WO2020215446A1 - 一种双功能核苷水凝胶及其制备方法和用途 - Google Patents

一种双功能核苷水凝胶及其制备方法和用途 Download PDF

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WO2020215446A1
WO2020215446A1 PCT/CN2019/089636 CN2019089636W WO2020215446A1 WO 2020215446 A1 WO2020215446 A1 WO 2020215446A1 CN 2019089636 W CN2019089636 W CN 2019089636W WO 2020215446 A1 WO2020215446 A1 WO 2020215446A1
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hydrogel
isoguanosine
borate
guanosine
nucleoside
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PCT/CN2019/089636
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French (fr)
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赵行
曾昕
吉宁
冯慧
刘江
陈谦明
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四川大学
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Priority to US17/594,657 priority Critical patent/US20220218605A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A61K31/708—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid having oxo groups directly attached to the purine ring system, e.g. guanosine, guanylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H23/00—Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12

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  • the invention belongs to the field of biomedical materials, and specifically relates to a bifunctional nucleoside hydrogel and a preparation method and application thereof.
  • the present invention provides a bifunctional nucleoside hydrogel and its preparation method and application.
  • the present invention provides a bifunctional nucleoside hydrogel, which is formed by cross-linking isoguanosine, guanosine and borate dissolved in water or aqueous solution.
  • the molar ratio of the isoguanosine, guanosine and borate is 1:1 to 5:1 to 5; preferably, the isoguanosine, guanosine and boron
  • the molar ratio of the acid salt is 1:1:1.
  • the concentration of isoguanosine is 0.005 to 5.6% by weight; preferably, isoguanosine
  • concentration of purine nucleosides is 0.3-5.6 wt%; more preferably, the concentration of isoguanosine nucleosides is 1.4 wt%.
  • the borate is selected from LiB(OH) 4 , NaB(OH) 4 , KB(OH) 4 , RbB(OH) 4 or CsB(OH) 4 ; preferably, the borate is NaB (OH) 4 .
  • the water or aqueous solution is ultrapure water or phosphate buffer.
  • isoguanosine, guanosine and borate are dissolved in water or aqueous solution and then cross-linked into isoguanosine, guanosine and borate after being dissolved in water or aqueous solution by heating, Cool to crosslink.
  • isoguanosine, guanosine and borate are dissolved in water or an aqueous solution and then cross-linked by adding isoguanosine and borate to the water or aqueous solution, heating to dissolve, and then adding guanosine Purine nucleosides are dissolved by heating and then gradually cooled to crosslink at room temperature.
  • the present invention also provides a method for preparing the aforementioned bifunctional nucleoside hydrogel, which includes the following steps:
  • the isoguanosine, purine nucleoside and borate are heated and dissolved in water or aqueous solution, and then cooled and cross-linked.
  • the aforementioned preparation method includes the following steps:
  • the present invention also provides the use of the aforementioned bifunctional nucleoside hydrogel in the preparation of anti-tumor drugs; preferably, the tumor is lung cancer, glioma, osteoma, colon cancer, breast cancer, oral squamous cell carcinoma , Tongue squamous cell carcinoma; more preferably, the tumor is oral squamous cell carcinoma.
  • the bifunctional nucleoside hydrogel of the present invention integrates carrier and drug effect, and has obvious inhibitory effect on tumor cell activity, especially for lung cancer, glioma, osteoma, colon cancer, breast cancer, and oral squamous cell carcinoma , Tongue squamous cell carcinoma related cell activity has a significant inhibitory effect. Among them, the inhibitory effect on the activity of oral squamous cell carcinoma is the best.
  • the bifunctional nucleoside hydrogel of the present invention can inhibit the growth of the transplanted tumor of oral squamous cell carcinoma HSC-3 in vivo. Therefore, the bifunctional nucleoside hydrogel of the present invention has potential application prospects in preparing anti-tumor drugs. In particular, it can provide a new way for the treatment of oral squamous cell carcinoma.
  • Figure 1 shows the preparation process of the bifunctional nucleoside hydrogel of the present invention.
  • Figure 2 shows the verification of the chemical structure of isoGBG hydrogel;
  • Figure 3 shows the effect of isoGBG hydrogel on cell viability of A549, U251, U2OS, HCT-116, MCF-7, HSC-3 and UM1 malignant tumors detected by CCK8 method.
