娄筱叮、夏帆教授课题组Angew. Chem. Int. Ed.: 多功能性多肽类聚集诱导发光探针实现基因药物高效可控的细胞核靶向性运输和实时追踪

【引言】

基因干扰疗法（也称为基于寡核苷酸的疗法）可以在哺乳动物细胞中稳定地沉默或抑制序列特异性基因的表达。其在病原体、癌症和代谢疾病的治疗中获得了显著的成果。在基于寡核苷酸的疗法中，反义单链DNA寡核苷酸（ASO）疗法和RNA干扰（RNAi）在过去几十年中在胞质内和内核干扰方面取得了显著成果。然而，由于负电荷和核苷酸易于酶促降解以及生物体的多种生物屏障的限制，基于寡核苷酸的疗效受到严重阻碍。

为了解决基于寡核苷酸疗法的局限性，多种载体被进行了深入研究。其中，多肽连接的载体由于其有利的生物相容性和多功能组份而被认为是优良的基因递送体系之一。这些复合物通常通过静电相互作用，分子自组装或共价结合形成稳定的纳米颗粒。此外，通过利用各种合成肽，已经开发出用于亚细胞器的有效基因传送系统。Tseng及其同事使用具有多步修饰的自组装超分子系统，包含与DNA质粒复合的转录因子，细胞穿透肽，整合型肽，聚乙烯亚胺，聚乙二醇，聚酰胺胺树枝状大分子，能够将DNA质粒以精确和高效的方式传递到细胞核。此外，已经证实具有复杂和多功能肽的多结构域设计载体可以通过合理排列保留其成分的个体功能。Hilvert及其同事开发了一种简单有效的模块化系统，用于体内包裹RNA。然而为了实现高效和准确的治疗，寡核苷酸疗法应该将亚细胞器靶向、模块化设计和实时长期跟踪一起考虑。因此，可实现有效基因传递和实时长期追踪的多功能多肽连接的载体在治疗中很有需求。

【成果简介】

近日，中国地质大学（武汉）娄筱叮、夏帆教授课题组报导了一种多功能的基因传递策略，可以以高效和可视化的方式将治疗基因传递到靶向的细胞核中。作者开发了一种整合素靶向、细胞渗透性和核质体运输肽连接的AIEgen（TDNCP），用于有效和顺序靶向传递反义单链DNA寡核苷酸（ASO），并进行实时追踪。与TDNCP/siRNA-NPs相比，TDNCP/ASO-NPs表现出更好地干扰效应，进一步证明TDNCP是细胞核靶向载体。此外，TDNCP/ASO-NPs在体内显示出良好的肿瘤抑制作用，证明多功能肽连接的AIEgen可用于顺序靶向基因传递，并实现有效的基因干扰治疗。该成果以题为”A Multifunctional Peptide-Conjugated AIEgen for Efficient and Sequential Targeted Gene Delivery into the Nucleus”发表在Angew. Chem. Int. Ed.上。

华中科技大学博士后程勇和孙春丽以及中国地质大学（武汉）博士后刘瑞为论文的共同第一作者，中国地质大学（武汉）娄筱叮教授和夏帆教授为论文的共同通讯作者。该研究工作得到了国家重点研发计划（2017YFA0208000, 2016YFF0100800）国家自然科学基金（21525523, 21722507, 21574048, 21605053）中国博士后科学基金（2017M620309, 2017M610492）等项目的资助。 

【图文导读】

Scheme 1.TNCP和TNCP/ASO自组装的纳米粒子的示意图

(a).TNCP分子结构

(b).TNCP/ASO-NPs用于实时追踪和靶向传递

Figure 1.TNCP及其衍生物的表征

(a).多肽序列

(b).高效液相色谱表征

(c,e).DGRKRRRR和RGDKRRRR对整合素的分子结合能力与氢键作用

(d,f).不同探针与整合素结合的荧光变化

(g,h).用TNCP处理细胞后的细胞荧光强度

Figure 2.细胞实验

(a-c).TNCP的ASO负载能力

(d).细胞成像

(e).细胞的平均荧光强度和Pearson相关系数

(f).western blot分析

(g).Bio-TEM图像

图3.动物实验

(a).小鼠体重变化

(b).小鼠肿瘤体积变化

(c).Bcl-2基因干扰效率

【小结】

在这个工作中，作者利用多功能肽和AIEgen来实现有效和顺序靶向递送以及跟踪细胞核。通过整合素靶向肽、细胞渗透性肽和核定位序列的组合，TDNCP比其他衍生物显示出更优异的生物相容性、核选择性成像和光稳定性。与TDNCP/siRNA-NPs，lipo/ASONPs和lipo/siRNA-NPs相比，TDNCP/ASO-NPs在整合素过表达的细胞系中显示出优异的基因干扰效应。由于顺序和靶向细胞穿透，TDNCP/ASO-NPs通过瘤内注射显示出比体内静脉注射更好的干扰效果。

A Multifunctional Peptide-Conjugated AIEgen for Efficient and Sequential Targeted Gene Delivery into the Nucleus

(Angew. Chem. Int. Ed., 2019, DOI: 10.1002/anie.201901527)

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