Department of Biomedical Engineering, Rutgers University, Piscataway, NJ 08854, USA.
J Control Release. 2009 Dec 3;140(2):134-40. doi: 10.1016/j.jconrel.2009.08.008. Epub 2009 Aug 20.
Antisense technology holds tremendous potential in the research and clinical settings. However, successful delivery of antisense oligodeoxynucleotides (ODNs) to the intracellular site of action requires the passage of many barriers, including survival against extracellular serum nucleases and escape from endolysosomal degradation. Previous work has shown that the effectiveness of antisense delivery by the cationic liposome, dioleoyl-3-trimethylammonium-propane (DOTAP), is enhanced substantially by the incorporation of a pH-sensitive polymer, poly (propylacrylic acid) (PPAA), in serum-free media. To improve this system for application in serum-containing media conditions, PPAA was modified in this work by grafting onto it either poly(ethylene oxide) (PEO) or a more hydrophobic analog, poly (oxyalkylene amine), known as Jeffamine. The ternary formulation of DOTAP/ODN/PPAA-g-Jeffamine resulted in 8-fold increased uptake of fluorescently-labeled ODNs compared to DOTAP/ODN/PPAA and ~80% silencing of green fluorescent protein (GFP) expression in CHO-d1EGFP cells treated in the presence of 10% FBS-containing media. In contrast, the carrier systems that contained PPAA or PPAA-g-PEO failed to display any significant antisense activity in the presence of serum, even though all of the delivery systems displayed moderate to high levels of antisense activity in serum-free conditions. The results reveal that the carrier system with the Jeffamine graft copolymer effectively mediates specific gene silencing in the presence of serum, while the system with the PEO graft copolymer fails to do so. While the pH-dependent lytic functionality of PPAA was found to be lost upon grafting with PEO or Jeffamine, the hydrophobicity of the latter was sufficient to mediate cellular internalization and endosomal escape. Thus, the PPAA-g-Jeffamine copolymers hold substantial promise as agents for controlled therapeutic delivery of antisense oligonucleotides.
反义技术在研究和临床环境中具有巨大的潜力。然而，将反义寡核苷酸（ODN）递送到细胞内作用部位需要通过许多屏障，包括抵抗细胞外血清核酶和逃避内溶酶体降解。以前的工作表明，在无血清介质中，通过阳离子脂质体二油酰基-3-三甲基铵丙烷（DOTAP）掺入 pH 敏感聚合物聚丙基丙烯酸（PPAA），可以大大提高反义递药的效果。为了改进该系统在含血清介质条件下的应用，本工作通过接枝聚氧乙烯（PEO）或更疏水的类似物聚氧烷基亚胺（Jeffamine）对 PPAA 进行了修饰。DOTAP/ODN/PPAA-g-Jeffamine 的三元配方导致与 DOTAP/ODN/PPAA 相比，荧光标记的 ODN 的摄取增加了 8 倍，并且在用 10%含 FBS 的培养基处理的 CHO-d1EGFP 细胞中 GFP 表达的沉默率约为 80%。相比之下，即使在无血清条件下，所有递药系统都显示出中等至高的反义活性，含血清时含有 PPAA 或 PPAA-g-PEO 的载体系统均未能显示出任何明显的反义活性。结果表明，含有 Jeffamine 接枝共聚物的载体系统在含血清的情况下能够有效地介导特定的基因沉默，而含有 PEO 接枝共聚物的系统则不能。虽然发现与 PEO 或 Jeffamine 接枝后 PPAA 的 pH 依赖性溶酶体功能丧失，但后者的疏水性足以介导细胞内化和内体逃逸。因此，PPAA-g-Jeffamine 共聚物有望成为控制反义寡核苷酸治疗性递药的有效试剂。
