Limits...
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.

Show MeSH

Related in: MedlinePlus

Figure 3. Relative cellular luciferase antisense activity of PNA conjugated to cholesteryl hemisuccinate (Chol-hs-PNA). HeLa pLuc705 cells were trypsinized and seeded (8 × 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: (A) 30 nM and (B) 100 nM PNA complexed with 2, 4 or 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
getmorefigures.php?uid=PMC3368813&req=5

Figure 3: Figure 3. Relative cellular luciferase antisense activity of PNA conjugated to cholesteryl hemisuccinate (Chol-hs-PNA). HeLa pLuc705 cells were trypsinized and seeded (8 × 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: (A) 30 nM and (B) 100 nM PNA complexed with 2, 4 or 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: As an example of a PNA polyplex for which the zeta potential is only marginally affected by the PNA we chose a cholesteryl-PNA conjugate (PNA 2977, Table 1).6 For this PNA modification we also observed a clear volume-dependent antisense activity at both 30 and 100 nM concentration (Fig. 3). Furthermore, the activity increased nearly linearly with the LFA2000 concentrations (2, 4 and 6 µl/ml), whereas only minor effects were observed on cell viability even for the highest PNA dose (Fig. S3). However, while at 30 nM PNA concentration the transfection efficacy was increasing for all relative volumes, this was limited to three relative volumes at 100 nM (Fig. 3).


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 3. Relative cellular luciferase antisense activity of PNA conjugated to cholesteryl hemisuccinate (Chol-hs-PNA). HeLa pLuc705 cells were trypsinized and seeded (8 × 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: (A) 30 nM and (B) 100 nM PNA complexed with 2, 4 or 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 3: Figure 3. Relative cellular luciferase antisense activity of PNA conjugated to cholesteryl hemisuccinate (Chol-hs-PNA). HeLa pLuc705 cells were trypsinized and seeded (8 × 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: (A) 30 nM and (B) 100 nM PNA complexed with 2, 4 or 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: As an example of a PNA polyplex for which the zeta potential is only marginally affected by the PNA we chose a cholesteryl-PNA conjugate (PNA 2977, Table 1).6 For this PNA modification we also observed a clear volume-dependent antisense activity at both 30 and 100 nM concentration (Fig. 3). Furthermore, the activity increased nearly linearly with the LFA2000 concentrations (2, 4 and 6 µl/ml), whereas only minor effects were observed on cell viability even for the highest PNA dose (Fig. S3). However, while at 30 nM PNA concentration the transfection efficacy was increasing for all relative volumes, this was limited to three relative volumes at 100 nM (Fig. 3).

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