substituents (n ¼ 1–3) remained relatively inefficient regarding transfection, whereas
more efficiency was noticeable for n ¼ 4 and 5 (which could be due to the more
pronounced destabilizing properties of cell membranes), then decreased again for
n ¼ 6, which might be due to a certain hindrance of its interaction with DNA itself by
the presence of the long hydrophobic chain. The substitution of the ester moiety in the
side chain of Cn-PVP (n ¼ 4 and 5) by a carboxylic group gave the corresponding
polycarboxybetaines (Cn-PCB). After complexation with DNA at a charge ratio of
5, they showed far less transfection activity compared to the parent polycations. The
presence of the carboxylic group certainly weakened the interaction with DNA and
possibly required a higher charge ratio for complexation. For the Cn-PVP-β series
with various quaternization degrees, at a charge ratio of 5 there was a bell-shaped
dependency of the transfection efficiency as a function of the alkylation degree, with
the maximum at β ¼ 65% for n ¼ 5 (1000% increase in efficiency compared to
pDNA alone) (β ¼ 40% for n ¼ 6), despite the similar sizes and zeta potentials of
the polyplexes over all the β range. The explanation of the authors regarding this
increased transfection efficiency of the partially alkylated PVP, Cn-PVP-β, was the
presence of the pyridine groups, which could eventually be protonated in acidic
media and thus could play a role in the proton sponge effect. Interestingly, the further
methylation of these Cn-PVP-β derivatives led to negligible transfection efficiencies.
More recently, amphiphilic dimethylaminopyridinium-containing polymethacrylates with tail-end geometries with octyl, dodecyl, and hexadecyl spacers
(n ¼ 8, 12, 16) neutralized by bromide (Br) and octylsulfonate (S8) counterions
were studied (PnDMAP-X, Fig. 20f) [211]. This study allowed the comparison of
pyridinium-based derivatives according to the length of the spacer, counterion, and
geometry. These polymers possess two kinds of amino moieties: a tertiary amine
tail linked directly to the heterocycle (not protonated under physiological
conditions) and an ammonio group that forms part of the pyridinium heterocycle,
which is involved in the electrostatic binding with DNA. The amphiphiles
PnDMAP-X formed a mixture of worm-like and spherical micelles in water for
concentrations above 0.5 mg mL
À1 , with P 8 DMAP-X forming the loosest
structures. The weight ratios of PnDMAP-X:DNA needed to retard DNA in gel
electrophoresis were about 1.5 for n ¼ 8, 3–5 for n ¼ 12, and 2.5–5 for
P 16 DMAP-X for X ¼ Br and 5–7.5 for X ¼ S8. Thus, the decrease in charge
density for these derivatives with increasing spacer length could not only be
accounted for by this trend in the minimum charge ratio needed. The counterion
effect appeared for n ¼ 16, where a higher weight ratio for S8 than for Br is needed
to complex DNA, probably due to the reduced accessibility of the pyridinium group
for DNA with this alkyl counterion (due to its size and/or hydrophobicity). Moreover, the transfection efficiency as a function of the spacer length followed the same
trend in both series (X ¼ Br and S8), with the highest transfection efficiency being
obtained for n ¼ 12, followed by n ¼ 8 and finally n ¼ 16. Also, a bell-shaped
dependency of the transfection efficiency was observed as function of the length of
the spacer. As in the previous example, it seems that a compromise between
membrane destabilization (also reflected by increased cytotoxicity) and efficient
DNA complexation has to be found for this type of derivative.
Polyelectrolyte Complexes of DNA and Polycations as Gene Delivery Vectors
169
more efficiency was noticeable for n ¼ 4 and 5 (which could be due to the more
pronounced destabilizing properties of cell membranes), then decreased again for
n ¼ 6, which might be due to a certain hindrance of its interaction with DNA itself by
the presence of the long hydrophobic chain. The substitution of the ester moiety in the
side chain of Cn-PVP (n ¼ 4 and 5) by a carboxylic group gave the corresponding
polycarboxybetaines (Cn-PCB). After complexation with DNA at a charge ratio of
5, they showed far less transfection activity compared to the parent polycations. The
presence of the carboxylic group certainly weakened the interaction with DNA and
possibly required a higher charge ratio for complexation. For the Cn-PVP-β series
with various quaternization degrees, at a charge ratio of 5 there was a bell-shaped
dependency of the transfection efficiency as a function of the alkylation degree, with
the maximum at β ¼ 65% for n ¼ 5 (1000% increase in efficiency compared to
pDNA alone) (β ¼ 40% for n ¼ 6), despite the similar sizes and zeta potentials of
the polyplexes over all the β range. The explanation of the authors regarding this
increased transfection efficiency of the partially alkylated PVP, Cn-PVP-β, was the
presence of the pyridine groups, which could eventually be protonated in acidic
media and thus could play a role in the proton sponge effect. Interestingly, the further
methylation of these Cn-PVP-β derivatives led to negligible transfection efficiencies.
More recently, amphiphilic dimethylaminopyridinium-containing polymethacrylates with tail-end geometries with octyl, dodecyl, and hexadecyl spacers
(n ¼ 8, 12, 16) neutralized by bromide (Br) and octylsulfonate (S8) counterions
were studied (PnDMAP-X, Fig. 20f) [211]. This study allowed the comparison of
pyridinium-based derivatives according to the length of the spacer, counterion, and
geometry. These polymers possess two kinds of amino moieties: a tertiary amine
tail linked directly to the heterocycle (not protonated under physiological
conditions) and an ammonio group that forms part of the pyridinium heterocycle,
which is involved in the electrostatic binding with DNA. The amphiphiles
PnDMAP-X formed a mixture of worm-like and spherical micelles in water for
concentrations above 0.5 mg mL
À1 , with P 8 DMAP-X forming the loosest
structures. The weight ratios of PnDMAP-X:DNA needed to retard DNA in gel
electrophoresis were about 1.5 for n ¼ 8, 3–5 for n ¼ 12, and 2.5–5 for
P 16 DMAP-X for X ¼ Br and 5–7.5 for X ¼ S8. Thus, the decrease in charge
density for these derivatives with increasing spacer length could not only be
accounted for by this trend in the minimum charge ratio needed. The counterion
effect appeared for n ¼ 16, where a higher weight ratio for S8 than for Br is needed
to complex DNA, probably due to the reduced accessibility of the pyridinium group
for DNA with this alkyl counterion (due to its size and/or hydrophobicity). Moreover, the transfection efficiency as a function of the spacer length followed the same
trend in both series (X ¼ Br and S8), with the highest transfection efficiency being
obtained for n ¼ 12, followed by n ¼ 8 and finally n ¼ 16. Also, a bell-shaped
dependency of the transfection efficiency was observed as function of the length of
the spacer. As in the previous example, it seems that a compromise between
membrane destabilization (also reflected by increased cytotoxicity) and efficient
DNA complexation has to be found for this type of derivative.
Polyelectrolyte Complexes of DNA and Polycations as Gene Delivery Vectors
169