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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 2. Relative cellular luciferase antisense activity of phosphonate-conjugated PNA (bP6-PNA). HeLa pLuc705 cells were trypsinized and seeded (2.4 × 104 cells/well) 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 solutions for 24 h: 0.25 to 2nM PNA complexed with 6 µl/ml LFA2000. Cell samples were then subjected to luciferase analysis and cellular viability test. All tests were performed in triplicate 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 2: Figure 2. Relative cellular luciferase antisense activity of phosphonate-conjugated PNA (bP6-PNA). HeLa pLuc705 cells were trypsinized and seeded (2.4 × 104 cells/well) 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 solutions for 24 h: 0.25 to 2nM PNA complexed with 6 µl/ml LFA2000. Cell samples were then subjected to luciferase analysis and cellular viability test. All tests were performed in triplicate 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: Next we analyzed an anionic PNA conjugate which can form a complex with cationic lipids in analogy to negatively charged nucleic acids.3 A bis-phosphonate-PNA conjugate was transfected at different concentrations (0–2 nM) using varying volumes of the transfection solution/well in a 96 well plate seeded at 24 × 103 cells/well. Results showed that antisense activity increased in a PNA dose-dependent manner at all transfection volumes, and in parallel, increasing volume also showed a higher antisense activity up to four relative volumes where a saturation was reached. We also observe from these experiments that the volume-effect is more pronounced at higher PNA concentrations. Whereas antisense activity at 1 and 2 nM PNA doses showed an approximately 2-fold increase at 4 relative volumes, at 0.5 and 0.25 nM PNA doses it was only about 1.75- and 1.5-fold, respectively (Fig. 2). However, the difference in PNA-Lipofectamine ratio, resulting in decreasing zeta potential as the PNA concentration increases at fixed Lipofectamine concentration, may also play a role, although an opposite effect could be expected as lowering of the zeta potential would decrease transfection efficacy. Cell viability exhibited a slight decrease (< 30%) with increasing transfection volume, but showed no dependence on PNA concentration (Fig. S2).


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 2. Relative cellular luciferase antisense activity of phosphonate-conjugated PNA (bP6-PNA). HeLa pLuc705 cells were trypsinized and seeded (2.4 × 104 cells/well) 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 solutions for 24 h: 0.25 to 2nM PNA complexed with 6 µl/ml LFA2000. Cell samples were then subjected to luciferase analysis and cellular viability test. All tests were performed in triplicate 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 2: Figure 2. Relative cellular luciferase antisense activity of phosphonate-conjugated PNA (bP6-PNA). HeLa pLuc705 cells were trypsinized and seeded (2.4 × 104 cells/well) 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 solutions for 24 h: 0.25 to 2nM PNA complexed with 6 µl/ml LFA2000. Cell samples were then subjected to luciferase analysis and cellular viability test. All tests were performed in triplicate 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: Next we analyzed an anionic PNA conjugate which can form a complex with cationic lipids in analogy to negatively charged nucleic acids.3 A bis-phosphonate-PNA conjugate was transfected at different concentrations (0–2 nM) using varying volumes of the transfection solution/well in a 96 well plate seeded at 24 × 103 cells/well. Results showed that antisense activity increased in a PNA dose-dependent manner at all transfection volumes, and in parallel, increasing volume also showed a higher antisense activity up to four relative volumes where a saturation was reached. We also observe from these experiments that the volume-effect is more pronounced at higher PNA concentrations. Whereas antisense activity at 1 and 2 nM PNA doses showed an approximately 2-fold increase at 4 relative volumes, at 0.5 and 0.25 nM PNA doses it was only about 1.75- and 1.5-fold, respectively (Fig. 2). However, the difference in PNA-Lipofectamine ratio, resulting in decreasing zeta potential as the PNA concentration increases at fixed Lipofectamine concentration, may also play a role, although an opposite effect could be expected as lowering of the zeta potential would decrease transfection efficacy. Cell viability exhibited a slight decrease (< 30%) with increasing transfection volume, but showed no dependence on PNA concentration (Fig. S2).

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