Llovera Laia, Berthold Peter, Nielsen Peter E, Shiraishi Takehiko
Department of Cellular and Molecular Medicine, Faculty of Health Sciences, The Panum Institute, University of Copenhagen, Copenhagen, Denmark.
Artif DNA PNA XNA. 2012 Jan-Mar;3(1):22-7. doi: 10.4161/adna.19906.
Efficient intracellular delivery is essential for high activity of nucleic acids based therapeutics, including antisense agents. Several strategies have been developed and practically all rely on auxiliary transfection reagents such as cationic lipids, cationic polymers and cell penetrating peptides as complexing agents and carriers of the nucleic acids. However, uptake mechanisms remain rather poorly understood, and protocols always require optimization of transfection parameters. Considering that cationic transfection complexes bind to and thus may up-concentrate on the cell surface, we have now quantitatively compared the cellular activity (in the pLuc705 HeLa cell splice correction system) of PNA antisense oligomers using lipoplex delivery of cholesterol- and bisphosphonate-PNA conjugates, polyplex delivery via a PNA-polyethyleneimine conjugate and CPP delivery via a PNA-octaarginine conjugate upon varying the cell culture transfection volume (and cell density) at fixed PNA concentration. The results show that for all delivery modalities the cellular antisense activity increases (less than proportionally) with increasing volume (in some cases accompanied with increased toxicity), and that this effect is more pronounced at higher cell densities. These results emphasize that transfection efficacy using cationic carriers is critically dependent on parameters such as transfection volume and cell density, and that these must be taken into account when comparing different delivery regimes.
高效的细胞内递送对于基于核酸的治疗药物（包括反义药物）的高活性至关重要。已经开发了几种策略，并且实际上所有策略都依赖于辅助转染试剂，如阳离子脂质、阳离子聚合物和细胞穿透肽，作为核酸的络合剂和载体。然而，摄取机制仍知之甚少，并且方案总是需要优化转染参数。考虑到阳离子转染复合物会结合并因此可能在细胞表面上浓缩，我们现在在固定PNA浓度下改变细胞培养转染体积（和细胞密度）时，定量比较了使用胆固醇和双膦酸盐-PNA缀合物的脂质体递送、通过PNA-聚乙烯亚胺缀合物的多聚体递送以及通过PNA-八聚精氨酸缀合物的细胞穿透肽递送的PNA反义寡聚体在pLuc705 HeLa细胞剪接校正系统中的细胞活性。结果表明，对于所有递送方式，细胞反义活性随着体积增加（小于成比例增加）而增加（在某些情况下伴随着毒性增加），并且这种效应在较高细胞密度下更明显。这些结果强调，使用阳离子载体的转染效率严重依赖于转染体积和细胞密度等参数，并且在比较不同递送方案时必须考虑这些参数。
