CN113082226B - 一种载药纳米笼的制备方法与其靶向循环肿瘤细胞释放雷公藤甲素的应用 - Google Patents
一种载药纳米笼的制备方法与其靶向循环肿瘤细胞释放雷公藤甲素的应用 Download PDFInfo
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Abstract
本发明公开了一种介孔结构载药纳米笼的制备方法，具体包括下列步骤：制备氨基介孔硅纳米笼，将功能化的DNA序列、氨基介孔硅纳米笼和药物雷公藤甲素在分散液中持续搅拌，经沉淀、离心清洗两次后，实现在氨基介孔硅纳米笼的介孔封装嵌插抗癌药物雷公藤甲素，即可得到载药纳米笼，同时提供了其靶向循环肿瘤细胞释放雷公藤甲素的应用。本发明属于医学技术领域，具体指一种载药纳米笼的制备方法与其靶向循环肿瘤细胞释放雷公藤甲素的应用。
Description
技术领域
本发明属于医学技术领域，具体为一种载药纳米笼的制备方法与其靶向循环肿瘤细胞释放雷公藤甲素的应用。
背景技术
还原型谷胱甘肽(GSH)是一种由谷氨酸、半胱氨酸及甘氨酸缩合而成的含有γ－酰胺键和巯基的三肽，也是细胞里被发现的主要非蛋白巯基化合物，它是细胞里含量最丰富的抗氧化因子。还原型谷胱甘肽(GSH)在细胞代谢、细胞增殖、细胞内环境稳定以及抗病性上起着重要作用。谷胱甘肽存在于二硫化合物(GSSG氧化型的)与巯基化合物(GSH还原型的)之前的氧化还原平衡中，活细胞内的还原型谷胱甘肽的水平显著的改变了相应的氧化应激，这种氧化应激已经在很多疾病和加速老化过程中有涉及。在氧化应激时，还原型谷胱甘肽可以被转化成氧化型(GSSG)去保护细胞免受氧化应激和协助捕获那种伤害RNA或DNA的自由基。作为一种敏感的指示剂，细胞内GSH和GSSG的最佳比例的任何变化能够导致人类的疾病比如：癌症、心脏病、中风和许多神经***疾病。尤其是在一些癌细胞内，细胞质内还原型谷胱甘肽的浓度与正常细胞相比普遍更高。因此，对体内谷胱甘肽的检测在疾病诊断中起着非常重要的作用。
目前，荧光检测、电化学、高效液相色谱法、化学发光法和分光光度法被广泛的用来研究GSH。然而，这些方法存在衍生化作用、复杂的实验程序、耗时、体内应用受限，从而不利于实际操作的发展，并且难以实现有效地靶向给药。石英晶体微天平(QCM)因其对质量变化的高敏感性，传感器具有特异性好、灵敏度高、成本低廉和操作简单的优势，越来越受到了科学界的关注，在疾病分析检测领域中具有很好的应用潜力。此外，近些年的研究中，作为纳米载体的介孔硅纳米笼引起广泛关注，该纳米笼表面可以进行化学修饰、较高的比表面积、特征纳米结构、生物相容性及高负载功能。作为一种纳米级别的分子，DNA以其自身的可编程性、结构的多样性和变化的可控性等诸多优点而活跃于纳米科学、生物科学和材料科学。
因此，有必要利用碱基的精确配对和序列可设计的特性，设计一种DNA功能化的纳米笼，以实现对循环肿瘤细胞的高特异性和高灵敏性的检测，以及向循环肿瘤细胞内靶向还原型谷胱甘肽释放抗癌药物。
发明内容
针对上述情况，为弥补上述现有缺陷，本发明基于一种DNA功能化的纳米笼，并以此纳米笼为抗癌药物载体，制备了一种介孔结构载药纳米笼；并基于修饰循环肿瘤细胞适体的芯片，通过石英晶体微天平技术实现了对循环肿瘤细胞的高特异性和高灵敏性的检测，同时细胞内靶向还原型谷胱甘肽释放抗癌药物，减少对正常细胞不必要的损伤，本发明提供的介孔结构载药纳米笼制备方法简单，对循环肿瘤细胞的检测高灵敏高，具有靶向释放药物的性能，适用于各种人体内的循环肿瘤细胞的快速检测和靶向释放药物。
本发明提供如下的技术方案：
一种载药纳米笼的制备方法，具体包括下列步骤：制备氨基介孔硅纳米笼，将功能化的DNA序列、氨基介孔硅纳米笼和药物雷公藤甲素在分散液中持续搅拌，经沉淀、离心清洗两次后，实现在氨基介孔硅纳米笼的介孔封装嵌插抗癌药物雷公藤甲素，即可得到载药纳米笼。