  • FIG. 4 shows that isoGBG hydrogel inhibits the growth of transplanted tumors of oral squamous cell carcinoma HSC-3 in nude mice under the route of administration around the tumor;
  • D) Weight change curve of each treatment group; (*)P ⁇ 0.05, (**)P ⁇ 0.01, (***)P ⁇ 0.001, n 10.
  • concentration of isoguanosine is It is 1.4wt%.
  • G guanosine
  • the molar ratio of guanosine to isoguanosine is 1:1, that is, the concentration of guanosine is 1.4wt%, and it is heated to fully dissolved at room temperature.
  • a transparent and colorless isoGBG hydrogel that is, the bifunctional nucleoside hydrogel of the present invention, is obtained, and the concentration of the isoGBG hydrogel is 14000 ⁇ g/ml.
  • Example 1 Using the preparation method of Example 1, only changing the concentration of isoguanosine and guanosine in the solution can obtain isoGBG hydrogels with different concentrations (Examples 2-10).
  • the concentration of isoguanosine and guanosine in the solution was 0.8 wt%, and isoGBG hydrogel with a concentration of 8000 ⁇ g/ml was prepared.
  • the concentration of isoguanosine and guanosine in the solution was 0.08 wt%, and isoGBG hydrogel with a concentration of 800 ⁇ g/ml was prepared.
  • the concentration of isoguanosine and guanosine in the solution was 0.04 wt%, and isoGBG hydrogel with a concentration of 400 ⁇ g/ml was prepared.
  • the concentration of isoguanosine and guanosine in the solution was 0.02 wt%, and isoGBG hydrogel with a concentration of 200 ⁇ g/ml was prepared.
  • the concentration of isoguanosine and guanosine in the solution was 0.01 wt%, and isoGBG hydrogel with a concentration of 100 ⁇ g/ml was prepared.
  • the concentration of isoguanosine and guanosine in the solution is 0.0075wt%, and isoGBG hydrogel with a concentration of 75 ⁇ g/ml is prepared.
  • the concentration of isoguanosine and guanosine in the solution was 0.005% by weight, and isoGBG hydrogel with a concentration of 50 ⁇ g/ml was prepared.
  • the concentration of isoguanosine and guanosine in the solution was 0.5 wt%, and isoGBG hydrogel with a concentration of 5000 ⁇ g/ml was prepared.
  • the concentration of isoguanosine and guanosine in the solution was 1.0 wt%, and isoGBG hydrogel with a concentration of 10000 ⁇ g/ml was prepared.
  • the bifunctional nucleoside hydrogel of the present invention can also be obtained (Example 11 ⁇ 14).
  • Example 1 The NaB(OH) 4 in Example 1 was replaced with LiB(OH) 4 to prepare the bifunctional nucleoside hydrogel of the present invention.
  • Example 1 NaB(OH) 4 was replaced with KB(OH) 4 to prepare the bifunctional nucleoside hydrogel of the present invention.
  • Example 1 The NaB(OH) 4 in Example 1 was replaced with RbB(OH) 4 to prepare the bifunctional nucleoside hydrogel of the present invention.
  • Example 1 NaB(OH) 4 was replaced with CsB(OH) 4 to prepare the bifunctional nucleoside hydrogel of the present invention.
  • the inventors used other nucleosides to prepare hydrogels, and the experimental results showed that other nucleosides could not form hydrogels.
  • isoguanosine (isoG) and NaB(OH) 4 Dissolve isoguanosine (isoG) and NaB(OH) 4 in a molar ratio of 1:1 in PBS, and heat to fully dissolve to obtain a clear and transparent solution.
  • concentration of isoguanosine is 0.5 wt%.
  • isoG hydrogel with a concentration of 5 mg/ml was obtained.
  • isoguanosine (isoG) and NaB(OH) 4 Dissolve isoguanosine (isoG) and NaB(OH) 4 in a molar ratio of 1:1 in PBS, and heat to fully dissolve to obtain a clear and transparent solution.
  • concentration of isoguanosine is 1.0 wt%.
  • isoG hydrogel with a concentration of 10 mg/ml was obtained.
  • Test Example 1 Verification of the molecular structure of the bifunctional nucleoside hydrogel of the present invention
  • the structure of the isoGBG hydrogel of the present invention is determined by nuclear magnetic resonance 11 B NMR, 1 H NMR, and infrared spectroscopy (IR).
