CN112641760A - 二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物、制备方法与应用 - Google Patents
二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物、制备方法与应用 Download PDFInfo
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- CN112641760A CN112641760A CN202110023249.4A CN202110023249A CN112641760A CN 112641760 A CN112641760 A CN 112641760A CN 202110023249 A CN202110023249 A CN 202110023249A CN 112641760 A CN112641760 A CN 112641760A
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- berberine
- ferrocene
- drug
- glucose oxidase
- hyaluronic acid
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Abstract
本发明公开了一种二茂铁‑黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸自组装靶向抗癌纳米药物的方法，通过设计二茂铁‑黄连素偶联药物分子，与吲哚美辛一起诱导葡萄糖氧化酶自组装，然后被透明质酸包裹，得到抗癌纳米药物。本发明提供所述的二茂铁‑黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物的制备方法及其在癌症治疗中的应用。本发明提供的纳米药物，能够靶向递送，并且对肝癌细胞体现出显著的生长和转移抑制效果。
Description
技术领域
本发明涉及药物合成技术，具体涉及一种靶向的二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物及其制备方法与应用。
背景技术
随着纳米技术的飞速发展，传统的癌症的化学疗法，取得了越来越大的进步，大大提高了药物的生物利用度和靶向能力，但是大多数纳米药物使用的单一疗法仍然存在耐药性频发以及疾病的反复复发和转移等不足。将两种或多种治疗策略相结合的组合治疗作为一种有前途的方法有潜力改善单一治疗的效果并克服它们的缺点。
与单线态氧(1O2)相比，羟基自由基(·OH)是另一种具有更高氧化能力的活性氧(ROS)，大量的·OH会对生物大分子或细胞器造成不可逆转的破坏。近年来，由于针对肿瘤细胞中·OH的特殊生成策略，Fenton反应驱动的化学动力学疗法(CDT)作为一种有前途的癌症治疗方法受到了广泛关注。
由过渡金属离子(尤其是Fe2+)介导，癌细胞中的H2O2可以通过Fenton反应产生高水平的·OH，从而破坏细胞的氧化还原稳态，并最终诱导细胞凋亡。然而，通过芬顿反应产生的CDT在肿瘤微环境中需要高水平的酸和H2O2，这限制了CDT的进一步使用。作为内源性耗氧脱氢酶，葡萄糖氧化酶(GOD)已用于协同癌症治疗。GOD可催化葡萄糖被氧气降解为有毒的H2O2和葡萄糖酸，从而获得酸性更大的肿瘤微环境和有效的抗癌饥饿治疗。在Fe2+存在的情况下，它还促进了Fenton反应的动力学，并增强了·OH的产生，实现了化学动力学治疗。因此，通过纳米递送系统共同递送Fe2+和GOD有望触发癌细胞中基于Fe的酶/Fenton级联反应，旨在获得具有显著功效的化学动力学治疗和饥饿治疗。
在淋巴器官和肿瘤部位合成的前列腺素(PG)在产生肿瘤免疫抑制微环境中起关键作用。在多种癌细胞中，如乳腺癌、结肠癌、肺癌、胃癌和胰腺癌等，PG均存在过度表达。吲哚美辛(IND)作为一种非甾体抗炎药，不仅可以缓解疼痛和炎症，更重要的是，最近的文献发现IND也可以抑制PG的合成，降低了从PG介导的由正常单核细胞转化而来的M2巨噬细胞的比例(促进肿瘤生长和逃逸)，从而增加了M1巨噬细胞的比例(显示出抗肿瘤作用)并增强了对肿瘤细胞的免疫反应。另外，IND还可以使耐药的肿瘤细胞对巨噬细胞敏感。基于以上讨论，IND具有巨大的潜力，可以与其他疗法联合用作免疫协同治疗。
