representative one of OPMDs, and OLK microenvironment is similar to that in tumors, and is a high oxidative stress environment. OLK has a higher risk of canceration.

For OLK, there is currently no standard intervention. Although OLK is located on the oral surface, topical chemical drugs to prevent their canceration are an important treatment option, their administration in clinical studies such as vitamin AD drops, tretinoin, etc. are mostly ointments or pastes, lack of adhesiveness, have a short residence time in the oral cavity, and have limited effectiveness. The research on a novel antioxidant which has good effect, long retention time in the oral cavity and aims at a local high active oxygen microenvironment has great clinical significance for delaying OLK canceration and preventing cancer.

Patent CN111557948A discloses a nucleoside hydrogel for preventing or delaying the canceration of potential malignant diseases of oral mucosa. The hydrogel is formed by dissolving isoguanosine, guanosine and borate in deionized water and then crosslinking, and can effectively inhibit the proliferation of cells of precancerous lesion of human oral mucosa and delay the cancerization of oral leukoderma. However, the nucleoside hydrogel lacks adhesiveness and targeting property, the injection mode may cause secondary damage to tongue, and the gel may obstruct local blood flow before degradation to cause local blood supply insufficiency and influence food intake.

Therefore, the research on the medicine which has good adhesiveness, targeting property, convenient use and small secondary injury and can prevent or delay the canceration of the potential malignant diseases of the oral mucosa has important significance.

Disclosure of Invention

In order to solve the problems, the invention provides a nucleoside hydrogel for preventing and/or delaying oral leukoplakia canceration, a preparation method and application thereof.

The invention provides a nucleoside hydrogel, which is formed by cross-linking raw materials of guanosine, borate, alkali metal ions and tannic acid in a solvent.

Further, the mole ratio of the guanosine, the borate, the alkali metal ions and the tannic acid is (5-20): (5-20): (5-20): 1.

further, the mole ratio of the guanosine, the borate, the alkali metal ion and the tannic acid is 10: 10: 10: 1.

further, the concentration of the guanosine is 1-2% w/v.

Further, the concentration of guanosine was 1.4% w/v.

Further, the guanosine is D-configuration guanosine, and the structural formula is as follows:

further, the borate is p-phenylboronic acid or boric acid;

and/or, the alkali metal ion is selected from KOH, LiOH, NaOH, RbOH or CsOH;

and/or the solvent is deionized water.

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

heating and dissolving guanosine, borate, alkali metal ions and tannic acid in deionized water, and cooling to obtain the product.

Further, the preparation method comprises the following steps:

(1) preparing a guanosine aqueous solution;

(2) adding borate and alkali metal ions into the guanosine aqueous solution, and heating for dissolving;

(3) adding tannic acid into the solution obtained in the step (2), and heating for dissolving;

(4) and cooling the solution to obtain the hydrogel.

The invention also provides application of the nucleoside hydrogel in preparing a medicament for preventing and/or delaying oral leukoplakia canceration.

The meaning of preventing oral leukoplakia canceration is to prevent oral leukoplakia canceration and avoid oral leukoplakia canceration.

Compared with the prior art, the invention has the beneficial effects that:

the DG-TA hydrogel integrating the carrier and the drug effect is successfully constructed on the basis of DG and TA through a boric acid diester bond. The hydrogel has good oxidation characteristic, can effectively prevent and/or delay oral leukoplakia canceration, and has excellent effect. Meanwhile, the hydrogel has excellent in-vivo degradation performance, good injectability and excellent skin adhesion performance; not only can inject and administer, but also can administer by smearing mode simultaneously, and the medicine is safer to use, and the effect is more excellent. In addition, the hydrogel is simple to prepare and has a good application prospect.

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 is a schematic diagram of the preparation process of a nucleoside hydrogel (DG-TA hydrogel).

FIG. 2 shows the rheological properties, macroscopic gelling results and injectable results of the nucleoside hydrogels of the present invention.

FIG. 3 shows the in vitro adhesion and antioxidant properties of DG-TA hydrogel: a is in vitro adhesiveness; b is antioxidant property, and p is less than 0.05.

FIG. 4 is the establishment of model OLK and histopathological analysis after drug administration: a is a model establishing process; b is the macroscopic result of the tongue backs of all groups after the medicine is taken; c is the remission rate results for each group.

Detailed Description

The preparation process of the nucleoside hydrogel (DG-TA hydrogel) of the invention is shown in FIG. 1.