  • This experiment uses the CCK8 experiment to detect the effects of isoGBG hydrogels (isoGBG hydrogels prepared in Examples 3 to 8 of the present invention) of different concentrations (50, 75, 100, 200, 400, 800 ⁇ g/ml) on human lung cancer Cell A549, glioma cell U251, osteoma cell U2OS, colon cancer cell HCT-116, breast cancer cell MCF-7, oral squamous cell HSC-3 and tongue squamous cell carcinoma cell UM1 influences.
  • the control group did not add isoguanosine and guanosine, it was NaB(OH) 4 ultrapure aqueous solution (that is, isoGBG hydrogel concentration is 0 ⁇ g/ml).
  • the NaB(OH) 4 ultrapure aqueous solution contained NaB(OH) )
  • the concentration of 4 is the same as in Example 3.
  • the blank control group is a PBS solution.
  • isoGBG hydrogel of the present invention The effect of isoGBG hydrogel of the present invention on the activity of A549, U251, U2OS, HCT-116, MCF-7, HSC-3 and UM1 malignant tumor cells is shown in Figure 3. It can be seen from Figure 3 that isoGBG hydrogel has an inhibitory effect on the above-mentioned tumor cells, indicating that isoGBG hydrogel has anti-tumor activity on the above-mentioned tumor cells. The increase in anti-tumor activity increases. Among them, isoGBG has the strongest inhibitory effect on oral squamous cell carcinoma cells, and the cell inhibitory effect can be as high as 90%.
  • isoGBG hydrogel has certain in vitro anti-tumor activity against common clinical malignancies such as lung cancer, glioma, osteoma, colon cancer, breast cancer, oral squamous cell carcinoma, tongue squamous cell carcinoma, etc., and has potential Application prospects. Among them, isoGBG hydrogel has the best in vitro anti-tumor activity against oral squamous cell carcinoma.
  • Test Example 3 The bifunctional nucleoside hydrogel of the present invention inhibits the growth of transplanted tumor of oral squamous cell carcinoma HSC-3
  • HSC-3 nude mouse xenograft model was established: the oral squamous cell carcinoma HSC-3 cells in logarithmic growth phase were collected and serum-free medium was used. The cells were washed with DMEM 3 times and resuspended, the cells were counted, and the cell concentration was adjusted to 2 ⁇ 10 7 cell/ml. 100 ⁇ l of cell suspension was subcutaneously inoculated on the right flank of nude mice to construct oral squamous cell carcinoma HSC-3 tumors model. 5 days after inoculation of oral squamous cell carcinoma HSC-3 cells, tumor nodules can be seen with naked eyes, and subcutaneous masses can be palpated.
  • the isoGBG hydrogels prepared in 9 and 10 are both experimental groups, and the corresponding treatments are given to each group by per-tumor injection, once a week, with an injection volume of 100 ⁇ l each time for 3 weeks.
  • the concentration of the hydrogel of Example 9 and Comparative Example 1 was 25 mg ⁇ kg -1 after being injected into nude mice, and the concentration of the hydrogel of Example 10 and Comparative Example 2 was 50 mg ⁇ kg -1 after being injected into nude mice.
  • tumor volume (V) ⁇ /6 ⁇ maximum diameter ⁇ (minimum diameter) 2 .
  • the results are expressed as mean ⁇ standard error.
  • the tumor inhibition rate was the ratio of the tumor volume between the experimental group and the blank control group.
  • Figure 4 shows the effect of isoGBG hydrogel on the growth of transplanted tumor of oral squamous cell carcinoma HSC-3. It can be seen from Figure 4 that isoGBG hydrogel at a concentration of 5mg/ml inhibited the growth of transplanted tumors by about 69.10%, and its inhibition on the growth of transplanted tumors was significantly better than that of the PBS group and isoG hydrogel group; and with isoGBG When the concentration of the hydrogel was increased to 10mg/ml, the tumor inhibition rate of isoGBG hydrogel was better, which was significantly increased to 78.80%.
  • isoGBG hydrogel as a dual-functional hydrogel that integrates carrier and pharmacodynamics, can inhibit the growth of oral squamous cell carcinoma xenografts in vivo, which may provide a new approach for the treatment of oral squamous cell carcinoma.
  • the bifunctional nucleoside hydrogel of the present invention integrates carrier and drug effect, and has obvious inhibitory effect on tumor cell activity, especially for lung cancer, glioma, osteoma, colon cancer, breast cancer,
  • the activity of oral squamous cell carcinoma and tongue squamous cell carcinoma related cells has a significant inhibitory effect.