黄连素是具有线粒体靶向抗癌活性的中药提取物，更重要的是，它还可以抑制癌细胞的扩散转移和疾病的复发。具有夹心结构的二茂铁(FC)衍生物具有广泛的生物活性，如抗癌、抗细菌、抗真菌和抗疟疾等。由二茂铁和苯酚形成的杂合体不同于顺铂，它不仅可以作用于DNA，还可以通过作用于蛋白质和不同的酶(例如硫氧还蛋白还原酶)来抑制癌细胞的繁殖，目前，该杂合体已进入临床研究。另外，由Fe2+组成的二茂铁由于其高稳定性和可逆的氧化还原特性而引起了广泛的关注，其在生理环境中可实现快速的芬顿反应。众所周知，透明质酸(HA)具有高度的生物相容性和免疫惰性，因此已在生物医学领域得到了广泛的应用。此外，HA可以与多种癌细胞过度表达的CD44受体特异性结合，因此可以用作药物递送和癌症治疗的活性靶向载体。
本专利制备了二茂铁-黄连素偶联药物，在水中与吲哚美辛共同诱导葡萄糖氧化酶自组装，然后由HA封装形成纳米药物，以实现癌症的靶向组合治疗以及抑制癌细胞的转移。
发明内容
发明目的：鉴于现有单一治疗的问题以及靶向治疗和组合治疗的优势，本发明提供了一种二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物；本发明还提供了该纳米药物的制备方法以及应用。
技术方案：本发明所述二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物的制备方法，包括如下步骤：
(1)盐酸黄连素在高温真空条件下得到去甲基黄连素；
(2)去甲基黄连素与溴乙醇反应得到羟乙基取代的去甲基黄连素；
(3)羟乙基取代的去甲基黄连素溶解于甲醇中，然后加入硼氢化钠固体，室温反应12～24h后得到还原的羟乙基黄连素；
(4)在有机溶剂中，脱水剂在催化剂催化下，对二茂铁甲酸与还原的羟乙基黄连素进行缩合得到二茂铁-黄连素偶联药物前体；
(5)二茂铁-黄连素偶联药物前体与碘反应得到二茂铁-黄连素偶联药物；
(6)二茂铁-黄连素偶联药物与吲哚美辛溶解在有机溶剂中，滴到葡萄糖氧化酶储备液中，然后加入透明质酸，超纯水中透析，即得到二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸自组装纳米药物。
进一步的，步骤(3)中，羟乙基取代的去甲基黄连素与硼氢化钠的物质的量之比为1：1.5～3，优选1:2。
进一步的，步骤(4)中，有机溶剂为吡啶、二氯甲烷、二甲基亚砜、四氢呋喃中任意一种或几种；脱水剂为N,N-二环己基碳二亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐中任意一种或几种；催化剂为4-二甲氨基吡啶；二茂铁甲酸、还原的羟乙基黄连素、脱水剂、催化剂的物质的量之比为1:1:2～3.5:0.2～0.8，优选1:1:2.5:0.5。
进一步的，步骤(5)中，二茂铁-黄连素偶联药物前体与碘的物质的量之比为1:1～2.5，优选1:2。
进一步的，步骤(6)中，有机溶剂为二甲基亚砜、四氢呋喃、甲醇、N,N-二甲基甲酰胺中任意一种或几种。
进一步的，步骤(6)中，二茂铁-黄连素偶联药物、吲哚美辛、葡萄糖氧化酶、透明质酸的质量之比为1～3:0.5～3:10:1～3，优选3:1.5:10:1.5。
进一步的，步骤(6)中，透析的时间为6～24h。
按照上述方法制备二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物也在本发明的保护范围内。
本发明还进一步公开了所述纳米药物在制备抗癌药物中的应用。其中，所述肿瘤为肝癌。所述药物能够诱导癌细胞凋亡，抑制癌细胞转移。
本发明中二茂铁-黄连素偶联药物合成路线如下：
有益效果：本发明利用超分子自组装技术制备了二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物，该纳米药物既通过靶向的组合治疗抑制了癌细胞的增殖，又能抑制癌细胞的转移。本发明所设计的新药物分子，结果确定，易于进行重复和表征，并且具有较好的治疗作用。
附图说明
图1为二茂铁-黄连素偶联药物前体的氢谱；
图2为二茂铁-黄连素偶联药物前体的高分辨质谱；
图3为二茂铁-黄连素偶联药物的氢谱；
图4为二茂铁-黄连素偶联药物的高分辨质谱；
图5为二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物的透射电镜图(A)；二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶纳米药物的粒径分布(B)；二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物的粒径分布(C)；
