CN115105606A - 透明质酸-芒果苷-甲氨蝶呤抗肿瘤偶联药物及其制备方法 - Google Patents
透明质酸-芒果苷-甲氨蝶呤抗肿瘤偶联药物及其制备方法 Download PDFInfo
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- CN115105606A CN115105606A CN202210809819.7A CN202210809819A CN115105606A CN 115105606 A CN115105606 A CN 115105606A CN 202210809819 A CN202210809819 A CN 202210809819A CN 115105606 A CN115105606 A CN 115105606A
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Abstract
本发明公开了一种透明质酸‑芒果苷‑甲氨蝶呤抗肿瘤偶联药物及其制备方法。所述方法先将透明质酸进行羧酸活化，再加入芒果苷，反应得到透明质酸‑芒果苷偶联物，然后将羧酸活化的甲氨蝶呤与透明质酸‑芒果苷偶联物反应，制得透明质酸‑芒果苷‑甲氨蝶呤抗肿瘤偶联药物。本发明的制备方法简单，通过引入芒果苷，三种药物在各自发挥自身作用的同时，透明质酸和芒果苷的联用能够提高药物的靶向抗肿瘤效果，且进一步降低甲氨蝶呤的毒副作用，提高药物的生物相容性、生物利用度及降解性。
Description
技术领域
本发明属于药物制备技术领域，涉及一种透明质酸-芒果苷-甲氨蝶呤抗肿瘤偶联药物及其制备方法。
背景技术
化学治疗是治疗癌症的主要手段，虽然可以缓解疼痛、达到一定的治疗效果，但伴随着不可避免的副作用，并不是所有的癌症患者都能从恶性肿瘤和化学治疗的副作用中生存下来。多聚物-药物偶合物可以克服化学抗癌药的上述缺点，多聚物作为载体并与化学抗癌药物连接，多聚物大分子能够滞留在肿瘤细胞内并且增强肿瘤细胞的渗透作用，从而实现肿瘤的靶向治疗。
甲氨蝶呤(MTX)是一种抗代谢类肿瘤药物，其结构与叶酸(FA)相似，是二氢叶酸还原酶(DHFR)的抑制剂。MTX与DHFR具有高亲和力，当它被内化到细胞后，可以参与FA代谢循环过程并抑制DHFR的形成，阻止二氢叶酸还原为四氢叶酸，能够终断DNA和RNA的生物合成。MTX渗透到细胞质中，会导致肿瘤细胞死亡，达到抗肿瘤效果。然而，MTX具有大多数抗代谢药物都具有的特点，即无组织选择性、高剂量、大的毒副作用等。应用纳米药物载体将MTX主动靶向病灶一直是研究人员关注的焦点，这种给药方式为MTX靶向治疗提供了新的途径。
透明质酸(HyaluronicAcid，HA)具有良好的生物相容性，被美国FDA认证无毒无害，常常被用作人体内部填充体，其衍生物常被用作药物载体材料，透明质酸可以通过其在细胞膜上的受体蛋白CD44介导而参与众多关键而复杂的生物功能。据研究报道很多肿瘤细胞表面都会不同程度地过量表达CD44，为靶向载药体系的设计提供新的发展方向。CD44是哺乳动物细胞表面普遍存在的糖蛋白，在多种实体瘤(如胰腺癌、乳腺癌和肺癌等)中过度表达。透明质酸是主要的CD44结合分子，利用透明质酸靶向能力可将药物有效递送到肿瘤组织(Hyaluronic acid targeting of CD44 for cancer therapy:from receptorbiology to nanomedicine,Journal of Drug Targeting,Volume 23,2015,605-618)。中国专利CN 108888775 A公开了一种透明质酸-甲氨蝶呤自组装纳米胶束及其制备方法，通过桥梁(二硫键的胱胺链接)将透明质酸和甲氨蝶呤进行偶联链接。