  • the inhibitory effect on the activity of oral squamous cell carcinoma is the best.
  • the bifunctional nucleoside hydrogel of the present invention can inhibit the growth of the transplanted tumor of oral squamous cell carcinoma HSC-3 in vivo. Therefore, the bifunctional nucleoside hydrogel of the present invention has potential application prospects in preparing anti-tumor drugs. In particular, it can provide a new way for the treatment of oral squamous cell carcinoma.

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Abstract

一种双功能核苷水凝胶，其由异鸟嘌呤核苷、鸟嘌呤核苷和硼酸盐溶于水或水溶液后交联而成。该双功能核苷水凝胶集载体和药效于一体，对肿瘤细胞活性具有明显的抑制作用，特别是对于肺癌、脑胶质瘤、骨质瘤、结肠癌、乳腺癌、口腔鳞癌、舌鳞癌相关细胞的活性有显著的抑制作用。其中，对口腔鳞癌细胞活性的抑制效果最好。因此，该双功能核苷水凝胶在制备抗肿瘤药物方面具有潜在的应用前景。特别是可以为口腔鳞癌的治疗提供了新途径。

Description

一种双功能核苷水凝胶及其制备方法和用途 技术领域
本发明属于生物医用材料领域，具体涉及一种双功能核苷水凝胶及其制备方法和用途。
背景技术
临床上多数恶性肿瘤患者就诊时处于中晚期，化疗是其综合治疗的主要手段之一，但因传统化疗药物存在生物利用度低、毒副作用大、易产生耐药等缺陷，严重限制了其在临床上的广泛应用。为了克服此类药物的缺陷，水凝胶药物递送系统应用而生。其中，天然产物及其衍生物自组装形成的超分子水凝胶材料因具有良好的生物相容性、高负载能力、定点释放及良好的缓释、控释效果等优势，近年来在药物递送及肿瘤治疗中引起了人们的广泛关注及研究。然而，目前大多数超分子水凝胶只能作为载体传递药物，本身并不具有抗肿瘤活性；而一些天然产物虽有抗肿瘤活性，但由于其自组装性能差，很难形成作为递送系统应用的水凝胶。因此，构建一种兼具载体及药效于一体的双功能超分子水凝胶，并研究其在肿瘤治疗中的应用，具有极其重要的科研价值及潜在的临床意义。
发明内容
为了解决上述问题，本发明提供了一种双功能核苷水凝胶及其制备方法和用途。
本发明提供了一种双功能核苷水凝胶，它是由异鸟嘌呤核苷、鸟嘌呤核苷和硼酸盐溶于水或水溶液后交联而成。
进一步地，所述异鸟嘌呤核苷、鸟嘌呤核苷和硼酸盐的摩尔比为1:1～5:1～5；优选地，所述异鸟嘌呤核苷、鸟嘌呤核苷和硼酸盐的摩尔比为1:1:1。
进一步地，所述异鸟嘌呤核苷、鸟嘌呤核苷和硼酸盐溶于水或水溶液后得到的混合溶液中，异鸟嘌呤核苷的浓度为0.005～5.6wt％；优选地，异鸟嘌呤核苷的浓度为0.3～5.6wt％；更优选地，异鸟嘌呤核苷的浓度为1.4wt％。
进一步地，所述硼酸盐选自LiB(OH) 4、NaB(OH) 4、KB(OH) 4、RbB(OH) 4或CsB(OH) 4；优选地，所述硼酸盐为NaB(OH) 4。
进一步地，所述水或水溶液为超纯水或磷酸盐缓冲液。