图6为二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物(FC-BBR/IND@GOD@HA NPs)和二茂铁-黄连素(FC-BBR)抗HepG2细胞效果；
图7为二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物中的吲哚美辛药物累积释放图；
图8为二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物(FC-BBR/IND@GOD@HA NPs)和二茂铁-黄连素(FC-BBR)与线粒体荧光探针共定位的激光共聚焦图；
图9为药物抑制HepG2细胞移动的结果；其中(A)对照，(B)二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物，(C)葡萄糖氧化酶，(D)二茂铁-黄连素，(E)吲哚美辛；
图10为药物抑制HepG2细胞迁移的结果；其中，(A)对照，(B)二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物，(C)葡萄糖氧化酶，(D)二茂铁-黄连素，(E)吲哚美辛。
具体实施方式
为了清楚了解本发明的技术方案，下面通过附图和实施例，对本发明的技术方案做进一步的详细描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
实施例1合成去甲基黄连素
在无溶剂以及真空和190℃条件下，5g黄连素置于100mL洁净圆底烧瓶中搅拌45min，得到的黄连素粗产品通过柱层析(洗脱机体积比：氯仿/甲醇＝20/1)分离得到去甲基黄连素(3.75g，收率79％)。
实施例2合成羟乙基取代的黄连素
在乙腈(20mL)为溶剂、碳酸钾为碱(1.29g)条件下，将实施例1中制备的去甲基黄连素(3g)置于100mL的圆底烧瓶中搅拌25min，然后分批加入溴乙醇(2.33g)，继续回流反应12h，去除反应溶剂并通过柱层析(洗脱机体积比：氯仿/甲醇＝20/1)分离，得到羟乙基黄连素(3.53g，收率85％)。
实施例3合成还原的羟乙基黄连素
在甲醇(20mL)为溶剂条件下，将实施例2中制备的羟乙基黄连素(2g,4.49mmol)置于250mL的圆底烧瓶中搅拌，然后分批加入硼氢化钠(0.34g,8.98mmol)，继续反应12h，去除反应溶剂并通过柱层析(洗脱机体积比：氯仿/甲醇＝30/1)分离，得到还原的羟乙基黄连素(1.54g，收率93％)。
实施例4合成还原的羟乙基黄连素
在甲醇(20mL)为溶剂条件下，将实施例2中制备的羟乙基黄连素(2g,4.49mmol)置于250mL的圆底烧瓶中搅拌，然后分批加入硼氢化钠(0.17g,4.49mmol)，继续反应12h，去除反应溶剂并通过柱层析(洗脱机体积比：氯仿/甲醇＝30/1)分离，得到还原的羟乙基黄连素(1.34g，收率81％)。
实施例5合成还原的羟乙基黄连素
在甲醇(20mL)为溶剂条件下，将实施例2中制备的羟乙基黄连素(2g,4.49mmol)置于250mL的圆底烧瓶中搅拌，然后分批加入硼氢化钠(0.51g,13.47mmol)，继续反应12h，去除反应溶剂并通过柱层析(洗脱机体积比：氯仿/甲醇＝30/1)分离，得到还原的羟乙基黄连素(1.57g，收率95％)。
实施例6合成二茂铁-黄连素偶联药物前体
向150mL圆底烧瓶中加入二茂铁甲酸(0.89g,3.86mmol)、4-二甲氨基吡啶(0.24g,1.93mmol)、二环己基碳二亚胺(2.00g,9.65mmol)、吡啶(10mL)，氮气保护，于冰浴下搅拌30min，向反应体系中加入实施例3中合成的还原的羟乙基黄连素(1.42g,3.86mmol)的二氯甲烷溶液，继续在冰浴下反应48h，得到二茂铁-黄连素偶联药物前体粗产品，减压蒸馏除去溶剂吡啶，然后柱层析(洗脱机体积比：氯仿/甲醇＝40/1)分离，得二茂铁-黄连素偶联药物前体1.70g，收率76％。1H NMR(600MHz,DMSO-d6,ppm)δ6.91(t,J＝4.1Hz,2H),6.86(d,J＝8.4Hz,1H),6.66(s,1H),5.95(d,J＝3.3Hz,2H),4.78(d,J＝10.0Hz,2H),4.52–4.48(m,2H),4.47–4.40(m,2H),4.31–4.27(m,1H),4.23(s,5H),4.20–4.15(m,2H),3.79(s,3H),3.39(t,J＝13.9Hz,2H),3.30(d,J＝3.3Hz,1H),3.01(dd,J＝11.0,3.5Hz,1H),2.88(dd,J＝19.1,7.9Hz,1H),2.56(dd,J＝21.3,9.7Hz,2H),2.43–2.38(m,1H).；ESI-MS m/z[M+H]+＝582.15889。
