CN112439078B - 一种dna四面体与替莫唑胺的纳米复合物 - Google Patents
一种dna四面体与替莫唑胺的纳米复合物 Download PDFInfo
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Abstract
本发明提供了一种DNA四面体与替莫唑胺形成的纳米复合物，优选地，它还共价连接了核酸适配体AS1411和/或核酸适配体GS24。本发明还提供了前述复合物的制备方法，它是通过DNA四面体与替莫唑胺振荡孵育，离心后取上清，即得。本发明还提供了前述复合物在制备抗肿瘤药物特别是抗胶质瘤药物中的用途。本发明相比替莫唑胺，具有更高的药效，且能逆转肿瘤细胞对替莫唑胺的耐药性，具有良好的应用前景。
Description
技术领域
本发明属于抗肿瘤药物领域，尤其涉及一种DNA四面体与替莫唑胺的纳米复合物。
背景技术
DNA四面体(Tetrahedral DNA，TDN)，又称四面体框架核酸(TetrahedralFramework Nucleic Acid，tFNA)，是近年来新兴的纳米材料。DNA四面体是在特定反应条件下，让四条特定序列的DNA单链根据碱基互补配对的原则自组装成具有四面体形状的三维纳米结构。因为tFNA具有良好的生物兼容性、可编辑性、低毒副性和机械刚性，使得它在生物检测、基因传递、活体成像和药物运输等领域有广泛的应用前景。
替莫唑胺(Temozolomide，TMZ)是目前临床上用于治疗恶性胶质母细胞瘤(Glioblastoma，GBM)的一线靶向化疗药。虽然TMZ能有效透过血脑屏障并靶向杀伤胶质瘤细胞，但是长期使用TMZ容易产生骨髓抑制，且有接近50％的患者在使用TMZ会快速产生耐药。目前有几项研究正尝试运用不同的纳米载体运载TMZ治疗GBM，但是暂未发现有效的手段能克服替莫唑胺的获得性耐药。
核酸适配体是一类具有特异性结合目标物质的DNA、RNA或核酸类似物。核酸适配体AS1411是可以与核仁素特异性结合的DNA单链；核酸适配体GS24是可以与转铁蛋白受体特异性结合的DNA单链。
发明内容
本发明的目的在于提供一种新的抗肿瘤纳米复合物，可以克服GBM耐药的问题。
本发明的技术方案包括：
一种抗肿瘤复合物，它是DNA四面体与替莫唑胺形成的纳米复合物。
优选地，如前述的复合物，所述DNA四面体共价连接了核酸适配体AS1411和/或核酸适配体GS24；
进一步优选地：DNA四面体与核酸适配体AS1411的摩尔比是1∶1；和/或，DNA四面体与核酸适配体GS24的摩尔比是1∶1。
如前述的复合物，所述DNA四面体共价连接有荧光标记基团，优选地，所述荧光标记基团是Cy5。
前述复合物的制备方法，它是通过DNA四面体与替莫唑胺振荡孵育，离心后取上清制备得到。
如前述的制备方法，其特征在于，包括如下步骤：
1)将替莫唑胺与DNA四面体混合后在100-1000rpm的振荡速率下孵育3-9小时；
2)将混合液离心后，取上清即得。
如前述的制备方法，步骤1)的振荡速率是600-700rpm。
如前述的制备方法，步骤1)的孵育时间是6小时。
如前述的制备方法，步骤2)的离心转速是6000rpm。
如前述的制备方法，所述DNA四面体共价连接了核酸适配体AS1411和/或核酸适配体GS24。
如前述的制备方法，所述DNA四面体还共价连接有荧光标记基团，优选地，所述荧光标记基团是Cy5。
DNA四面体与替莫唑胺的纳米复合物在制备抗肿瘤药物中的用途。
如前述的用途，其特征在于，所述DNA四面体共价连接了核酸适配体AS1411和/或核酸适配体GS24。
如前述的用途，所述DNA四面体共价连接有荧光标记基团，优选地，所述荧光标记基团是Cy5。
如前述的用途，所述肿瘤为胶质瘤。
本发明克服了如下技术障碍：