进一步地，所述异鸟嘌呤核苷、鸟嘌呤核苷和硼酸盐溶于水或水溶液后交联为异鸟嘌呤核苷、鸟嘌呤核苷和硼酸盐加热溶于水或水溶液后，冷却交联。
进一步地，所述异鸟嘌呤核苷、鸟嘌呤核苷和硼酸盐溶于水或水溶液后交联为将异鸟嘌呤核苷和硼酸盐加入水或水溶液中，加热溶解，再加入鸟嘌呤核苷，加热溶解，室温下逐渐冷却交联。
本发明还提供了一种前述的双功能核苷水凝胶的制备方法，它包括如下步骤：
将异鸟嘌呤核苷、嘌呤核苷和硼酸盐加热溶于水或水溶液后，冷却交联即得。
进一步地，前述的制备方法包括如下步骤：
将异鸟嘌呤核苷和硼酸盐加入水或水溶液中，加热溶解，再加入鸟嘌呤核苷，加热溶解，室温下逐渐冷却交联后即得。
本发明还提供了前述的双功能核苷水凝胶在制备抗肿瘤药物中的用途；优选地，所述肿瘤为肺癌、脑胶质瘤、骨质瘤、结肠癌、乳腺癌、口腔鳞癌、舌鳞癌；更优选地，所述肿瘤为口腔鳞癌。
本发明双功能核苷水凝胶集载体和药效于一体，对肿瘤细胞活性具有明显的抑制作用，特别是对于肺癌、脑胶质瘤、骨质瘤、结肠癌、乳腺癌、口腔鳞癌、舌鳞癌相关细胞的活性有显著的抑制作用。其中，对口腔鳞癌细胞活性的抑制效果最好。此外，本发明双功能核苷水凝胶能在体内制口腔鳞癌细胞HSC-3移植瘤的生长。因此，本发明双功能核苷水凝胶在制备抗肿瘤药物方面具有潜在的应用前景。特别是可以为口腔鳞癌的治疗提供了新途径。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为本发明双功能核苷水凝胶的制备过程。
图2为isoGBG水凝胶的化学结构的验证；A)NaB(OH) 4、isoGBG的 11B NMR谱图及在DMSO溶液中isoG、isoGBG 1H NMR谱图；B)isoG和isoGBG的IR图；C)和D)在温度从25℃升至65℃的变化过程中，isoGBG在D 2O溶液中的VT 11B NMR、VT 1H NMR谱图。
图3为CCK8法检测isoGBG水凝胶对A549、U251、U2OS、HCT-116、MCF-7、HSC-3和UM1恶性肿瘤细胞活性的影响。
图4为在瘤周给药的途径下，isoGBG水凝胶抑制裸鼠口腔鳞状癌细胞HSC-3移植瘤的生长；A)各处理组HSC-3肿瘤的生长曲线；B)在21天时，各治疗组裸鼠的HSC-3移植瘤重量；C)在21天时，各组肿瘤的直观图；D)各治疗组的体重变化曲线；(*)P<0.05,(**)P<0.01,(***)P<0.001，n＝10。
具体实施方式
1、本发明主要原料及试剂
异鸟嘌呤核苷(分析纯，国药集团)：结构式为
Figure PCTCN2019089636-appb-000001
鸟嘌呤核苷(分析纯，国药集团)：
Figure PCTCN2019089636-appb-000002
NaB(OH) 4(分析纯，国药集团)、H 2O(实验室自制超纯水)。
2、本发明双功能核苷水凝胶的制备过程如图1所示。
实施例1、本发明双功能核苷水凝胶的制备
将摩尔比1:1的异鸟嘌呤核苷(isoG)和NaB(OH) 4溶于超纯水中，加热至充分溶解后得到澄清透明的溶液，得到的溶液中，异鸟嘌呤核苷浓度为1.4wt％。再在溶液中加入的鸟嘌呤核苷(G)，鸟嘌呤核苷与异鸟嘌呤核苷的摩尔比为1:1，即鸟嘌呤核苷浓度为1.4wt％，加热至充分溶解，室温下溶液自然冷却后得到透明无色的isoGBG水凝胶，即本发明双功能核苷水凝胶，该isoGBG水凝胶的浓度为14000μg/ml。
利用实施例1的制备方法，仅改变溶液中异鸟嘌呤核苷和鸟嘌呤核苷的浓度，可以得到不同浓度的isoGBG水凝胶(实施例2～10)。
实施例2、本发明双功能核苷水凝胶的制备