图1和图2分别是二茂铁-黄连素偶联药物前体的氢谱和高分辨质谱，证明了该化合物制备成功。
实施例7合成二茂铁-黄连素偶联药物前体
向150mL圆底烧瓶中加入二茂铁甲酸(0.89g,3.86mmol)、4-二甲氨基吡啶(0.24g,1.93mmol)、二环己基碳二亚胺(0.8g,3.86mmol)、吡啶(10mL)，氮气保护，于冰浴下搅拌30min，向反应体系中加入实施例3中合成的还原的羟乙基黄连素(1.42g,3.86mmol)的二氯甲烷溶液，继续在冰浴下反应48h，得到二茂铁-黄连素偶联药物前体粗产品，减压蒸馏除去溶剂吡啶，然后柱层析(洗脱机体积比：氯仿/甲醇＝40/1)分离，得二茂铁-黄连素偶联药物前体1.48g，收率66％。
实施例8合成二茂铁-黄连素偶联药物前体
向150mL圆底烧瓶中加入二茂铁甲酸(0.89g,3.86mmol)、4-二甲氨基吡啶(0.38g,3.08mmol)、二环己基碳二亚胺(0.8g,3.86mmol)、吡啶(10mL)，氮气保护，于冰浴下搅拌30min，向反应体系中加入实施例3中合成的还原的羟乙基黄连素(1.42g,3.86mmol)的二氯甲烷溶液，继续在冰浴下反应48h，得到二茂铁-黄连素偶联药物前体粗产品，减压蒸馏除去溶剂吡啶，然后柱层析(洗脱机体积比：氯仿/甲醇＝40/1)分离，得二茂铁-黄连素偶联药物前体1.53g，收率68％。
实施例9合成二茂铁-黄连素偶联药物
向50mL圆底烧瓶中加入实施例6中合成的二茂铁-黄连素偶联药物前体(0.29g,0.50mmol)、甲醇(20mL)，室温下搅拌，然后向反应体系加入碘单质(0.25g,1.00mmol)的氯仿溶液，室温下继续反应24h，得到二茂铁-黄连素偶联药物粗产品，柱层析(洗脱机体积比：氯仿/甲醇＝7/1)分离，得二茂铁-黄连素偶联药物0.22g，收率61％。1H NMR(600MHz,DMSO-d6,ppm)δ9.85(s,1H),8.95(s,1H),8.23(d,J＝9.2Hz,1H),8.02(d,J＝9.1Hz,1H),7.79(s,1H),7.08(s,1H),6.18(s,2H),4.89–4.85(m,2H),4.67–4.62(m,6H),4.48–4.45(m,2H),4.19(s,5H),4.08(s,3H),3.19–3.15(m,2H).ESI-MS m/z[M-I]+＝578.11740。
图3和图4分别是二茂铁-黄连素偶联药物的氢谱和高分辨质谱，证明了该化合物制备成功。
实施例10合成二茂铁-黄连素偶联药物
向50mL圆底烧瓶中加入实施例6中合成的二茂铁-黄连素偶联药物前体(0.29g,0.50mmol)、甲醇(20mL)，室温下搅拌，然后向反应体系加入碘单质(0.13g,0.50mmol)的氯仿溶液，室温下继续反应24h，得到二茂铁-黄连素偶联药物粗产品，柱层析(洗脱机体积比：氯仿/甲醇＝7/1)分离，得二茂铁-黄连素偶联药物0.19g，收率53％。
实施例11合成二茂铁-黄连素偶联药物
向50mL圆底烧瓶中加入实施例6中合成的二茂铁-黄连素偶联药物前体(0.29g,0.50mmol)、甲醇(20mL)，室温下搅拌，然后向反应体系加入碘单质(0.63g,2.50mmol)的氯仿溶液，室温下继续反应24h，得到二茂铁-黄连素偶联药物粗产品，柱层析(洗脱机体积比：氯仿/甲醇＝7/1)分离，得二茂铁-黄连素偶联药物0.22g，收率63％。
实施例12制备二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物
配置1mg/mL的葡萄糖氧化酶储备溶液。将实施例9中得到的二茂铁-黄连素(3mg)和吲哚美辛(1.5mg)溶于0.3mL的二甲基亚砜中，然后将该混合溶液滴加到10mL的葡萄糖氧化酶储备液中，搅拌2小时，形成二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶纳米药物，向该体系进一步加入1.5mg的透明质酸钠，将该混合溶液透析6h，得到二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物。
实施例13制备二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物