其一方面利用透明质酸可以与肿瘤细胞特异性结合，使聚合物胶束具有靶向性，能够使药物有目标的载入病灶部位，另一方面利用二硫键的谷胱甘肽响应性，实现药物的控制释放。中国专利CN 107049955A通过透明质酸包裹聚酞胺-胺进行甲氨蝶呤载药的方法具有较好的多级靶向性，但是毒副作用较大且制备复杂。中国专利CN 110237266 A公开了一种透明质酸-阿仑膦酸钠-甲氨蝶呤纳米粒的制备方法，通过酰胺键和酯键共价连接方法制备透明质酸-阿仑膦酸钠-甲氨蝶呤偶联物，这种偶联物能够靶向性治疗癌细胞，由于阿仑膦酸钠易吸附于骨组织，因此该偶联物局限于靶向骨肿瘤，应用范围有限。
芒果苷是一种具有药理活性的天然多酚类化合物，具有良好的抗炎作用、抗过敏性能、抗肿瘤和抗氧化活性。芒果苷的应用范围广，能够治疗糖尿病、神经退行性疾病和预防衰老症状。目前国内还没有将芒果苷与甲氨蝶呤偶联制备抗肿瘤药物的文献报道。
发明内容
本发明的目的在于提供一种靶向性优异且毒副作用低的透明质酸-芒果苷-甲氨蝶呤抗肿瘤偶联药物及其制备方法。
实现本发明目的的技术方案如下：
透明质酸-芒果苷-甲氨蝶呤抗肿瘤偶联药物的制备方法，包括步骤如下：
步骤1，将透明质酸(HA)溶于二甲基亚砜(DMSO)中，加入1-乙基-3-(3-二甲氨基丙基)碳二亚胺(EDC)和4-二甲氨基吡啶(DMAP)的DMSO溶液，搅拌使羧酸活化，再加入芒果苷(MA)，继续搅拌反应至完全，透析并冷冻干燥，得到透明质酸-芒果苷(HA-MA)偶联物；
步骤2，将甲氨蝶呤溶于二甲基亚砜(DMSO)中，加入1-乙基-3-(3-二甲氨基丙基)碳二亚胺和4-二甲氨基吡啶的DMSO溶液，搅拌使羧酸活化，再加入透明质酸-芒果苷偶联物，继续搅拌反应至完全，透析并冷冻干燥，得到透明质酸-芒果苷-甲氨蝶呤抗肿瘤偶联药物(HA-MA-MTX)。
优选地，步骤1中，透明质酸与芒果苷的质量比为1:2。
优选地，步骤1中，搅拌反应时间为1h，继续搅拌反应时间为24h，冷冻干燥时间为24h。
优选地，步骤2中，透明质酸-芒果苷偶联物与甲氨蝶呤的质量比为1:4。
优选地，步骤2中，1-乙基-3-(3-二甲氨基丙基)碳二亚胺和4-二甲氨基吡啶的质量比为2:1。
与现有技术相比，本发明具有以下优点：
(1)本发明通过酯键将芒果苷、甲氨蝶呤与透明质酸结合，透明质酸可以和癌细胞中过度表达CD44受体结合，具有肿瘤细胞靶向性，同时其大分子结构也可以作为药物载体运载甲氨蝶呤，芒果苷除了发挥其自身的药用价值外，还与透明质酸协同作用，提高药物的靶向抗肿瘤效果的同时，进一步降低了甲氨蝶呤的毒副作用，提高药物的生物相容性、生物利用度及降解性。
(2)本发明中，透明质酸、芒果苷、甲氨蝶呤三种原料之间本身结构不需要通过桥梁(二硫键的胱胺链接)进行偶联，偶联药物结构稳定且制备方法简单。
附图说明
图1为实施例中透明质酸-芒果苷-甲氨蝶呤体系的合成示意图；
图2为实施例中制备的HA、MA、MTX、HA-MA、HA-MTX、HA-MA-MTX偶联物的核磁共振谱；
图3为实施例中制备的HA、MA、MTX、HA-MA、HA-MTX、HA-MA-MTX偶联物的傅里叶红外光谱；
图4为实施例中HA-MA、HA-MTX和HA-MA-MTX NPs的粒径分布和形貌图；
图5为实施例中制备的HA、MA、MTX、HA-MA、HA-MTX、HA-MA-MTX在K7癌细胞培养之后的细胞活死染色图；
图6为实施例中制备的HA、MA、MTX、HA-MA、HA-MTX、HA-MA-MTX在K7癌细胞共培养之后的cck-8实验结果图；
图7为实施例中制备的HA、MA、MTX、HA-MA、HA-MTX、HA-MA-MTX在MC3T3-E1正常细胞共培养之后的细胞活死染色图；
图8为实施例中制备的HA、MA、MTX、HA-MA、HA-MTX、HA-MA-MTX在MC3T3-E1正常细胞共培养之后的cck-8实验结果图。
具体实施方式
下面结合具体实施例和附图对本发明作进一步详述。
实施例
1.HA-MTX偶联物的制备：将42mg MTX溶于3mL DMSO中，加入75mg EDC和46mgDMAP，搅拌使羧酸活化；随后加入50mg HA和3mL DMSO进行酯化反应。用铝箔包裹溶液避免光照，让其充分进行反应48h，透析2d并冷冻干燥24h，得到HA-MTX偶联物。
2.HA-MA偶联物和HA-MA-MTX偶联物的制备：
(1)将200mg HA溶于混有800mg EDC和400mg DMAP的60mL DMSO中，混合搅拌1h，随后在反应混合液中加入400mg MA，搅拌24h并透析2d，冷冻干燥得到HA-MA偶联物。
(2)将400mg MTX溶于混有800mg EDC和400mg DMAP的60mLDMSO中，搅拌反应后，加入100mg HA-MA，继续搅拌24h，透析2d并冷冻干燥24h，得到HA-MA-MTX偶联物。
图1是透明质酸-芒果苷-甲氨蝶呤体系的合成示意图，HA-MA-MTX偶联物的结构式如图所示。
图2是HA、MA、MTX、HA-MA、HA-MTX和HA-MA-MTX偶联物的核磁共振谱，可以确定它们的化学结构。HA中的乙酰基(-NHCOCH3)含量为1.85ppm，糖苷H的含量为3.0-4.0ppm。MA在7.04ppm、3.38ppm和0.97ppm处有特征峰。与MTX光谱相比，HA-MTX光谱在6.8-8.6ppm的微弱特征峰对应于MTX的芳香质子，光谱中还显示了HA的N-乙酰基(1.85ppm)和糖苷H(3.0-4.0ppm)，证实了MTX与HA的成功连接。MTX的芳香族质子相对应的6.8-8.6ppm的特征峰表明MTX成功地偶联到HA-MA偶联物上，表明HA-MA-MTX偶联物制备成功。
图3是HA-MA-MTX偶联物的傅里叶红外光谱。HA在1625cm-1(-CO-)、1125cm-1(C-O-C)、1050cm-1(-C-OH)处具有特征透过率；MA在2920cm-1(-CH2)，1720cm-1(-C＝O)，1250cm-1(Ar-O-Ar)和1050cm-1(-C-OH)有特征透过率；MTX在3500cm-1(-COOH)，2960cm-1(-CH3)，1720cm-1(-C＝O)，1600cm-1和1500cm-1和820cm-1(对位苯)处有特征透过率。在MTX和HA-MTX偶联物的红外光谱中发现1600cm-1和1500cm-1的FT-IR峰，这归因于对苯环的伸缩，结果表明MTX已成功偶联到透明质酸上。在HA-MA-MTX的光谱中同时出现了MTX和MA的特征峰，表明HA-MA-MTX偶联成功。
图4是利用原子力显微镜(AFM)对合成的HA-MA(A、B)、HA-MTX(C、D)和HA-MA-MTX(E、F)NPs的粒径分布和形貌进行表征。这些纳米颗粒呈球形，大小在100nm左右。这些自组装的纳米颗粒由MA、MTX或组合的内部疏水核心和HA多糖链的OH、COOH等亲水基团组成。MA和MTX的疏水分子形成结构的内核以充当药物库。糖胺聚糖HA的外部亲水外壳可以通过其CD44结合能力特异性靶向癌细胞。
图5分别为HA(A)、MA(B)、MTX(C)、HA-MA(D)、HA-MTX(E)、HA-MA-MTX(F)在K7癌细胞共培养后的活死染色图，结果表明MTX、HA-MTX和HA-MA-MTX能够有效的抑制K7癌细胞的增殖。
图6分别为HA、MA、MTX、HA-MA、HA-MTX、HA-MA-MTX在K7癌细胞共培养后的cck-8测试结果图，结果表明HA-MTX和HA-MA-MTX偶联物纳米粒子与游离的MTX一样对肿瘤细胞的生长具有抑制作用，这与cck-8实验结果一致。随着浓度升高，抑制作用明显增强，并且HA-MA-MTX比游离MTX和HA-MTX的抑制作用更强。当给定的药物浓度为100μg/mL时，HA-MA-MTX的抑制作用均可达到72％左右，而MTX抑制率60％左右，HA-MTX抑制率65％左右。因此，对于K7癌细胞，HA-MA-MTX的抑制作用高于游离的MTX和HA-MTX，说明了MA的加入，能够进一步提高HA-MTX偶联物的抑制肿瘤的作用。