溶液中异鸟嘌呤核苷和鸟嘌呤核苷浓度为0.8wt％，制备得到浓度为8000μg/ml的isoGBG水凝胶。
实施例3、本发明双功能核苷水凝胶的制备
溶液中异鸟嘌呤核苷和鸟嘌呤核苷浓度为0.08wt％，制备得到浓度为800μg/ml的isoGBG水凝胶。
实施例4、本发明双功能核苷水凝胶的制备
溶液中异鸟嘌呤核苷和鸟嘌呤核苷浓度为0.04wt％，制备得到浓度为400μg/ml的isoGBG水凝胶。
实施例5、本发明双功能核苷水凝胶的制备
溶液中异鸟嘌呤核苷和鸟嘌呤核苷浓度为0.02wt％，制备得到浓度为200μg/ml的isoGBG水凝胶。
实施例6、本发明双功能核苷水凝胶的制备
溶液中异鸟嘌呤核苷和鸟嘌呤核苷浓度为0.01wt％，制备得到浓度为100μg/ml的isoGBG水凝胶。
实施例7、本发明双功能核苷水凝胶的制备
溶液中异鸟嘌呤核苷和鸟嘌呤核苷浓度为0.0075wt％，制备得到浓度为75μg/ml的isoGBG水凝胶。
实施例8、本发明双功能核苷水凝胶的制备
溶液中异鸟嘌呤核苷和鸟嘌呤核苷浓度为0.005wt％，制备得到浓度为50μg/ml的isoGBG水凝胶。
实施例9、本发明双功能核苷水凝胶的制备
溶液中异鸟嘌呤核苷和鸟嘌呤核苷浓度为0.5wt％，制备得到浓度为5000μg/ml的isoGBG水凝胶。
实施例10、本发明双功能核苷水凝胶的制备
溶液中异鸟嘌呤核苷和鸟嘌呤核苷浓度为1.0wt％，制备得到浓度为10000μg/ml的isoGBG水凝胶。
利用实施例1的制备方法，仅改变溶液中的硼酸盐的种类(实施例1中硼酸盐为NaB(OH) 4)，也可得到本发明双功能核苷水凝胶(实施例11～14)。
实施例11、本发明双功能核苷水凝胶的制备
将实施例1中NaB(OH) 4替换为LiB(OH) 4，制备得到本发明双功能核苷水凝胶。
实施例12、本发明双功能核苷水凝胶的制备
将实施例1中NaB(OH) 4替换为KB(OH) 4，制备得到本发明双功能核苷水凝胶。
实施例13、本发明双功能核苷水凝胶的制备
将实施例1中NaB(OH) 4替换为RbB(OH) 4，制备得到本发明双功能核苷水凝胶。
实施例14、本发明双功能核苷水凝胶的制备
将实施例1中NaB(OH) 4替换为CsB(OH) 4，制备得到本发明双功能核苷水凝胶。
本发明人利用其它核苷制备水凝胶，实验结果表明其它核苷均无法形成水凝胶。
对比例1、isoG水凝胶的制备
将摩尔比1:1的异鸟嘌呤核苷(isoG)和NaB(OH) 4溶于PBS中，加热至充分溶解后得到澄清透明的溶液，得到的溶液中，异鸟嘌呤核苷浓度为0.5wt％。室温下溶液自然逐渐冷却后得到浓度为5mg/ml的isoG水凝胶。
对比例2、isoG水凝胶的制备
将摩尔比1:1的异鸟嘌呤核苷(isoG)和NaB(OH) 4溶于PBS中，加热至充分溶解后得到澄清透明的溶液，得到的溶液中，异鸟嘌呤核苷浓度为1.0wt％。室温下溶液自然逐渐冷却后得到浓度为10mg/ml的isoG水凝胶。
以下通过具体的试验例证明本发明的有益效果。
试验例1、本发明双功能核苷水凝胶分子结构的验证
1、试验方法
利用核磁共振 11B NMR、 1H NMR、红外光谱(IR)确定本发明isoGBG水凝胶的结构。
2、试验结果
为了验证isoGBG水凝胶的结构和性质，通过核磁共振 11B NMR、 1H NMR、红外光谱(IR)对其化学结构进行分析，实验结果表明isoGBG水凝胶中存在硼酸二酯键(图2所示)。
试验例2、本发明双功能核苷水凝胶体外抗肿瘤活性的研究
1、试验方法
