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

Llovera L, Berthold P, Nielsen PE, Shiraishi T - Artif DNA PNA XNA (2012 Jan-Mar)

Bottom Line: However, uptake mechanisms remain rather poorly understood, and protocols always require optimization of transfection parameters.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Faculty of Health Sciences, The Panum Institute, University of Copenhagen, Copenhagen, Denmark.

ABSTRACT
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.

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Figure 1. Relative cellular luciferase antisense activity in HeLa pLuc 705 cells of PNA conjugated to octa-arginine [(D-Arg)8-PNA]. Different cell numbers were plated in a 96-well plate the day before transfection. Cells were treated with different volumes (50 µl/well for a relative volume 1) of the transfection solution for 24 h: 4 µM PNA and 120 µM CQ. Cell samples were then subjected to luciferase analysis and cellular viability test. All tests were performed in duplicate and the results are given as average values ± standard deviations (SD). Luciferase activity was analyzed using Bright-Glo reagent (Promega), normalized to cell viability (Figs. S1–5) and given as relative light units (RLU/cell).
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Figure 1: Figure 1. Relative cellular luciferase antisense activity in HeLa pLuc 705 cells of PNA conjugated to octa-arginine [(D-Arg)8-PNA]. Different cell numbers were plated in a 96-well plate the day before transfection. Cells were treated with different volumes (50 µl/well for a relative volume 1) of the transfection solution for 24 h: 4 µM PNA and 120 µM CQ. Cell samples were then subjected to luciferase analysis and cellular viability test. All tests were performed in duplicate and the results are given as average values ± standard deviations (SD). Luciferase activity was analyzed using Bright-Glo reagent (Promega), normalized to cell viability (Figs. S1–5) and given as relative light units (RLU/cell).

Mentions: In view of the previous evidence of the up-concentration of cell-penetrating peptides on the cell surface,17 our first choice for studying the volume effect of transfection solutions was the CPP-PNA conjugate. First we examined the effect of the CPP-PNA modification on HeLa pLuc705 cells at 4 µM PNA and 8 × 103 cells/well (in order to have 50% confluency at the transfection time, which is the typical cell confluency for ordinary transfection). Under these conditions hardly any volume effect was observed (Fig. 1). This lack of effect could be due to saturation of the cells at this PNA concentration, and we therefore decided to increase the cell number range in steps from 4 to 32 × 103 cells/well in order to obtain lower PNA-to-cell ratios. By using 24 or 32 × 103 cells/well we observed around 2-fold efficacy increase at six relative volumes (Fig. 1), but cell viability was also reduced comparably (Fig. S1). These results do confirm that transfection efficacy depends on PNA-to-cell ratio, in accordance with previous conclusions,16,17 and also indicate that saturation may be possible at lower cell numbers.


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

Llovera L, Berthold P, Nielsen PE, Shiraishi T - Artif DNA PNA XNA (2012 Jan-Mar)

Figure 1. Relative cellular luciferase antisense activity in HeLa pLuc 705 cells of PNA conjugated to octa-arginine [(D-Arg)8-PNA]. Different cell numbers were plated in a 96-well plate the day before transfection. Cells were treated with different volumes (50 µl/well for a relative volume 1) of the transfection solution for 24 h: 4 µM PNA and 120 µM CQ. Cell samples were then subjected to luciferase analysis and cellular viability test. All tests were performed in duplicate and the results are given as average values ± standard deviations (SD). Luciferase activity was analyzed using Bright-Glo reagent (Promega), normalized to cell viability (Figs. S1–5) and given as relative light units (RLU/cell).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3368813&req=5

Figure 1: Figure 1. Relative cellular luciferase antisense activity in HeLa pLuc 705 cells of PNA conjugated to octa-arginine [(D-Arg)8-PNA]. Different cell numbers were plated in a 96-well plate the day before transfection. Cells were treated with different volumes (50 µl/well for a relative volume 1) of the transfection solution for 24 h: 4 µM PNA and 120 µM CQ. Cell samples were then subjected to luciferase analysis and cellular viability test. All tests were performed in duplicate and the results are given as average values ± standard deviations (SD). Luciferase activity was analyzed using Bright-Glo reagent (Promega), normalized to cell viability (Figs. S1–5) and given as relative light units (RLU/cell).
Mentions: In view of the previous evidence of the up-concentration of cell-penetrating peptides on the cell surface,17 our first choice for studying the volume effect of transfection solutions was the CPP-PNA conjugate. First we examined the effect of the CPP-PNA modification on HeLa pLuc705 cells at 4 µM PNA and 8 × 103 cells/well (in order to have 50% confluency at the transfection time, which is the typical cell confluency for ordinary transfection). Under these conditions hardly any volume effect was observed (Fig. 1). This lack of effect could be due to saturation of the cells at this PNA concentration, and we therefore decided to increase the cell number range in steps from 4 to 32 × 103 cells/well in order to obtain lower PNA-to-cell ratios. By using 24 or 32 × 103 cells/well we observed around 2-fold efficacy increase at six relative volumes (Fig. 1), but cell viability was also reduced comparably (Fig. S1). These results do confirm that transfection efficacy depends on PNA-to-cell ratio, in accordance with previous conclusions,16,17 and also indicate that saturation may be possible at lower cell numbers.

Bottom Line: However, uptake mechanisms remain rather poorly understood, and protocols always require optimization of transfection parameters.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Faculty of Health Sciences, The Panum Institute, University of Copenhagen, Copenhagen, Denmark.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus