of 0.55 per unit (not influenced by the molecular weight) and had negligible
cytotoxicity and hemolytic properties (synonymous of membrane damage) up to
concentrations of 7 and 15 mg mL
À1 , respectively. But, as argued by the authors,
the lack of membrane damaging properties did not necessarily imply lack of
interactions with membranes, which is of importance for intracellular trafficking
properties. At pH 7.4, complete retardation of DNA was achieved at N:P ratio of
15 for intermediate molecular weights (M n ¼ 4.8 kDa). The size of the polyplexes
decreased with increasing molecular weight until M n ¼ 10 kDa, being less than
200 nm for this M n and around 270 nm for a polymer with M n ¼ 20 kDa. At
pH 5, compared to the values at pH 7.4, the size of the polyplexes decreased
markedly and the zeta potential values became slightly more positive, which was
due to an increase in average excess positive charge per polymer unit at this
pH. The same rule as before seemed to apply: the smallest size and comparatively
highest zeta potential helped the polyplexes based on polymers with molecular
weight of 7–10 kDa to transfect cells more efficiently than polyplexes based on
polymers with higher or lower molecular weights.
4 Conclusion
This review has shown that the design of polycations for gene delivery must take
into account a balance between protection of DNA versus loss of efficiency for
DNA condensation and efficient condensation versus hindering of DNA release,
and that parameters leading to transfection efficiency in vivo still need to be
optimized. Indeed, if the IPEC are not stable enough, premature dissociation will
occur before delivery of the genetic material at the desired place, resulting in low
transfection efficiency; on the other hand, a complex that is too stable will not
release the DNA, also resulting in low gene expression. To determine these
properties, gel electrophoresis to test the DNA/polymer complexation, EtBr or
polyanion displacement to test the affinity of a polymer for DNA, and DLS to
determine the extent of DNA condensation, are well-adapted techniques.
Polymers without steric stabilizer components were abandoned relatively early
due to the inherent cytotoxicity of the permanent charges (even if these facilitate
cellular entry of the polyplexes) and the propensity to be easily destabilized and
precipitate. Strong complexation can also mean difficulty of release of the genetic
material and, consequently, low transfection efficiency. The presence of steric
stabilizers in the polyplexes results in an increased solubility under physiological
conditions, but the problem of finding the right balance between steric stabilization
and shielding of charges (that lowers the affinity of the polymer for DNA) is
nevertheless present. On the other hand, this steric barrier and the shielding of the
charges help the polymer to protect DNA from nuclease attacks, i.e., limit protein
adsorption.
Unfortunately, until now, most of the polyplexes (if not all) presented in the
studies reviewed here need to be prepared at high N:P ratio (higher than 10 and
182
A. Bertin
cytotoxicity and hemolytic properties (synonymous of membrane damage) up to
concentrations of 7 and 15 mg mL
À1 , respectively. But, as argued by the authors,
the lack of membrane damaging properties did not necessarily imply lack of
interactions with membranes, which is of importance for intracellular trafficking
properties. At pH 7.4, complete retardation of DNA was achieved at N:P ratio of
15 for intermediate molecular weights (M n ¼ 4.8 kDa). The size of the polyplexes
decreased with increasing molecular weight until M n ¼ 10 kDa, being less than
200 nm for this M n and around 270 nm for a polymer with M n ¼ 20 kDa. At
pH 5, compared to the values at pH 7.4, the size of the polyplexes decreased
markedly and the zeta potential values became slightly more positive, which was
due to an increase in average excess positive charge per polymer unit at this
pH. The same rule as before seemed to apply: the smallest size and comparatively
highest zeta potential helped the polyplexes based on polymers with molecular
weight of 7–10 kDa to transfect cells more efficiently than polyplexes based on
polymers with higher or lower molecular weights.
4 Conclusion
This review has shown that the design of polycations for gene delivery must take
into account a balance between protection of DNA versus loss of efficiency for
DNA condensation and efficient condensation versus hindering of DNA release,
and that parameters leading to transfection efficiency in vivo still need to be
optimized. Indeed, if the IPEC are not stable enough, premature dissociation will
occur before delivery of the genetic material at the desired place, resulting in low
transfection efficiency; on the other hand, a complex that is too stable will not
release the DNA, also resulting in low gene expression. To determine these
properties, gel electrophoresis to test the DNA/polymer complexation, EtBr or
polyanion displacement to test the affinity of a polymer for DNA, and DLS to
determine the extent of DNA condensation, are well-adapted techniques.
Polymers without steric stabilizer components were abandoned relatively early
due to the inherent cytotoxicity of the permanent charges (even if these facilitate
cellular entry of the polyplexes) and the propensity to be easily destabilized and
precipitate. Strong complexation can also mean difficulty of release of the genetic
material and, consequently, low transfection efficiency. The presence of steric
stabilizers in the polyplexes results in an increased solubility under physiological
conditions, but the problem of finding the right balance between steric stabilization
and shielding of charges (that lowers the affinity of the polymer for DNA) is
nevertheless present. On the other hand, this steric barrier and the shielding of the
charges help the polymer to protect DNA from nuclease attacks, i.e., limit protein
adsorption.
Unfortunately, until now, most of the polyplexes (if not all) presented in the
studies reviewed here need to be prepared at high N:P ratio (higher than 10 and
182
A. Bertin