进一步地，所述功能化的DNA序列为三种DNA序列发生链式杂交反应的含双硫键的链式杂交产物。
进一步地，三种所述DNA序列分别为序列一、序列二和序列三，所述序列一为：5’-ATC AGA CTG ATG TTG A CAA AGT-/HS-SH/-T CAA CAT CAG TCT-(BHQ)-GAT AAG CTA-3’；所述序列二为：5’-ACT TTG TCA ACA TCA GTC TGA T TAG CT-/HS-SH/-T ATCA GAC TGATGT TGA-3’，所述序列三为：5’-COOH-TTT TTT TTT TTT TAG CTT ATC AGA CTG ATG TTGA-3’。
作为优选地，所述载药纳米笼为介孔结构载药纳米笼。
一种载药纳米笼靶向循环肿瘤细胞释放雷公藤甲素的应用，具体包括下述过程：
(1)以载药纳米笼为功能探针，石英晶体微天平芯片上修饰功能核酸，介孔结构载药纳米笼孵育进入循环肿瘤细胞，基于适体识别原理捕获细胞，石英晶体微天平芯片捕获循环肿瘤细胞，介孔结构载药纳米笼提高了石英晶体微天平检测循环肿瘤细胞的灵敏度，实现了对循环肿瘤细胞的高灵敏检测；
(2)将结合了循环肿瘤细胞的石英晶体微天平芯片加入切刻内切酶，基于硫醇交换反应，介孔结构载药纳米笼与被测物循环肿瘤细胞内的还原型谷胱甘肽反应，在循环肿瘤细胞内靶向性地释放抗癌药物雷公藤甲素。
采用上述结构本发明取得的有益效果如下：本发明一种载药纳米笼及其制备方法与其靶向循环肿瘤细胞释放雷公藤甲素的应用，与现有技术相比，本发明的主要创新性和优越性如下：
1.本发明采用“二步法”制备了介孔结构载药纳米笼，制备方法操作简单；
2.本发明以介孔结构载药纳米笼为探针，提高石英晶体微天平(QCM)检测循环肿瘤细胞的灵敏度，实现了对循环肿瘤细胞的高灵敏检测；
3.本发明以介孔结构载药纳米笼为载药探针，实现了高特异选择性的在循环肿瘤细胞释放抗癌药物，减少对正常细胞不必要的损伤。
附图说明
图1为本发明介孔结构载药纳米笼的扫描电镜图；
图2为本发明循环肿瘤细胞个数与石英晶体微天平(QCM)信号响应的曲线关系图；
图3为本发明循环肿瘤细胞个数与石英晶体微天平(QCM)频率强度的线性关系图；
图4为本发明介孔结构载药纳米笼(a)、正常细胞(b)和循环肿瘤细胞(c)的药物释放量的荧光强度对比图；
图5为本发明介孔结构载药纳米笼用于靶向循环肿瘤细胞释放药物的成像图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例；基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
1.介孔结构载药纳米笼的制备：
(1)将70克三乙醇胺、390克十六烷基三甲基苯磺酸、200mL超纯水、有机胺小分子溶液、2200g正硅酸乙酯混合80℃搅拌3小时；
(2)将步骤(1)的混合物经过滤、洗涤，95℃烘箱过夜烘烤24小时，合成得到介孔硅；
(3)将合成的介孔硅称取10克溶解于1000毫升的无水乙醇，加入3-氨丙基三乙氧基硅烷37℃搅拌6小时；
(4)将步骤(3)制备的产物经过滤、无水乙醇洗涤、60℃烘干得到氨基介孔硅纳米笼；
(5)DNA序列发生链式杂交，其杂交产物在分散液中与氨基介孔硅纳米笼(30mg/ml)、药物雷公藤甲素37℃下持续搅拌3小时，经沉淀、离心洗两次后，得到介孔结构载药纳米笼的悬浮液。
2.石英晶体微天平(QCM)检测循环肿瘤细胞：
QCM芯片上修饰适体，介孔结构载药纳米笼的悬浮液(20mg/ml)孵育进入循环肿瘤细胞MDA-MB-231，在37℃的QCM反应池中，基于适体识别原理QCM芯片捕获循环肿瘤细胞MDA-MB-231，进行QCM测试，实验结果如图2和图3所示。
如图2所示，随着循环肿瘤MDA-MB-231添加细胞个数的增加，QCM频率强度增加。