DG nucleoside is guanosine with D configuration, and the structural formula is as follows:

example 1 preparation of nucleoside hydrogel

Firstly, preparing a DG nucleoside aqueous solution with the concentration of 1.4% w/v, then adding para-phenyl diboronic acid (PBA) and a KOH solution with the concentration of 0.5M, wherein the molar equivalent of the DG nucleoside, the para-phenyl diboronic acid and the KOH is 1:1:1, and heating until the solution is fully dissolved to obtain a clear and transparent liquid. Adding 1/10 molar equivalents of Tannic Acid (TA) to the clear and transparent liquid, heating to dissolve completely, and naturally and gradually cooling the solution at room temperature to obtain a transparent yellow DG-TA hydrogel.

Example 2 preparation of nucleoside hydrogel

Firstly, preparing a DG nucleoside aqueous solution with the concentration of 1.4% w/v, then adding para-phenyl diboronic acid (PBA) and a KOH solution with the concentration of 0.5M, wherein the molar equivalent of the DG nucleoside, the para-phenyl diboronic acid and the KOH is 1:1:1, and heating until the solution is fully dissolved to obtain a clear and transparent liquid. Adding 1/5 molar equivalents of Tannic Acid (TA) to the clear and transparent liquid, heating to dissolve completely, and naturally and gradually cooling the solution at room temperature to obtain a transparent yellow DG-TA hydrogel.

Example 3 preparation of nucleoside hydrogel

Firstly, preparing a DG nucleoside aqueous solution with the concentration of 1.4% w/v, then adding para-phenyl diboronic acid (PBA) and a KOH solution with the concentration of 0.5M, wherein the molar equivalent of the DG nucleoside, the para-phenyl diboronic acid and the KOH is 1:1:1, and heating until the solution is fully dissolved to obtain a clear and transparent liquid. Adding 1/15 molar equivalents of Tannic Acid (TA) to the clear and transparent liquid, heating to dissolve completely, and naturally and gradually cooling the solution at room temperature to obtain a transparent yellow DG-TA hydrogel.

Example 4 preparation of nucleoside hydrogel

Firstly, preparing a DG nucleoside aqueous solution with the concentration of 1.4% w/v, then adding para-phenyl diboronic acid (PBA) and a KOH solution with the concentration of 0.5M, wherein the molar equivalent of the DG nucleoside, the para-phenyl diboronic acid and the KOH is 1:1:1, and heating until the solution is fully dissolved to obtain a clear and transparent liquid. Adding 1/20 molar equivalents of Tannic Acid (TA) to the clear and transparent liquid, heating to dissolve completely, and naturally and gradually cooling the solution at room temperature to obtain a transparent yellow DG-TA hydrogel.

Example 5 preparation of nucleoside hydrogel

Firstly, preparing a DG nucleoside aqueous solution with the concentration of 2% w/v, then adding para-phenyl diboronic acid (PBA) and a KOH solution with the concentration of 0.5M, wherein the molar equivalent of the DG nucleoside, the para-phenyl diboronic acid and the KOH is 1:1:1, and heating until the solution is fully dissolved to obtain a clear and transparent liquid. Adding 1/10 molar equivalents of Tannic Acid (TA) to the clear and transparent liquid, heating to dissolve completely, and naturally and gradually cooling the solution at room temperature to obtain a transparent yellow DG-TA hydrogel.

Example 6 preparation of nucleoside hydrogel

Firstly, preparing a DG nucleoside aqueous solution with the concentration of 2% w/v, then adding para-phenyl diboronic acid (PBA) and a KOH solution with the concentration of 0.5M, wherein the molar equivalent of the DG nucleoside, the para-phenyl diboronic acid and the KOH is 1:1:1, and heating until the solution is fully dissolved to obtain a clear and transparent liquid. Adding 1/5 molar equivalents of Tannic Acid (TA) to the clear and transparent liquid, heating to dissolve completely, and naturally and gradually cooling the solution at room temperature to obtain a transparent yellow DG-TA hydrogel.

Example 7 preparation of nucleoside hydrogel

Firstly, preparing a DG nucleoside aqueous solution with the concentration of 2% w/v, then adding para-phenyl diboronic acid (PBA) and a KOH solution with the concentration of 0.5M, wherein the molar equivalent of the DG nucleoside, the para-phenyl diboronic acid and the KOH is 1:1:1, and heating until the solution is fully dissolved to obtain a clear and transparent liquid. Adding 1/15 molar equivalents of Tannic Acid (TA) to the clear and transparent liquid, heating to dissolve completely, and naturally and gradually cooling the solution at room temperature to obtain a transparent yellow DG-TA hydrogel.

