at +/À ratio of 4, except for PTBP, which condensed DNA into polyplexes of this
size at +/À ratio of 6, meaning that the binding is less tight. PTEA, PTBA, and
PTEP also exhibited a plateau in their zeta potential (positive) without significant
change from a +/À ratio of 2. PTBP polyplexes generated at +/À ratio of 2 had
zeta potentials near neutral, and then a positive plateau starting at +/À ratio of 4.
The presence of a plateau in the polyplex diameter and zeta potential suggests
that the additional polymer remained as free polymer in the solution, uncomplexed
to DNA. The zeta potentials of the triethyl-based polyplexes were more positive
than those of the tributyl-based polyplexes due to hydrophobic screening of the
cationic charge with longer alkyl chains. All polymers exhibited similar toxicities
due to their 100% charge densities, approximately like that of the transfection
reagent jetPEI. PTEA and PTEP displayed poor transfection efficiency compared
with SuperFect, whereas PTBA and PTBP exhibited excellent transfection, similar
to SuperFect. Given that the entry into the cell of all polyplexes was successful,
the higher transfection efficiency of tributyl-containing polyelectrolytes over
triethyl-based polyelectrolytes could be due to a higher endosomolytic activity.
Ionene are polycations with charged quaternized nitrogen atoms in the polymer
backbone (Fig. 3d). Izumrudov and colleagues synthesized ionenes via Menshutkin
polyaddition reaction between N,N,N
0 ,N
0 -tetramethylethylenediamine (TMED) and
dibromoalkanes such as 1,4-dibromobutane and 1,8-dibromooctane [126]. For
[ionene]/[DNA] < 1, the increase in ionene content was accompanied by a substantial decrease in PEC particle size (from 500 to 100 nm), up to a charge ratio
of unity, where the particles were neutral and formed aggregates. With excess
polycation, the positively charged PEC did not aggregate, and at charge ratios of
the polymers of 2:1 the particle size was again ~100 nm, regardless of the charge
density or chain length of the polycation. Nevertheless, a difference could be
observed in the protection of DNA against nuclease attack: the polymers with
the highest DP offered better protection and, at a given DP, the shortest spacer
(i.e., the highest charge density) was preferred. These results correlated with the
stability of the polyplexes, even if upon lengthening of the ionene chains
(DP > 20), the difference in PEC stability between ionenes with different spacers
became relatively small. The transfection efficiency in COS-7 cells followed the
same trend for the ionenes as the PEC stability.
By comparing the DNA/polycation complexes based on various architectures
such as PEVP (Fig. 3a), ionene (Fig. 3d) and PDMDAAC, which is a polycation of
pendant type (Fig. 3e), Galaev and colleagues suggested that phase separation in
solutions of DNA-containing PECs (with strong polycations without steric stabilization) follows the general rules ascertained from PECs formed by flexible vinyl
polyanions. However, the high rigidity of the double helix of native DNA appears to
be responsible for significant extension of the region of insoluble PECs at the
expense of the region in which soluble PECs are formed [127].
Polyelectrolyte Complexes of DNA and Polycations as Gene Delivery Vectors
135
size at +/À ratio of 6, meaning that the binding is less tight. PTEA, PTBA, and
PTEP also exhibited a plateau in their zeta potential (positive) without significant
change from a +/À ratio of 2. PTBP polyplexes generated at +/À ratio of 2 had
zeta potentials near neutral, and then a positive plateau starting at +/À ratio of 4.
The presence of a plateau in the polyplex diameter and zeta potential suggests
that the additional polymer remained as free polymer in the solution, uncomplexed
to DNA. The zeta potentials of the triethyl-based polyplexes were more positive
than those of the tributyl-based polyplexes due to hydrophobic screening of the
cationic charge with longer alkyl chains. All polymers exhibited similar toxicities
due to their 100% charge densities, approximately like that of the transfection
reagent jetPEI. PTEA and PTEP displayed poor transfection efficiency compared
with SuperFect, whereas PTBA and PTBP exhibited excellent transfection, similar
to SuperFect. Given that the entry into the cell of all polyplexes was successful,
the higher transfection efficiency of tributyl-containing polyelectrolytes over
triethyl-based polyelectrolytes could be due to a higher endosomolytic activity.
Ionene are polycations with charged quaternized nitrogen atoms in the polymer
backbone (Fig. 3d). Izumrudov and colleagues synthesized ionenes via Menshutkin
polyaddition reaction between N,N,N
0 ,N
0 -tetramethylethylenediamine (TMED) and
dibromoalkanes such as 1,4-dibromobutane and 1,8-dibromooctane [126]. For
[ionene]/[DNA] < 1, the increase in ionene content was accompanied by a substantial decrease in PEC particle size (from 500 to 100 nm), up to a charge ratio
of unity, where the particles were neutral and formed aggregates. With excess
polycation, the positively charged PEC did not aggregate, and at charge ratios of
the polymers of 2:1 the particle size was again ~100 nm, regardless of the charge
density or chain length of the polycation. Nevertheless, a difference could be
observed in the protection of DNA against nuclease attack: the polymers with
the highest DP offered better protection and, at a given DP, the shortest spacer
(i.e., the highest charge density) was preferred. These results correlated with the
stability of the polyplexes, even if upon lengthening of the ionene chains
(DP > 20), the difference in PEC stability between ionenes with different spacers
became relatively small. The transfection efficiency in COS-7 cells followed the
same trend for the ionenes as the PEC stability.
By comparing the DNA/polycation complexes based on various architectures
such as PEVP (Fig. 3a), ionene (Fig. 3d) and PDMDAAC, which is a polycation of
pendant type (Fig. 3e), Galaev and colleagues suggested that phase separation in
solutions of DNA-containing PECs (with strong polycations without steric stabilization) follows the general rules ascertained from PECs formed by flexible vinyl
polyanions. However, the high rigidity of the double helix of native DNA appears to
be responsible for significant extension of the region of insoluble PECs at the
expense of the region in which soluble PECs are formed [127].
Polyelectrolyte Complexes of DNA and Polycations as Gene Delivery Vectors
135