Mumcuoglu Didem, Sardan Melis, Tekinay Turgay, Guler Mustafa O, Tekinay Ayse B
†Institute of Materials Science and Nanotechnology, National Nanotechnology Research Center (UNAM), Bilkent University, 06800 Ankara, Turkey.
‡Department of Medical Biology and Genetics, Faculty of Medicine, Life Sciences Application and Research Center, Gazi University, 06500 Ankara, Turkey.
Mol Pharm. 2015 May 4;12(5):1584-91. doi: 10.1021/acs.molpharmaceut.5b00007. Epub 2015 Apr 9.
A drug delivery system designed specifically for oligonucleotide therapeutics can ameliorate the problems associated with the in vivo delivery of these molecules. The internalization of free oligonucleotides is challenging, and cytotoxicity is the main obstacle for current transfection vehicles. To develop nontoxic delivery vehicles for efficient transfection of oligonucleotides, we designed a self-assembling peptide amphiphile (PA) nanosphere delivery system decorated with cell penetrating peptides (CPPs) containing multiple arginine residues (R4 and R8), and a cell surface binding peptide (KRSR), and report the efficiency of this system in delivering G-3129, a Bcl-2 antisense oligonucleotide (AON). PA/AON (peptide amphiphile/antisense oligonucleotide) complexes were characterized with regards to their size and secondary structure, and their cellular internalization efficiencies were evaluated. The effect of the number of arginine residues on the cellular internalization was investigated by both flow cytometry and confocal imaging, and the results revealed that uptake efficiency improved as the number of arginines in the sequence increased. The combined effect of cell penetration and surface binding property on the cellular internalization and its uptake mechanism was also evaluated by mixing R8-PA and KRSR-PA. R8 and R8/KRSR decorated PAs were found to drastically increase the internalization of AONs compared to nonbioactive PA control. Overall, the KRSR-decorated self-assembled PA nanospheres were demonstrated to be noncytotoxic delivery vectors with high transfection rates and may serve as a promising delivery system for AONs.
一种专门为寡核苷酸治疗药物设计的给药系统可以改善与这些分子体内递送相关的问题。游离寡核苷酸的内化具有挑战性，细胞毒性是当前转染载体的主要障碍。为了开发用于高效转染寡核苷酸的无毒递送载体，我们设计了一种自组装肽两亲物（PA）纳米球递送系统，该系统装饰有含有多个精氨酸残基（R4和R8）的细胞穿透肽（CPP）和细胞表面结合肽（KRSR），并报告了该系统递送Bcl-2反义寡核苷酸（AON）G-3129的效率。对PA/AON（肽两亲物/反义寡核苷酸）复合物的大小和二级结构进行了表征，并评估了它们的细胞内化效率。通过流式细胞术和共聚焦成像研究了精氨酸残基数量对细胞内化的影响，结果表明，随着序列中精氨酸数量的增加，摄取效率提高。还通过混合R8-PA和KRSR-PA评估了细胞穿透和表面结合特性对细胞内化及其摄取机制的联合作用。与无生物活性的PA对照相比，发现R8和R8/KRSR修饰的PA能显著提高AON的内化。总体而言，KRSR修饰的自组装PA纳米球被证明是具有高转染率的无细胞毒性递送载体，可能成为一种有前景的AON递送系统。
