positive charge of the guanidine functions, pK a ¼ 12.5, in comparison with tertiary
amines, pK a ¼ 10).
Other imidazole derivatives based on poly(L-histidine) partially grafted with
gluconic acid (PHis-g-Glu, Fig. 10i) to impart solubility at physiological pH
(polyhistidine is insoluble in aqueous solutions at pH >6.0) were reported by
Pack et al. [164]. Approximately one carbohydrate–imidazole substitution out of
every 23 imidazole groups was enough to confer solubility of at least 100 mg mL
À1
at pH 7. Migration of DNA was completely retarded at a pHis-g-Glu:DNA weight
ratio of 3:1, which was consistent with the charge ratio causing 50% reduction of
EtBr fluorescence; the size of the polyplexes at this ratio was >500 nm. The DNA
seemed to be fully condensed at a pHis-g-Glu:DNA weight ratio of 5:1, given that
the size of the polyplexes was 240 nm at pH 7. Unfortunately, these polyplexes
were unable to transfect COS-7 cells, maybe because of their large size, despite
their negligible cytotoxicity.
Poly(L-ornithine) (PLO, Fig. 10g) has a structure similar to polylysine, except
that it possesses one CH 2 less in the side chain. In a publication by the group of
Gumbleton, polyplexes based on PLO, PLL and poly(D-lysine) (PDL) possessed
roughly similar physico-chemical characteristics; nevertheless, the polyplexes
based on PLO showed better transfection capacity in A459cells and COS-7 cells
[165]. It should be noted that condensation of pDNA occurred at the following
charge ratios (<10% EtBr fluorescence): 0.8:1 for PLO, 1.2:1 for PLL, and 1.5:1 for
PDL, and that PLO polyplexes also showed greater resistance to polyanion-induced
disruption. As explained by the authors, Blauer and Alfassi had previously suggested
that the additional CH 2 group contained within the lysine may make the α-helix
conformation in PLL more stable than in PLO [166]. Given the comparable pK a
values of the primary amine groups for lysine and ornithine (pK a ¼ 10.5–10.7) [167],
it is probable that conformational differences rather than protonation per se provides a
basis for the differential behavior of PLO-mediated pDNA condensation. The least
effective condensing polycation was PDL. Given that the L-isomer is the natural
orientation of nuclear enzymes and proteins, it is possible that DNA interactions with
other macromolecules are biased towards L-isomer conformations [168].
In order to determine the size of the polyplexes, most of the studies use light
scattering, which gives the R h as well as other information, but is not able to give the
exact structure of the polyplexes. Therefore, AFM is a complementary method to
light scattering and allows imaging matter at the nanoscale (possible interactions
with the surface of the wafers should nevertheless be taken into account). An AFM
study of the complexation of DNA by PLO was conducted by the group of Ganguli
and could provide insights into the mechanism of DNA condensation [169]. Based
on AFM images, the mechanism seemed to be different at low (<7 μg mL
À1 ) and
high DNA concentrations (>13 μg mL
À1 ), i.e., monomolecular and multimolecular
condensations, respectively (Scheme 16). It should nevertheless be noted that in
contrast to most of the studies where the polycation is added to DNA solution, in
this study DNA was added to the polymer solution, which could influence the
mechanism pathway.
Polyelectrolyte Complexes of DNA and Polycations as Gene Delivery Vectors
149
amines, pK a ¼ 10).
Other imidazole derivatives based on poly(L-histidine) partially grafted with
gluconic acid (PHis-g-Glu, Fig. 10i) to impart solubility at physiological pH
(polyhistidine is insoluble in aqueous solutions at pH >6.0) were reported by
Pack et al. [164]. Approximately one carbohydrate–imidazole substitution out of
every 23 imidazole groups was enough to confer solubility of at least 100 mg mL
À1
at pH 7. Migration of DNA was completely retarded at a pHis-g-Glu:DNA weight
ratio of 3:1, which was consistent with the charge ratio causing 50% reduction of
EtBr fluorescence; the size of the polyplexes at this ratio was >500 nm. The DNA
seemed to be fully condensed at a pHis-g-Glu:DNA weight ratio of 5:1, given that
the size of the polyplexes was 240 nm at pH 7. Unfortunately, these polyplexes
were unable to transfect COS-7 cells, maybe because of their large size, despite
their negligible cytotoxicity.
Poly(L-ornithine) (PLO, Fig. 10g) has a structure similar to polylysine, except
that it possesses one CH 2 less in the side chain. In a publication by the group of
Gumbleton, polyplexes based on PLO, PLL and poly(D-lysine) (PDL) possessed
roughly similar physico-chemical characteristics; nevertheless, the polyplexes
based on PLO showed better transfection capacity in A459cells and COS-7 cells
[165]. It should be noted that condensation of pDNA occurred at the following
charge ratios (<10% EtBr fluorescence): 0.8:1 for PLO, 1.2:1 for PLL, and 1.5:1 for
PDL, and that PLO polyplexes also showed greater resistance to polyanion-induced
disruption. As explained by the authors, Blauer and Alfassi had previously suggested
that the additional CH 2 group contained within the lysine may make the α-helix
conformation in PLL more stable than in PLO [166]. Given the comparable pK a
values of the primary amine groups for lysine and ornithine (pK a ¼ 10.5–10.7) [167],
it is probable that conformational differences rather than protonation per se provides a
basis for the differential behavior of PLO-mediated pDNA condensation. The least
effective condensing polycation was PDL. Given that the L-isomer is the natural
orientation of nuclear enzymes and proteins, it is possible that DNA interactions with
other macromolecules are biased towards L-isomer conformations [168].
In order to determine the size of the polyplexes, most of the studies use light
scattering, which gives the R h as well as other information, but is not able to give the
exact structure of the polyplexes. Therefore, AFM is a complementary method to
light scattering and allows imaging matter at the nanoscale (possible interactions
with the surface of the wafers should nevertheless be taken into account). An AFM
study of the complexation of DNA by PLO was conducted by the group of Ganguli
and could provide insights into the mechanism of DNA condensation [169]. Based
on AFM images, the mechanism seemed to be different at low (<7 μg mL
À1 ) and
high DNA concentrations (>13 μg mL
À1 ), i.e., monomolecular and multimolecular
condensations, respectively (Scheme 16). It should nevertheless be noted that in
contrast to most of the studies where the polycation is added to DNA solution, in
this study DNA was added to the polymer solution, which could influence the
mechanism pathway.
Polyelectrolyte Complexes of DNA and Polycations as Gene Delivery Vectors
149