众所周知，DNA四面体中，DNA的单链之间是通过严格的“碱基互补”的原则进行配对的。因此，任何单链之间，如果序列之间的碱基对的数量越大，搭载的适配体越多，单链之间的相互干扰就越大，能够完全互补配对的难度就越大。从既往的文献中看，当试图搭载超过1种适配体的情况下，tFNA产生的副产物就越多，甚至因为碱基对之间的相互干扰，难以将适配体搭载到tFNA上。
本发明具有如下有益效果：
1)发明中所采用tFNA作为TMZ的运载体，有效提高TMZ的入胞量和入核能力；在tFNA上修饰了AS1411适配体后，AS1411可进一步帮助tFNA靶向胶质瘤细胞，并增加TMZ进入胶质瘤细胞核的量；在tFNA上修饰了GS24适配体后，后者能够特异性识别脑微血管内皮细胞上的转铁蛋白受体，帮助tFNA透过血脑屏障，解决大部分药物无法穿透血脑屏障这一难题。本发明的两种核酸适配体相互配合，提高了药物进入作用靶点的效率。间接地降低了药物使用量，能在一定程度上降低耐药性和其它副作用。
2)MGMT与替莫唑胺的耐药相关，大量文献(例如：Temozolomide resistance inglioblastoma multiforme.Genes Dis.2016 May 11；3(3)：198-210.)已证实MGMT表达升高导致了胶质瘤细胞对替莫唑胺的耐药。本发明的纳米复合物中，tFNA载体、载体上的适配体以及替莫唑胺功能上彼此支持，相互促进，可以下调MGMT的表达，有效降低耐药性。进一步地，替莫唑胺能激活自噬和凋亡通路进而增强疗效。
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是本发明复合物的合成示意图。
图2是tFNA透射电镜检测图。
图3是TMZ定量标准曲线。
图4是本发明复合物的吸收光谱图。
图5是tFNA与替莫唑胺复合物的透射电镜检测图。
图6是CCK8细胞活性检测结果统计图。
图7是替莫唑胺敏感型细胞A172细胞中基因表达检测图；a，Western blot检测图，图中C列表示对照组，T列表示tFNA组，TT列表示tFNA与替莫唑胺复合物组；b，qPCR检测图；c，免疫荧光检测图。
图8是替莫唑胺耐药型细胞T98G细胞中基因表达检测图；a，Western blot检测图，图中C列表示对照组，T列表示tFNA组，TT列表示tFNA与替莫唑胺复合物组；b，qPCR检测图；c，免疫荧光检测图。
具体实施方式
实施例1本发明的复合物的制备方法
将带有Cy5荧光标记基团(仅负责标记)、核酸适配体AS1411和核酸适配体GS24的DNA单链组成四面体，与TMZ共孵育后得到本发明的复合物，过程如图1所示。
1 DNA四面体的合成
将四条特殊的DNA单链以相同的摩尔浓度加入TM buffer(含10mM Tris-Hcl，50mMMgCl2，PH＝8)中。将溶液涡旋振荡混匀，离心后置于热循环仪中。在95℃下反应10分钟，然后迅速冷却至4℃反应20分钟，tFNA合成完毕。tFNA溶液置于4℃冰箱保存。
涉及的四条单链的序列如表1所示，表1序列与说明书序列表的对应关系是：S1和Cy5-S1对应SEQ ID NO.1，AS1411-S2对应SEQ ID NO.2，GS24-S3对应SEQ ID NO.3，S4对应SEQ ID NO.4。
表1 DNA四面体单链序列
AS1411-S2是核酸适配体AS1411共价地连接于DNA单链S2上，GS24-S3是核酸适配体GS24共价地连接于DNA单链S3上。
AS1411序列：GGTGGTGGTGGTTGTGGTGGTGGTGGT(SEQ ID NO.5)；
GS24序列：
GAATTCCGCGTGTGCACACGGTCACAGTTAGTATCGCTACGTTCTTTGGTAGTCCGTTCGGGA(SEQID NO.6)。
四面体合成后，对其进行透射电镜(TEM)检测，结果如图2所示。
2TMZ的制备和标准曲线.
