Lutz Gordon J, Sirsi Shashank R, Williams Jason H
Basic and Clinical Myology Laboratory Department of Physiology, The University of Melbourne, Victoria, Australia.
Methods Mol Biol. 2008;433:141-58. doi: 10.1007/978-1-59745-237-3_9.
Inefficient delivery of antisense oligonucleotides (AO) to target cell nuclei remains as the foremost limitation to their usefulness. Copolymers of cationic poly(ethylene imine) (PEI) and polyethylene glycol (PEG) are extremely well-studied compounds that markedly improve the in vitro and in vivo delivery of AOs to cells and tissues. By varying the Mw of PEI, as well as the nature of PEG shielding, PEG-PEI-AO nanoparticulates can be prepared with a dynamic range of size, surface charge, and stability. Each of these properties in-turn influences the transfection capacity of the PEG-PEI-AO polyplexes. In addition, PEG-PEI copolymers are readily functionalized for enhanced efficacy and specificity of cellular and tissue targeting. The synthesis and functionalization of PEG-PEI copolymers is remarkably simple and requires very little specialized equipment. Thus, PEG-PEI copolymers represent a tractable and adaptable oligonucleotide delivery system that can be customized and optimized to the investigators' specific application. This chapter describes the step-by-step synthesis of several PEG-PEI copolymers that are specifically formulated to provide effective delivery of AOs using both in vitro and in vivo applications. We describe the preparation of the PEG-PEI-AO polyplexes and provide examples showing transfection of cultured cells in vitro, as well as skeletal muscles in vivo using both local and systemic delivery.
反义寡核苷酸（AO）向靶细胞核的低效递送仍然是其应用的首要限制。阳离子聚（乙烯亚胺）（PEI）和聚乙二醇（PEG）的共聚物是经过深入研究的化合物，可显著改善AO在体外和体内向细胞和组织的递送。通过改变PEI的分子量以及PEG屏蔽的性质，可以制备具有不同尺寸范围、表面电荷和稳定性的PEG-PEI-AO纳米颗粒。这些性质中的每一个又反过来影响PEG-PEI-AO多聚体的转染能力。此外，PEG-PEI共聚物易于功能化，以提高细胞和组织靶向的功效和特异性。PEG-PEI共聚物的合成和功能化非常简单，所需的专业设备很少。因此，PEG-PEI共聚物代表了一种易于处理和适应性强的寡核苷酸递送系统，可以根据研究者的具体应用进行定制和优化。本章描述了几种PEG-PEI共聚物的逐步合成方法，这些共聚物经过专门配制，可通过体外和体内应用有效地递送AO。我们描述了PEG-PEI-AO多聚体的制备，并提供了在体外培养细胞以及通过局部和全身递送在体内骨骼肌中转染的示例。