配置1mg/mL的葡萄糖氧化酶储备溶液。将实施例9中得到的二茂铁-黄连素(1mg)和吲哚美辛(1.5mg)溶于0.3mL的二甲基亚砜中，然后将该混合溶液滴加到10mL的葡萄糖氧化酶储备液中，搅拌2小时，形成二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶纳米药物，向该体系进一步加入1.5mg的透明质酸钠，将该混合溶液透析6h，得到二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物。
实施例14制备二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物
配置1mg/mL的葡萄糖氧化酶储备溶液。将实施例9中得到的二茂铁-黄连素(2mg)和吲哚美辛(1.5mg)溶于0.3mL的二甲基亚砜中，然后将该混合溶液滴加到10mL的葡萄糖氧化酶储备液中，搅拌2小时，形成二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶纳米药物，向该体系进一步加入1.5mg的透明质酸钠，将该混合溶液透析6h，得到二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物。
实施例15纳米药物的粒径和形貌表征
采用激光粒度仪对实施例12所得二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶纳米药物的粒径进行了表征，图5B表明，其粒径大小在126纳米左右。对二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物的粒径也采用激光粒度仪和透射电子显微镜进行了检测，图5A和5C表明，该纳米药物的粒径在156纳米左右。
实例16MTT法癌细胞活力实验
HepG2人肝癌细胞接种于96孔培养板，5％CO2、37℃培养箱中培养24h后，每孔加入不同浓度的实施例12的二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物溶液、二茂铁-黄连素，使最终的药物浓度分别为0.625、1.25、2.5、5、10、20、40μM，继续培养48或72h，分别加入50μL的MTT，于5％CO2、37℃培养箱中继续培养4h，弃去培养基，加入150μL的DMSO，在平板摇床上摇匀，酶标仪读板，根据测得的吸光度值计算细胞抑制率。数据表示为平均数±SD(n＝3)。
结果发现(如图6所示)，A图为培养48h的结果，B图为培养72h的结果。图中横坐标为药物浓度，纵坐标为癌细胞存活率。在培养48或72小时后，二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物抗HepG2的IC50值分别为，1.106和0.689μM，二茂铁-黄连素抗HepG2的IC50值分别为19.306和15.637μM。
实施例17纳米药物释放实验
通过透析法测量吲哚美辛从纳米药物中的释放。将1mL实施例12的二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物溶液置于透析袋(MwCO＝2000Da)中，然后浸没于30mL的磷酸缓冲溶液(pH＝7.4)中，在37℃下，以150转/分钟速率震荡。释放的吲哚美辛采用高效液相色谱进行测量，检测波长为260nm，流动相的流速1.0mL/min，流动相为甲醇:0.05％磷酸溶液＝70:30。
结果发现(如图7所示，图中横坐标为时间，纵坐标为累计释放的药物)，吲哚美辛从纳米药物中能缓慢释放，在48小时内，有大约70％的药物被释放。
实施例18纳米药物的线粒体共定位实验
激光共聚焦显微镜被用来观察二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物的线粒体共定位特性。HepG2肝癌细胞接种于玻璃小皿中，5％CO2、37℃培养箱中培养12h后，每个皿中分别加入5μM实施例12的二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸纳米药物或二茂铁-黄连素继续培养60min。然后使用缓冲溶液将玻璃皿洗涤3次后，加入线粒体共定位荧光探针，继续培养30min，最后使用激光共聚焦显微镜观察。
结果发现(如图8所示，图中第一列为黄连素片段的共聚焦图，第二列为线粒体荧光探针的共聚焦图，第三列为第一列和第二列合并的图)，纳米药物和二茂铁-黄连素有较好的线粒体靶向效果。