Example 8 preparation of nucleoside hydrogel

Firstly, preparing a DG nucleoside aqueous solution with the concentration of 2% w/v, then adding para-phenyl diboronic acid (PBA) and a KOH solution with the concentration of 0.5M, wherein the molar equivalent of the DG nucleoside, the para-phenyl diboronic acid and the KOH is 1:1:1, and heating until the solution is fully dissolved to obtain a clear and transparent liquid. Adding 1/20 molar equivalents of Tannic Acid (TA) to the clear and transparent liquid, heating to dissolve completely, and naturally and gradually cooling the solution at room temperature to obtain a transparent yellow DG-TA hydrogel.

In examples 1 to 8, terephthalic diboronic acid (PBA) was replaced by boric acid; KOH can be replaced by LiOH, NaOH, RbOH or CsOH.

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

Experimental example 1 in vivo application potential study of nucleoside hydrogel

1. Experimental methods

The nucleoside hydrogel prepared in example 1 was subjected to rheological experiments to study the shear thinning characteristics; after gelling, verifying whether the nucleoside hydrogel is formed by using a vial inversion test, namely inverting the vial loaded with the hydrogel at room temperature, and determining the formation of the hydrogel if no flow of the sample is observed; meanwhile, the injectability of the gel is researched through an injection experiment of in vitro visualization.

2. Results of the experiment

As shown in fig. 2: the DG-TA hydrogel is successfully established through the boric acid diester bond, and has the characteristic that the viscosity is reduced along with the increase of the shear rate, namely the shear thinning characteristic. Successful gel formation was confirmed using a vial inversion test. The injection experiment in vitro by visualization proves that the gel can be applied to local parts by injection and can continuously maintain the gel state after injection.

The above results illustrate that: the nucleoside hydrogel prepared by the invention has good injectability and in-vivo application potential.

Experimental example 2 in vitro adhesion and antioxidant Properties of nucleoside hydrogel

1. Experimental methods

Fresh dorsal skin tissue was obtained from male BALB/cJ Gpt mice and the hydrogel prepared in example 1 (DG-TA hydrogel) was injected onto the skin (10 spots, 20. mu.L/spot) with DG hydrogel as a control. The skin was fixed and immersed in phosphate buffer in a polystyrene cassette while shaking at 100 rpm at 37 ℃. The retention of DG-TA hydrogel and DG hydrogel on the skin in vitro was recorded at specific time intervals to characterize the adhesion properties.

Experimental methods for antioxidant Properties: the total antioxidant activity of DG-TA hydrogel is determined by adding a sample into a ferric iron reduction/antioxidant capacity (FRAP) working solution by using an Abcam detection kit (Abcam), standing and reacting for 0.5h in a 96-well plate at normal temperature (22 ℃), and then measuring the absorbance at 593nm in an enzyme-linked immunosorbent assay (ELIASA). The standard curve is FeSO with different concentrations₄Solutions were made and then used to calibrate the antioxidant activity of each sample.

The preparation method of the DG hydrogel is as follows: firstly, preparing a DG nucleoside aqueous solution with the concentration of 1.4% w/v, then adding para-phenyl diboronic acid (PBA) and a KOH solution with the concentration of 0.5M, wherein the molar equivalent of the DG nucleoside, the para-phenyl diboronic acid and the KOH is 1:1:1, heating to fully dissolve to obtain a clear and transparent liquid, and naturally and gradually cooling the solution at room temperature to obtain the DG hydrogel.

2. Results of the experiment

As shown in fig. 3A: the TA is introduced to obviously enhance the adhesiveness of the hydrogel, and the DG-TA hydrogel can keep the adhesiveness on the surface of the skin for more than 12 hours; i.e., DG-TA hydrogel adhered to the skin surface for up to 12 hours, still showed good adhesion. And after the DG hydrogel without TA introduced is adhered to the skin surface for 2.5 hours, the adhesiveness begins to be reduced, part of the gel falls off, and all the gel falls off in the experiment after 7.5 hours.

And (3) determining the antioxidant performance of each component of the hydrogel by using an iron ion reduction/antioxidant capacity method. The results are shown in FIG. 3B: the raw materials DG, PBA and KOH have no oxidation resistance, and the nucleoside hydrogel prepared by using DG also has no oxidation resistance; the raw material TA has obvious oxidation resistance, and the oxidation resistance of the DG-TA hydrogel is derived from the introduction of the TA. And after the DG-TA hydrogel is placed in vitro for 10 days, the oxidation resistance of the DG-TA hydrogel is not obviously lost.

The above experimental results demonstrate that: the DG-TA hydrogel prepared by the method disclosed by the invention has obviously better adhesion performance and oxidation resistance on the skin surface than the DG hydrogel.

