Cationic lipid mediated transfection of plasmid DNA-IBB peptide covalent chimera
The IBB peptide was covalently coupled to plasmid DNA by photoactivation at various IBB peptide / plasmid molar ratios. We first tried to evaluate the direct effect of IBB peptide on nuclear import using transfection since our final aim was to enhance gene transfer by transfection. When using RPR120535 cationic lipid to mediate plasmid-IBB peptide chimera transfection, we observed a partial dose-dependent inhibition of reporter gene expression, which reached 88 % at 50 IBB peptide / plasmid (mol / mol), and 99 % at 100 IBB peptide / plasmid (mol / mol) (Figure 1). At 1 to 10 molar ratios, reporter gene expression following transfection was neither inhibited, nor increased (data not shown). The same result was obtained using confluent cells, i.e. non-dividing cells (data not shown). Thus the plasmid DNA was still biologically active but transfection efficiency was not enhanced by covalently coupled IBB peptides. At high IBB peptide / plasmid ratios, the inhibition of transfection was a consequence of the photoactivation as non-photoactivated IBB / plasmid complexes of the same high ratio exhibited the same transfection activity as the control plasmid (Figure 1). In an effort to determine why covalent coupling of IBB peptide to DNA did not give the effect we had expected, we examined the binding characteristics of IBB peptide.
Interaction of free and covalently coupled IBB peptide with importin β – Nuclear import assay
Interaction of the free IBB peptide with its intracellular target, namely importin β, was studied using recombinant importin β-GST fixed on glutathione-sepharose beads. In a first series of assays, we tested the interaction of either free IBB peptide or, as a control that of cytochrome c, with importin β (Figures 2A and 2B). The IBB peptide was detected in the bound fraction (Figure 2B), whereas cytochrome c was almost completely recovered in the unbound fraction (Figure 2A). This suggested that the synthetic IBB peptide was still able to interact with its nuclear import receptor, namely importin β, as the hSRP1α IBB domain described by Weis et al. [21].
However, this interaction was shown to be non-specific in a series of control experiments as the IBB peptide also interacted with importin α and GST (Figures 2C and 2D). No interaction could be detected between the non-functionalized glutathione-sepharose beads and the IBB peptide (data not shown). Thus, the IBB peptide was shown to be able to interact with cytoplasmic receptors that mediate karyopheric proteins nuclear import, but not specifically.
We then coupled IBB peptide to plasmid DNA. We first verified the formation of the IBB-TFPAM conjugate by HPLC (Figure 3A) followed by SDS-PAGE analysis of the eluted compounds (Figure 3B). At pH 2, the reaction cannot occur, peaks corresponding to non-coupled IBB (I) and TFPAM (T1, T2, T3) were observed. R1 and R2 peaks that appeared only when the reaction was performed at pH 7.5 have migration properties on SDS-PAGE consistent with a 7.5 kDa-peptide. Considering their respective retention times, R1 was probably non-reacting IBB and R2 the TFPAM-IBB conjugate. We then added IBB-TFPAM to plasmid DNA and photoactivated the mixture. We were unable to purify the conjugate IBB-TFPAM-plasmid DNA from free IBB-TFPAM because IBB peptide bound to every support, dialysis membrane, plastic tube, etc. Every experiment was performed in low binding tubes to prevent non-specific interaction of the IBB peptide with the wall of the tubes. We have then no direct proof of the formation of the conjugate IBB-TFPAM-DNA except the fact that photoactivation of the complex IBB plasmid led to an inhibition of transfection. We have previously shown that the covalent coupling of ligands to plasmid DNA could cause inhibition of transfection, probably by transcriptional inactivation [26].
We next examined whether IBB peptide was still able to interact with its receptor when it was coupled to DNA. IBB peptide was coupled to DNA at two ratios and applied to importin β-containing beads. Bound and unbound fractions were analyzed by electrophoresis on agarose gel. As appeared in Figure 3C, at the ratio of 25 IBB peptides / plasmid (mol / mol), the covalent chimera were retained on importin β beads. However, this retention was also observed with non-covalently associated IBB / plasmid (Figure 3C). We therefore verified that plasmid DNA alone (without IBB added) was unable to bind to these beads (Figure 3D). Taken together, these results proved that IBB was able to lead to the retention of the plasmid on importin β-conjugated beads even when non-covalent interactions occurred between the peptide and DNA. We hypothesized that covalent interactions occurred when photoactivation was applied, but we could not prove their existence due to the formation of strong non-covalent interactions between IBB and plasmid DNA.