先将TMZ溶于二甲基亚砜(DMSO)中配制成浓度为1mg/ml的储存液。用超纯水稀释TMZ制成浓度分别为20μg/ml、40μg/ml、50μg/ml和80μg/ml的工作液。分别取上述溶液10μl，在330nm波长处用超微量紫外分光光度计测量(UV5Nano)吸光度。制定出TMZ的标准曲线(图3)。
3 tFNA与TMZ结合
取不同浓度的TMZ溶液与tFNA混合，分三组制备本发明的复合物：
①取TMZ储存液稀释成40μg/ml，与250nM的tFNA(TMZ和tFNA的体积比为4：25)混合，以600rpm的振动速率在室温下孵育6h，随后以6000rpm将混合液离心10min，去除未结合的TMZ。取10ul混合液在紫外分光光度计中测量波长及吸光度。剩余混合液保存于4℃冰箱；
②取TMZ储存液稀释成50μg/ml，与250nM的tFNA(TMZ和tFNA的体积比为1∶5)混合，以600rpm的振动速率在室温下孵育6h，随后以6000rpm将混合液离心10min，去除未结合的TMZ。取10ul混合液在紫外分光光度计中测量波长及吸光度。剩余混合液保存于4℃冰箱；
③取TMZ储存液稀释成80μg/ml，与250nM的tFNA(TMZ和tFNA的体积比为8：25)混合，以700rpm的振动速率在室温下孵育6h，随后以6000rpm将混合液离心10min，去除未结合的TMZ。取10ul混合液在紫外分光光度计中测量波长及吸光度。剩余混合液保存于4℃冰箱。
三个组别的吸收峰图如图4所示，可见随着TMZ用量提高，复合物在330nm处吸收值提高。
合成的tFNA与TMZ复合物(tFNA-TMZ)的透射电镜检测图如图5所示。
4载药率计算
根据图4中混合液的在波长为330nm处的吸光度对应标准曲线中的浓度，计算出载药率(表2)。
载药率＝原始的TMZ浓度/搭载的TMZ浓度×100％。
表2 tFNA搭载TMZ的效率
实施例2本发明的复合物的制备方法
1 DNA四面体的合成
同实施例1的1.1节。
2 TMZ的制备和标准曲线
同实施例1的1.2节。
3 DNA四面体与TMZ结合
取TMZ储存液稀释成40μg/ml，与250nM的tFNA(TMZ和tFNA的体积比为4∶25)混合，以100rpm的振动速率在室温下孵育3h，随后以6000rpm将混合液离心10min，去除未结合的TMZ，即得。
实施例3本发明的复合物的制备方法
1 DNA四面体的合成
同实施例1的1.1节。
2 TMZ的制备和标准曲线
同实施例1的1.2节。
3 DNA四面体与TMZ结合
取TMZ储存液稀释成60μg/ml，与250nM的tFNA(TMZ和tFNA的体积比为4∶25)混合，以1000rpm的振动速率在室温下孵育9h，随后以6000rpm将混合液离心10min，去除未结合的TMZ，即得。
实施例4本发明复合物的制备方法
1 DNA四面体的合成
将GS24-S3改成S3序列，其余方法同实施例1的1.1节。
S3序列为SEQ ID NO.3除去SEQ ID NO.6以外的部分。
2 TMZ的制备和标准曲线
同实施例1的1.2节。
3 DNA四面体与TMZ结合
取TMZ储存液稀释成60μg/ml，与250nM的tFNA(TMZ和tFNA的体积比为4：25)混合，以1000rpm的振动速率在室温下孵育9h，随后以6000rpm将混合液离心10min，去除未结合的TMZ，即得。
实施例5本发明复合物的制备方法
1 DNA四面体的合成
将GS24-S3改成S3序列，将AS1411-S2改为S2序列，其余方法同实施例1的1.1节。
S3序列为SEQ ID NO.3除去SEQ ID NO.6以外的部分；S2序列是SEQ ID NO.2除去SEQ ID NO.5以外的部分。
2 TMZ的制备和标准曲线
同实施例1的1.2节。
3 DNA四面体与TMZ结合
取TMZ储存液稀释成60μg/ml，与250nM的tFNA(TMZ和tFNA的体积比为4∶25)混合，以1000rpm的振动速率在室温下孵育9h，随后以6000rpm将混合液离心10min，去除未结合的TMZ，即得。