Experimental example 3 investigation of nucleoside hydrogel for preventing OLK canceration in vivo

1. Experimental methods

The OLK model was generated by feeding C57 mice with drinking water containing 100. mu.g/mL 4-nitroquinoline-1-oxide (4-NQO). Mice were placed under isoflurane inhalation anesthesia every week to examine the oral mucosa of the mice. At week 12 of feeding, white lesions appeared on the dorsum of the tongue in all mice. One mouse was taken for tongue histopathological analysis, confirming the successful establishment of the OLK model.

Subsequently, mice were randomly grouped (n ═ 7):

control group (Control): model group, no drug treatment.

TA injection group: injecting TA at oral leukoplakia at multiple points once a week, wherein the administration dosage of TA is 8.5 mg/kg;

DG hydrogel injection group: injecting DG hydrogel at oral leukoplakia once a week, wherein the DG administration dose of each DG hydrogel is 14mg/kg (which is consistent with the DG dose in a DG-TA hydrogel administration group);

DG-TA hydrogel injection group: injecting DG-TA hydrogel at the oral leukoplakia at multiple points once a week, wherein the administration dose of TA in the DG-TA hydrogel is 8.5mg/kg each time;

DG-TA hydrogel application group: DG-TA hydrogel is smeared at the oral leukoplakia once every day, and the administration dose of TA in the DG-TA hydrogel is 8.5 mg/kg.

H & E histological examination was performed 6 weeks after administration to determine the progression of tongue lesions, which were classified as free of dysplasia, dysplasia (mild, mild-moderate, moderate-severe, severe) and OSCC (oral squamous cell carcinoma). The canceration process of the oral mucosa is normal epithelium, mild abnormal hyperplasia, moderate abnormal hyperplasia, severe abnormal hyperplasia and oral cancer. Mild abnormal hyperplasia is an early-stage lesion of oral leukoplakia, and the oral leukoplakia gradually enters a late-stage lesion along with the more severe abnormal hyperplasia, so that the oral leukoplakia finally becomes cancerate. Thereby delaying the abnormal proliferation of oral leukoplakia and effectively delaying the canceration of leukoplakia.

HE shows that: in all mice in the experiment, mild and above abnormal hyperplasia appeared after molding, and the OLK ratio of mild abnormal hyperplasia was recorded as remission rate (%). The higher the remission rate, the higher the proportion of early-stage lesion (mild abnormal hyperplasia) mice, and further the better the effect of delaying the progress of white spot cancer.

Remission rate (%). percent (n 1)/total (n). 100% of mildly abnormal hyperplastic mice

2. Results of the experiment

The results are shown in FIG. 4: it can be known that, as with the model group, the remission rate of the DG hydrogel or TA injected alone is 0%, which indicates that the oral leukoplakia of the mice is all abnormally proliferated more than slight degree after the DG hydrogel or TA injected alone, and the DG hydrogel or TA injected alone has no remission effect on the abnormal proliferation of the oral leukoplakia, further indicates that the DG hydrogel or TA injected alone has no delay effect on the canceration of the oral leukoplakia. The DG-TA hydrogel prepared by the invention can effectively relieve abnormal hyperplasia of oral leukoplakia of mice no matter injection administration or smearing administration, which shows that the DG-TA hydrogel prepared by the invention has obvious delay effect on oral leukoplakia canceration. Wherein, the DG-TA hydrogel prepared by the invention has a remission rate of 20 percent when injected and 42.8 percent when smeared, which shows that the smearing has better delay effect on the oral leukoplakia canceration. In addition, it was found that administration of DG-TA hydrogel by injection resulted in 28% of mice dying, probably because the gel injection locally blocked the blood flow of the tongue, affecting the feeding of the mice; while the DG-TA hydrogel-coated mice did not show significant wasting or death. Therefore, the DG-TA hydrogel can effectively delay oral leukoplakia canceration by adopting a smearing mode, and has better safety.

The above results indicate that DG-TA hydrogel prepared based on DG and TA has the potential to prevent OLK malignant change and can be treated by smearing with better safety.

In conclusion, the DG-TA hydrogel integrating the carrier and the drug effect is successfully constructed on the basis of DG and TA through the boric acid diester bond. The hydrogel has good antioxidant property, can effectively prevent and/or delay canceration of oral leukoplakia, and has excellent effect. Meanwhile, the hydrogel has excellent in-vivo degradation performance, good injectability and excellent skin adhesion performance; not only can inject and administer, but also can administer by smearing mode simultaneously, and the medicine is safer to use, and the effect is more excellent. In addition, the hydrogel is simple to prepare and has a good application prospect.