图7分别为HA(A)、MA(B)、MTX(C)、HA-MA(D)、HA-MTX(E)、HA-MA-MTX(F)在MC3T3-E1正常细胞共培养后的活死染色图，结果表明MTX、HA-MTX和HA-MA-MTX能够在一定程度上杀死正常细胞，但HA-MA-MTX偶联物杀死的正常细胞较少，即毒副作用小，证明了相较于HA-MTX，MA的加入能够进一步减少HA-MA-MTX对正常组织细胞的毒副作用。
图8分别为HA、MA、MTX、HA-MA、HA-MTX、HA-MA-MTX在MC3T3-E1正常细胞共培养后的cck-8测试结果图，结果表明游离MTX显著降低正常细胞的生存能力，毒副作用大，当给定的药物浓度为100μg/mL时，MC3T3-E1细胞的存活率为分别为57％、62％和68％左右，其中HA-MA-MTX偶联物纳米粒作用下的细胞成活率比游离MTX和HA-MTX药物要高。以上结果表明，相较于HA-MTX，HA-MA-MTX纳米粒子可以进一步减少MTX对正常细胞的毒副作用。
Claims (6)
1.透明质酸-芒果苷-甲氨蝶呤抗肿瘤偶联药物的制备方法，其特征在于，包括步骤如下：
步骤1，将透明质酸溶于二甲基亚砜中，加入1-乙基-3-(3-二甲氨基丙基)碳二亚胺和4-二甲氨基吡啶的二甲基亚砜溶液，搅拌使羧酸活化，再加入芒果苷，继续搅拌反应至完全，透析并冷冻干燥，得到透明质酸-芒果苷偶联物；
步骤2，将甲氨蝶呤溶于二甲基亚砜中，加入1-乙基-3-(3-二甲氨基丙基)碳二亚胺和4-二甲氨基吡啶的二甲基亚砜溶液，搅拌使羧酸活化，再加入透明质酸-芒果苷偶联物，继续搅拌反应至完全，透析并冷冻干燥，得到透明质酸-芒果苷-甲氨蝶呤抗肿瘤偶联药物。
2.根据权利要求1所述的制备方法，其特征在于，步骤1中，透明质酸与芒果苷的质量比为1:2。
3.根据权利要求1所述的制备方法，其特征在于，步骤1中，搅拌反应时间为1h，继续搅拌反应时间为24h，冷冻干燥时间为24h。
4.根据权利要求1所述的制备方法，其特征在于，步骤2中，透明质酸-芒果苷偶联物与甲氨蝶呤的质量比为1:4。
5.根据权利要求1所述的制备方法，其特征在于，步骤2中，1-乙基-3-(3-二甲氨基丙基)碳二亚胺和4-二甲氨基吡啶的质量比为2:1。
6.根据权利要求1至5任一所述的制备方法制得的透明质酸-芒果苷-甲氨蝶呤抗肿瘤偶联药物。
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| CN110237266A (zh) * | 2019-05-30 | 2019-09-17 | 扬州大学 | 透明质酸-阿仑膦酸钠-甲氨蝶呤纳米粒的制备方法 |
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| CN110237266A (zh) * | 2019-05-30 | 2019-09-17 | 扬州大学 | 透明质酸-阿仑膦酸钠-甲氨蝶呤纳米粒的制备方法 |
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| CN115920076B (zh) * | 2022-11-07 | 2024-05-31 | 盐城师范学院 | 一种马齿苋多糖-去甲基斑蝥素-甲氨蝶呤偶联物及其制备方法和应用 |
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