本试验运用CCK8实验检测不同浓度(50、75、100、200、400、800μg/ml)的isoGBG水凝胶(本发明实施例3～8所制备的isoGBG水凝胶)对人源性的肺癌细胞A549、脑胶质瘤细胞U251、骨质瘤细胞U2OS、结肠癌细胞HCT-116、乳腺癌细胞MCF-7、口腔鳞癌细胞HSC-3和舌鳞癌细胞UM1七株恶性肿瘤细胞活性的影响。对照组不加异鸟嘌呤核苷和鸟嘌呤核苷，为NaB(OH) 4超纯水溶液(即isoGBG水凝胶浓度为0μg/ml)，该NaB(OH) 4超纯水溶液中NaB(OH) 4的浓度与实施例3相同，空白对照组为PBS溶液。
具体操作方法如下：
(1)分别收集生长良好的A549、U251、U2OS、HCT-116、MCF-7、HSC-3和UM1，配制成细胞悬液，调整其细胞密度后接种于96孔板中，放于37℃、5％CO 2的细胞孵箱中孵育；
(2)培养4～6h，待细胞贴壁，分别加入100μl实施例3～8制备的不同浓度的isoGBG水凝胶(isoGBG水凝胶是一种超分子水凝胶，当在注射条件下给它剪切应力时，凝胶即可变为液体状态，把剪切应力去掉以后即刻恢复为凝胶状态。)、对照组NaB(OH) 4溶液和空白对照组PBS溶液，放入孵箱中继续孵育24h；
(3)孵育24h后，利用CCK8试剂检测细胞活性。取出96孔板，弃去培养基，加入10％CCK8试剂，使用酶标仪检测450nm波长的吸光度值(OD)。每组设计≥3个副孔，结果取平均值，实验独立重复3次。细胞活性百分率(％)＝实验组(OD)/对照组(OD)×100％。
2、试验结果
本发明isoGBG水凝胶对A549、U251、U2OS、HCT-116、MCF-7、HSC-3和UM1恶性肿瘤细胞活性的影响如图3所示。由图3可知，isoGBG水凝胶对上述肿瘤细胞均具有抑制作用，说明isoGBG水凝胶对上述肿瘤细胞均具有抗肿瘤活性，且随着isoGBG水凝胶浓度(0μg/mL～800μg/mL)的增加，抗肿瘤活性随之增高。其中，isoGBG对口腔鳞癌细胞抑制作用最强，细胞抑制作用可高达90％。试验结果表明，isoGBG水凝胶对肺癌、脑胶质瘤、骨质瘤、结肠癌、乳腺癌、口腔鳞癌、舌鳞癌等临床常见的恶性肿瘤均具有一定体外抗肿瘤活性，具有潜在的应用前景。其中，isoGBG水凝胶对口腔鳞癌的体外抗肿瘤活性最优。
试验例3、本发明双功能核苷水凝胶抑制口腔鳞癌细胞HSC-3移植瘤的生长
1、试验方法
为了研究isoGBG水凝胶对口腔鳞癌细胞HSC-3移植瘤生长的影响，建立了HSC-3裸鼠移植瘤模型：收集对数生长期的口腔鳞癌HSC-3细胞，用无血清培养基DMEM洗涤细胞3次后重悬，细胞计数，调整细胞浓度为2×10 7cell/ml，皮下接种100μl细胞悬液于裸鼠的右侧胁肋部，构建口腔鳞癌细胞HSC-3移植瘤模型。接种口腔鳞癌HSC-3细胞5天后肉眼可见肿瘤结节，可以扪及皮下肿块。
将造模后的裸鼠随机分组(n＝10只/组)，PBS溶液为空白对照组(PBS组)，对比例1和2制备的isoG水凝胶(isoG水凝胶组)、实施例9和10制备的isoGBG水凝胶(isoGBG水凝胶组)均为实验组，通过瘤周注射的方式给予各组相应的治疗，每周1次，每次注射量为100μl，共3周。实施例9和对比例1的水凝胶注入裸鼠体内后浓度为25mg·kg -1， 实施例10和对比例2的水凝胶注入裸鼠体内后浓度为50mg·kg -1。每隔3～4天用灭菌的游标卡尺测量肿瘤体积并称动物体重，同时观察裸鼠的毛发、行为及体重等有无异常。肿瘤的体积计算公式为：肿瘤体积(V)＝π/6×最大径×(最小径) 2。结果均以平均数±标准误来表示。肿瘤抑制率为实验组和空白对照组肿瘤体积的比。
第一次给药21天后，研究isoGBG水凝胶对HSC-3裸鼠移植瘤皮下模型的治疗效果。
