makes it quite difficult to compare these results with those previously reported. For
a grafting density of 50%, an increase in molecular weight of the polycation led to
an increase in DNA compaction ability of the polymer and slight increase in the
polyplex size (60–80 nm). An increase in the net positive charge density for a given
chain length also led to an increase in compaction ability (EtBr quenching) and,
correspondingly, in a decrease in size of the polyplex (from 67 to 32 nm for 0.49
and 0.94 net positive charge density, respectively). At similar N:P ratio (10 for
instance), it was remarkable that these polyplexes were much smaller than poly
(1,2-propylene H-phosphonate) modified with spermidine (which could be due to
other conditions of preparation, as these are not mentioned). These polyplexes were
relatively stable under physiological conditions as well as with time, except those
based on the shortest polymer (10 kDa) with a net positive charge density of 0.49.
The cellular uptake of these polyplexes was cell line-dependent. In HeLa cells, the
polyplexes with the second highest zeta potential but by far the smallest size were
preferentially taken up; in HEK293 cells, the difference in uptake was not significant when the M w of the polymers was higher than 25 kDa; in HepG2 cells, there
was no significant difference, showing that the zeta potential and size were not
determining factors in these last two cell lines. Nevertheless, the transfection
efficiency dramatically increased in all three cell lines with increasing molecular
weight and grafting rate. As in the previous case, the influence of the partially
negatively charged polymer backbone was not shown.
Poly(isobutylene-alt-maleic acid-g-oligoethyleneamine) grafted with diethylene
triamine P(Mal-g-DET), tetraethylenepentamine P(Mal-g-TEP), and pentaethylenehexamine P(Mal-g-PEH) (Fig. 24b) were studied by Yang and coworkers
[235]. Complete retardation was only observed for the polymers with the longest
oligoethylene amine chains at N:P ratio of 10. The polyplexes based on P(Mal-gTEP) and P(Mal-g-PEH) had a neutral zeta potential at N:P ratio of about 12 and
were positively charged for higher N:P ratio, but their size stayed in the micrometer
range until an N:P ratio of 16 for P(Mal-g-TEP) and 22 for P(Mal-g-PEH). This size
was not adequate for gene delivery and could be explained by the difficulty in
condensing the negatively charged DNA with the partially negatively charged
backbone. Above these critical ratios, the nanoparticles obtained were in the
range of 400–800 nm, P(Mal-g-TEP) being less efficient than P(Mal-g-PEH) for
condensing DNA, which could be explained by a greater number of protonated
amine groups in P(Mal-g-PEH). Due to the larger particle size and lower zeta
potential of P(Mal-g-TEP) compared to P(Mal-g-PEH), the polyplexes based on
the latter polymer were most efficient in transfecting cells, comparable to the
performance of PEI in some cases. The transfection efficiency was dependent
upon N:P ratio and also on the cell line but, most importantly, polyplexes based
on P(Mal-g-PEH) were localized to a large extent in the nucleus after 8 h.
At pH 7.4, prevailing negatively charged amphoteric PAAs were found to
be relatively cytotoxic [236], while positively charged PAAs were far less
cytotoxic [237]. Among them, PAgma (Fig. 24c) possesses three ionizable groups:
a strong acid (pK a ¼ 2.3), a medium-strength base (pK a ¼ 7.4) and a strong base
(pK a > 12.1) [238, 239]. At pH 7.4, PAgma has an excess average positive charge
Polyelectrolyte Complexes of DNA and Polycations as Gene Delivery Vectors
181
a grafting density of 50%, an increase in molecular weight of the polycation led to
an increase in DNA compaction ability of the polymer and slight increase in the
polyplex size (60–80 nm). An increase in the net positive charge density for a given
chain length also led to an increase in compaction ability (EtBr quenching) and,
correspondingly, in a decrease in size of the polyplex (from 67 to 32 nm for 0.49
and 0.94 net positive charge density, respectively). At similar N:P ratio (10 for
instance), it was remarkable that these polyplexes were much smaller than poly
(1,2-propylene H-phosphonate) modified with spermidine (which could be due to
other conditions of preparation, as these are not mentioned). These polyplexes were
relatively stable under physiological conditions as well as with time, except those
based on the shortest polymer (10 kDa) with a net positive charge density of 0.49.
The cellular uptake of these polyplexes was cell line-dependent. In HeLa cells, the
polyplexes with the second highest zeta potential but by far the smallest size were
preferentially taken up; in HEK293 cells, the difference in uptake was not significant when the M w of the polymers was higher than 25 kDa; in HepG2 cells, there
was no significant difference, showing that the zeta potential and size were not
determining factors in these last two cell lines. Nevertheless, the transfection
efficiency dramatically increased in all three cell lines with increasing molecular
weight and grafting rate. As in the previous case, the influence of the partially
negatively charged polymer backbone was not shown.
Poly(isobutylene-alt-maleic acid-g-oligoethyleneamine) grafted with diethylene
triamine P(Mal-g-DET), tetraethylenepentamine P(Mal-g-TEP), and pentaethylenehexamine P(Mal-g-PEH) (Fig. 24b) were studied by Yang and coworkers
[235]. Complete retardation was only observed for the polymers with the longest
oligoethylene amine chains at N:P ratio of 10. The polyplexes based on P(Mal-gTEP) and P(Mal-g-PEH) had a neutral zeta potential at N:P ratio of about 12 and
were positively charged for higher N:P ratio, but their size stayed in the micrometer
range until an N:P ratio of 16 for P(Mal-g-TEP) and 22 for P(Mal-g-PEH). This size
was not adequate for gene delivery and could be explained by the difficulty in
condensing the negatively charged DNA with the partially negatively charged
backbone. Above these critical ratios, the nanoparticles obtained were in the
range of 400–800 nm, P(Mal-g-TEP) being less efficient than P(Mal-g-PEH) for
condensing DNA, which could be explained by a greater number of protonated
amine groups in P(Mal-g-PEH). Due to the larger particle size and lower zeta
potential of P(Mal-g-TEP) compared to P(Mal-g-PEH), the polyplexes based on
the latter polymer were most efficient in transfecting cells, comparable to the
performance of PEI in some cases. The transfection efficiency was dependent
upon N:P ratio and also on the cell line but, most importantly, polyplexes based
on P(Mal-g-PEH) were localized to a large extent in the nucleus after 8 h.
At pH 7.4, prevailing negatively charged amphoteric PAAs were found to
be relatively cytotoxic [236], while positively charged PAAs were far less
cytotoxic [237]. Among them, PAgma (Fig. 24c) possesses three ionizable groups:
a strong acid (pK a ¼ 2.3), a medium-strength base (pK a ¼ 7.4) and a strong base
(pK a > 12.1) [238, 239]. At pH 7.4, PAgma has an excess average positive charge
Polyelectrolyte Complexes of DNA and Polycations as Gene Delivery Vectors
181