A large number of methods are available for nucleic acid cellular transfection and typically require careful optimization for use. However, despite the fact that cationic transfection reagents cause up-concentration of the (nucleic acid) cargo on the cell membrane, the effect of the volume of the transfection medium is rarely considered in experimental protocols. Thus in a comparative study across different cationic delivery modalities we set out to study this effect using different volumes of PNA-lipoplex, -polyplex or PNA-CPP preparations exploiting different PNA modifications () designed for use with these delivery modalities.
In order to define initial conditions, PNA doses, transfection reagents and incubation time were individually optimized for each PNA modification according to transfection efficacy and toxicity data. Conditions showing the highest antisense activity above 50% cell viability were selected for further volume-effect assays (data not shown). Subsequently, transfection solutions were prepared specifically for each PNA conjugation. PNAs were either complexed with a cationic lipid reagent (Lipofectamine 2000), chemically conjugated to a cationic polymer (PEI), or for CPP-PNAs, mixed with CQ as a singular preparation at their optimal concentrations and transfected to HeLa pLuc705 cells for 24 h. All tested PNA modifications contained a PNA oligomer of identical sequence targeting the cryptic splice site within the luciferase gene in HeLa pLuc705 cells and antisense activity (and thus be inference cellular uptake) was measured as induced luciferase activity.18
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 (). 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 (), 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.
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 (more ...)
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 (). 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
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 (more ...)
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, ).6
For this PNA modification we also observed a clear volume-dependent antisense activity at both 30 and 100 nM concentration (). 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 ().
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. (more ...)
In order to test the generality of the effect, two of the PNAs, CPP-PNA and cholesteryl-PNA, were transfected at the same conditions in a 24-well plate (instead of 96-well plate) with equivalent cell confluency. Both conjugates showed an increase of the antisense activity with increasing transfection volume (Fig. S5
), fully consistent with the results obtained in the 96 well plates.
Finally, we studied the volume-effect for a PNA-polyplex using a disulfide coupled PNA-PEI (polyethyleneimine) conjugate.8
A 4-fold antisense activity increase at 5 relative volumes was seen for both of the analyzed doses (1 and 2 µM), but in contrast to the other PNA conjugates, the transfection volume-effect for the PEI-PNA is not dependent on the PNA dose (at 1 and 2 µM), and showed a close to proportional dependence on the volume up to 4 relative volumes (), whereas only minor effect of the cell number was observed. Finally, the PNA-polyplexes exhibited a pronounced volume-dependent cellular toxicity at the lower cell densities (4 and 8 × 103
cells/well) and the higher concentration (2 µM) (Fig. S4
Figure 4. Relative cellular luciferase antisense activity of PNA conjugated to PEI [PEI25K-(PEG8-SS-PNA)15]. HeLa pLuc705 cells were trypsinized and seeded in a 96 well plate at different cell numbers the day before transfection. Cells were treated (more ...)