Cell number and transfection volume dependent peptide nucleic acid antisense activity by cationic delivery methods

TY - JOUR

T1 - Cell number and transfection volume dependent peptide nucleic acid antisense activity by cationic delivery methods

AU - Llovera Nadal, Laia

AU - Berthold, Peter

AU - Nielsen, Peter E

AU - Shiraishi, Takehiko

PY - 2012/1

Y1 - 2012/1

N2 - 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.

AB - 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.

U2 - 10.4161/adna.19906

DO - 10.4161/adna.19906

M3 - Journal article

C2 - 22679530

SN - 1949-095X

VL - 3

SP - 22

EP - 30

JO - Artificial DNA: PNA and XNA

JF - Artificial DNA: PNA and XNA

IS - 1

ER -