2、试验结果
isoGBG水凝胶对口腔鳞癌细胞HSC-3移植瘤生长的影响结果如图4所示。由图4可知，浓度为5mg/ml的isoGBG水凝胶对移植瘤生长的抑制率约为69.10％，其对移植瘤生长的抑制明显优于PBS组和isoG水凝胶组；而随着isoGBG水凝胶的浓度增加到10mg/ml时，isoGBG水凝胶的肿瘤抑制率更优，明显提高到78.80％。同时，由图4可知，isoGBG水凝胶治疗口腔鳞癌移植瘤后，肿瘤体积明显减小，且有浓度依赖性，随着水凝胶浓度的增大，其抑制肿瘤生长的疗效更明显(P<0.05)，且优于PBS组和isoG水凝胶组。因此，可以发现isoGBG水凝胶作为集载体和药效于一体的双功能水凝胶，在体内能够抑制口腔鳞癌移植瘤的生长，可能为口腔鳞癌的治疗提供了新途径。
综上，本发明双功能核苷水凝胶集载体和药效于一体，对肿瘤细胞活性具有明显的抑制作用，特别是对于肺癌、脑胶质瘤、骨质瘤、结肠癌、乳腺癌、口腔鳞癌、舌鳞癌相关细胞的活性有显著的抑制作用。其中，对口腔鳞癌细胞活性的抑制效果最好。此外，本发明双功能核苷水凝胶能在体内制口腔鳞癌细胞HSC-3移植瘤的生长。因此，本发明双功能核苷水凝胶在制备抗肿瘤药物方面具有潜在的应用前景。特别是可以为口腔鳞癌的治疗提供了新途径。

Claims (10)

  1. 一种双功能核苷水凝胶，其特征在于：它是由异鸟嘌呤核苷、鸟嘌呤核苷和硼酸盐溶于水或水溶液后交联而成。
  2. 根据权利要求1所述的双功能核苷水凝胶，其特征在于：所述异鸟嘌呤核苷、鸟嘌呤核苷和硼酸盐的摩尔比为1:1～5:1～5；优选地，所述异鸟嘌呤核苷、鸟嘌呤核苷和硼酸盐的摩尔比为1:1:1。
  3. 根据权利要求1所述的双功能核苷水凝胶，其特征在于：所述异鸟嘌呤核苷、鸟嘌呤核苷和硼酸盐溶于水或水溶液后得到的混合溶液中，异鸟嘌呤核苷的浓度为0.005～5.6wt％；优选地，异鸟嘌呤核苷的浓度为0.3～5.6wt％；更优选地，异鸟嘌呤核苷的浓度为1.4wt％。
  4. 根据权利要求1所述的双功能核苷水凝胶，其特征在于：所述硼酸盐选自LiB(OH) 4、NaB(OH) 4、KB(OH) 4、RbB(OH) 4或CsB(OH) 4；优选地，所述硼酸盐为NaB(OH) 4。
  5. 根据权利要求1所述的双功能核苷水凝胶，其特征在于：所述水或水溶液为超纯水或磷酸盐缓冲液。
  6. 根据权利要求1所述的双功能核苷水凝胶，其特征在于：所述异鸟嘌呤核苷、鸟嘌呤核苷和硼酸盐溶于水或水溶液后交联为异鸟嘌呤核苷、鸟嘌呤核苷和硼酸盐加热溶于水或水溶液后，冷却交联。
  7. 根据权利要求6所述的双功能核苷水凝胶，其特征在于：所述异鸟嘌呤核苷、鸟嘌呤核苷和硼酸盐溶于水或水溶液后交联为将异鸟嘌呤核苷和硼酸盐加入水或水溶液中，加热溶解，再加入鸟嘌呤核苷，加热溶解，室温下逐渐冷却交联。
  8. 一种权利要求1～7任一项所述的双功能核苷水凝胶的制备方法，其特征在于：它包括如下步骤：
    将异鸟嘌呤核苷、嘌呤核苷和硼酸盐加热溶于水或水溶液后，冷却交联即得。
  9. 根据权利要求8所述的制备方法，其特征在于：它包括如下步骤：
    将异鸟嘌呤核苷和硼酸盐加入水或水溶液中，加热溶解，再加入鸟嘌呤核苷，加热溶解，室温下逐渐冷却交联后即得。
  10. 权利要求1～7任一项所述的双功能核苷水凝胶在制备抗肿瘤药物中的用途；优选地，所述肿瘤为肺癌、脑胶质瘤、骨质瘤、结肠癌、乳腺癌、口腔鳞癌、舌鳞癌；更优选地，所述肿瘤为口腔鳞癌。
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