实施例19划痕愈合及细胞迁移实验
划痕测试用于分析细胞的体外运动。将HepG2细胞接种在6孔板中并培养过夜。细胞附着后，用100μL移液器吸头划6孔板。用磷酸缓冲溶液洗涤3次后，加入20μM实施例12的二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸、二茂铁-黄连素或等量的葡萄糖氧化酶及吲哚美辛继续培养24小时，并拍照观察划痕的愈合情况。
将20μM的二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸、二茂铁-黄连素或等量的葡萄糖氧化酶及吲哚美辛和HepG2细胞接种到孔径为8μm的细胞上室中，并将血清置于六孔板的下腔室中，培养24h。然后用酒精棉球擦拭上室中的细胞，用0.25％的结晶紫染色30min，并拍照观察细胞迁移。
结果发现(如图9和10所示)，纳米药物能够抑制划痕的愈合以及细胞的迁移，这表明制备的纳米药物有潜力阻止癌细胞的转移。
Claims (10)
1.一种二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸靶向抗癌纳米药物的合成方法，其特征在于，包括如下步骤：
(1)盐酸黄连素在高温真空条件下得到去甲基黄连素；
(2)去甲基黄连素与溴乙醇反应得到羟乙基黄连素；
(3)羟乙基黄连素溶解于甲醇中，然后加入硼氢化钠固体，室温反应得到还原的羟乙基黄连素；
(4)在有机溶剂中，脱水剂在催化剂催化下，对二茂铁甲酸与还原的羟乙基黄连素进行缩合得到二茂铁-黄连素偶联药物前体；
(5)二茂铁-黄连素偶联药物前体与碘反应得到二茂铁-黄连素偶联药物；
(6)二茂铁-黄连素偶联药物与吲哚美辛溶解在有机溶剂中，滴到葡萄糖氧化酶储备液中，然后加入透明质酸，超纯水中透析，即得到二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸自组装纳米药物。
2.根据权利要求1所述的二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸靶向抗癌纳米药物的合成方法，其特征在于，步骤(3)中，所述羟乙基黄连素与硼氢化钠的物质的量之比为1:1.5～3。
3.根据权利要求1所述的二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸靶向抗癌纳米药物的合成方法，其特征在于，步骤(4)中，有机溶剂为吡啶、二氯甲烷、二甲基亚砜、四氢呋喃中任意一种或几种；脱水剂为N,N-二环己基碳二亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐中任意一种或几种；催化剂为4-二甲氨基吡啶；所述二茂铁甲酸、还原的羟乙基黄连素、脱水剂、催化剂的物质的量之比为1:1:2～3.5:0.2～0.8。
4.根据权利要求1所述的二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸靶向抗癌纳米药物的合成方法，其特征在于，步骤(5)中，所述二茂铁-黄连素偶联药物前体与碘的物质的量之比为1:1～2.5。
5.根据权利要求1所述的二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸靶向抗癌纳米药物的合成方法，其特征在于，步骤(6)中，所述有机溶剂为二甲基亚砜、四氢呋喃、甲醇、N,N-二甲基甲酰胺中任意一种或几种。
6.根据权利要求1所述的二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸靶向抗癌纳米药物的合成方法，其特征在于，步骤(6)中，所述二茂铁-黄连素偶联药物、吲哚美辛、葡萄糖氧化酶、透明质酸的质量之比为1～3:0.5～3:10:1～3。
7.根据权利要求1所述的二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸靶向抗癌纳米药物的合成方法，其特征在于，步骤(6)中，透析的时间为6～24h。
8.权利要求1-6中任一方法制备所得二茂铁-黄连素/吲哚美辛@葡萄糖氧化酶@透明质酸靶向抗癌纳米药物。
9.权利要求8所述纳米药物在制备抗癌药物中的应用。
10.根据权利要求9所述应用，其特征在于，所述药物能够诱导癌细胞凋亡，抑制癌细胞转移。
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