如图3所示，当循环肿瘤MDA-MB-231细胞个数在10到9000的范围内，QCM强度响应与循环肿瘤MDA-MB-231细胞个数呈线性关系，线性回归方程为ΔF＝137.18lgN–122.19，其中N为循环肿瘤细胞MDA-MB-231个数，ΔF＝F–F0，F是循环肿瘤细胞MDA-MB-231作用后的QCM频率强度，F0为循环肿瘤细胞作用前的频率强度，R＝0.992，实验结果表明循环肿瘤的个数与QCM频率强度呈现很好的线性关系。
3.释放药物的荧光强度对比：
通过荧光分光光度计检测技术，以介孔结构载药纳米笼自身释放雷公藤甲素的荧光强度作为参照样，配合比较介孔结构载药纳米笼(20mg/ml)在正常细胞和循环肿瘤细胞MDA-MB-231中释放雷公藤甲素的荧光强度，该介孔结构载药纳米笼，可在循环肿瘤细胞内靶向还原型谷胱甘肽，引发硫醇交换反应，实现在循环肿瘤细胞内的释放抗癌药物雷公藤甲素，实验结果如图4所示。
图4中的数据显示了介孔结构载药纳米笼(a)、介孔结构载药纳米笼在正常细胞(b)和介孔结构载药纳米笼在循环肿瘤细胞MDA-MB-231(c)的三个药物释放量的荧光强度对比，对比数据显示：该介孔结构载药纳米笼，可在循环肿瘤细胞内靶向还原型谷胱甘肽，引发硫醇交换反应，实现循环肿瘤细胞内的有效释放抗癌药物雷公藤甲素，对正常细胞释放药物量较少，减少了对正常细胞的损伤。
4.靶向循环肿瘤细胞释放药物：
在结合了循环肿瘤细胞MDA-MB-231的QCM芯片反应池加入3μL切刻内切酶，QCM芯片与液体分离得到产物，进行激光共聚焦显微镜成像测试，实验结果如图5所示。
如图5所示为介孔结构载药纳米笼靶向循环肿瘤细胞MDA-MB-231释放药物的荧光成像图，通过可视化成像技术显示了循环肿瘤细胞内释放了大量的抗癌药物雷公藤甲素。
要说明的是，在本文中，诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系例要素的过程、方法、物料或者设备不仅包括那些要素，而且还包括没有明确例出的其他要素，或者是还包括为这种过程、方法、物料或者设备所固有的要素。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。
SEQUENCE LISTING
<110> 山东中医药大学
<120> 一种载药纳米笼的制备方法与其靶向循环肿瘤细胞释放雷公藤甲素的应用
<130> 山东中医药大学
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Claims (2)
1.一种载药纳米笼的制备方法，其特征在于，具体包括下列步骤：制备氨基介孔硅纳米笼，将功能化的DNA序列、氨基介孔硅纳米笼和药物雷公藤甲素在分散液中持续搅拌，经沉淀、离心清洗两次后，实现在氨基介孔硅纳米笼的介孔封装嵌插抗癌药物雷公藤甲素，即可得到载药纳米笼；
所述功能化的DNA序列为三种DNA序列发生链式杂交反应的含双硫键的链式杂交产物；
三种所述DNA序列分别为序列一、序列二和序列三，所述序列一为：5’-ATC AGA CTGATG TTG A CAA AGT-/HS-SH/-T CAA CAT CAG TCT-(BHQ)-GAT AAG CTA-3’；所述序列二为：5’-ACT TTG TCA ACA TCA GTC TGA T TAG CT-/HS-SH/-T A TCA GAC TGA TGT TGA-3’，所述序列三为：5’-COOH-TTT TTT TTT TTT TAG CTT ATC AGA CTG ATG TTG A-3’。
2.根据权利要求1所述的一种载药纳米笼的制备方法，其特征在于，所述载药纳米笼为介孔结构载药纳米笼。
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