Since IBB peptide associated with plasmid DNA was still able to bind importin β, we next evaluated whether IBB peptide was able to improve the nuclear import of plasmid DNA. We used the digitonin-permeabilized cells model with fluorescent plasmid. The IBB peptide was coupled to fluorescent plasmid at the molar ratio of 25 and incubated with digitonin-permeabilized HeLa cells. As shown in Figure 4, fluorescence was concentrated on the remaining cytoplasm and the nuclear envelope (Figure 4B), the staining pattern was the same with fluorescent plasmid alone, without IBB peptide covalent coupling (Figure 4A). No fluorescence was observed within cell nuclei.
It seemed that IBB peptide was still able to interact with its specific receptor, but despite this interaction no significant improvement of nuclear import of plasmid could be obtained.
Since IBB peptide was also able to interact non-specifically with other proteins, IBB-plasmid complexes might be sequestered by unspecific interactions with cytoplasmic proteins. Nevertheless, in an effort to obtain an improvement of gene transfer, we studied other formulations of DNA and IBB peptide and observed that IBB peptide / plasmid complexes formed by self assembly, without photoactivation, were able to increase gene transfer activity in particular conditions of transfection.
Gene transfer efficiency of self assembling complexes composed of IBB peptide / plasmid DNA / RPR120535 cationic lipid
We evaluated the effect of IBB peptide adjunction on transfection efficiency of DNA / cationic lipid complexes. These lipoplexes were prepared by mixing the lipofectant RPR120535 with plasmid DNA and IBB peptide in 150 mM NaCl in water and then diluted in serum containing cell culture medium. Without IBB, this formulation is known to lead to small lipoplexes and inefficient transfection [22–24].
The presence of IBB peptide greatly increased reporter gene expression. At ratios of 250 and 500 IBB peptide / plasmid DNA (mol / mol), i. e. 0.8 to 1.6 IBB peptide / plasmid DNA (w / w), we observed a 20-fold increase of luciferase gene expression (Figure 5). Enhancement of gene transfer efficiency depended on IBB peptide / plasmid DNA molar ratio: reporter gene expression increased with the amount of added IBB peptide. Transfection assays were also performed with lipoplexes prepared in water. In those assays, gene expression was increased 100-fold by adding 500 moles IBB peptide per mole plasmid DNA (data not shown).
We then wondered if this transfection enhancement could be attributed to a nuclear targeting of plasmid DNA by IBB peptide or to a change in the overall physico-chemical properties of plasmid DNA / RPR120535 lipoplexes.
Physico-chemical properties of the plasmid DNA / IBB complexes
The IBB peptide is globally basic, characterised by the presence of 17 R and K aminoacids, protonated at physiological pH, and of 10 acidic residues (D, E). Its charge at neutral pH is globally positive, for this reason it can potentially form complexes with negatively charged DNA molecules.
The formation of self-assembling IBB peptide / plasmid DNA complexes was examined by analysis of their electrophoretic mobility on agarose gel (Figure 6). At ratios of 100 and 250 IBB / plasmid (mol / mol), i. e. 0.3 and 0.8 IBB / plasmid (w / w), the migration of plasmid DNA was delayed. For ratios of 500 to 2000 IBB / plasmid (mol / mol), i.e. 1.6 to 6 IBB / plasmid (w / w), no DNA migration occurred. This lack of migration indicates neutralization of nucleic acids by cationic IBB peptide and / or formation of large complexes that cannot migrate through the gel.
Lipoplexes size determination
The size of the ternary complexes was monitored using dynamic light scattering and the size distribution of the complex's populations was determined by size distribution processor analysis. When compared to binary DNA / cationic lipid complexes prepared in water, the addition of 250 mol of IBB peptide per mol plasmid DNA induced a marked size increase, leading to 3000 nm sized complexes (Figure 7A). In the case of 500 IBB / plasmid DNA (mol / mol), the lipoplexes were totally aggregated, as eye-observable aggregates could be seen (data not shown). In this case, lipoplex size cannot be precisely defined due to the detection limits of the nanosizer.
Lipoplexes prepared in water or in 150 mM NaCl and then diluted in serum-containing cell culture medium are known to be small and to lead to inefficient transfection [22–24]. On the contrary, preparing lipoplexes in bicarbonate buffer (20 mM NaHCO3, 150 mM NaCl, pH 10), and diluting them in serum-containing cell culture medium leads to bigger lipoplexes and highly efficient transfection. The size of ternary IBB / DNA / RPR120535 complexes prepared in bicarbonate buffer (20 mM NaHCO3, 150 mM NaCl, pH 10) were around 2000–3000 nm (Figure 7B), which was comparable to the size of IBB / plasmid DNA / RPR120535 lipoplexes prepared in water with 250 and 500 IBB / plasmid (mol / mol) (Figure 7A). Transfection with these ternary lipoplexes prepared in bicarbonate buffer was as efficient as transfection with binary plasmid DNA / RPR120535 complexes prepared in bicarbonate buffer (data not shown).
IBB peptide / plasmid / lipofectant complexes that exhibited an improvement of transfection clearly had different physico-chemical characteristics.