以下将以实验例的形式对本发明有益效果进一步说明。
实验例1细胞实验
1.方法
将相同浓度(浓度均为34.77μg/ml)的TMZ与tFNA-TMZ分别作用于对TMZ敏感的胶质瘤细胞(A172和U87)以及对TMZ耐药的胶质瘤细胞(T98G和LN-18)。
采用CCK8方法，分别与药物作用后24h、36h、48h和72h检测细胞活力。
再使用Western blot、qPCR和免疫荧光检测凋亡相关基因(Casapase-3和Bax)、替莫唑胺耐药性相关基因(MGMT)、自噬相关基因(ATG7、LC3-B和Beclin-1)在A172和T98细胞中的表达。
2.结果
如图6所示，无论是对TMZ敏感的胶质瘤细胞还是对TMZ耐药的胶质瘤细胞，tFNA-TMZ的杀伤力均显著高于TMZ，结果具有统计学意义。
如图7(对应A127细胞)、8(对应T98G细胞)所示，凋亡相关基因(Casapase-3和Bax)和自噬相关基因(ATG7、LC3-B和Beclin-1)在tFNA-TMZ组的表达量均高于TMZ组和对照组。MGMT基因的表达量从高到低则依次是对照组、TMZ组和tFNA-TMZ组。
MGMT基因是导致替莫唑胺治疗耐药性(Temozolomide Resistance)的关键基因，在TMZ耐药胶质瘤细胞T98G中，本发明的tFNA-TMZ可显著降低MGMT的表达(图8)，证实了其具有增强替莫唑胺治疗敏感性的作用。而凋亡相关基因和自噬相关基因在tFNA-TMZ组表达量比单独使用TMZ更高，表明本发明的复合物可以通过增强凋亡和自噬两条通路，达到更好的肿瘤杀伤效果。
综上，本发明的DNA四面体与TMZ的纳米复合物，可以明显减弱TMZ耐药性；相比等浓度的TMZ，本发明的纳米复合物可以更有效地杀伤胶质瘤细胞。
SEQUENCE LISTING
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Claims (14)
1.一种抗肿瘤复合物，其特征在于，它是DNA四面体与替莫唑胺形成的纳米复合物；所述DNA四面体共价连接了核酸适配体AS1411和核酸适配体GS24；DNA四面体与核酸适配体AS1411的摩尔比是1:1；DNA四面体与核酸适配体GS24的摩尔比是1:1；所述DNA四面体的四条单链的序列如SEQ ID NO.1-4所示。
2.如权利要求1所述的复合物，其特征在于，所述DNA四面体共价连接有荧光标记基团。
3.如权利要求2所述的复合物，其特征在于，所述荧光标记基团是Cy5。
4.权利要求1~3任意一项所述的复合物的制备方法，其特征在于，它是通过DNA四面体与替莫唑胺振荡孵育，离心后取上清，即得；所述DNA四面体共价连接了核酸适配体AS1411和核酸适配体GS24。
5.如权利要求4所述的制备方法，其特征在于，包括如下步骤：
1）将替莫唑胺与DNA四面体混合后在100-1000rpm的振荡速率下孵育3-9小时；
2）将混合液离心后，取上清即得。
6.如权利要求5所述的制备方法，其特征在于，步骤1）的振荡速率是600-700rpm。
7.如权利要求5所述的制备方法，其特征在于，步骤1）的孵育时间是6小时。
8.如权利要求5所述的制备方法，其特征在于，步骤2）的离心转速是6000rpm。
9.如权利要求5所述的制备方法，其特征在于，所述DNA四面体还共价连接有荧光标记基团。
10.如权利要求9所述的制备方法，其特征在于，所述荧光标记基团是Cy5。
11.权利要求1-3任一项所述的复合物在制备抗肿瘤药物中的用途，其特征在于，所述DNA四面体共价连接了核酸适配体AS1411和核酸适配体GS24。
12.如权利要求11所述的用途，其特征在于，所述DNA四面体共价连接有荧光标记基团。
13.如权利要求12所述的用途，其特征在于，所述荧光标记基团是Cy5。
14.如权利要求11-13任一所述的用途，其特征在于，所述肿瘤为胶质瘤。
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