degradation in Lipofectamine/DNA complexes are controversial [95–98] and do
not allow a detailed discussion on the importance of DNase protection on the
transfection efficiency.
Whereas the release experiments clearly showed that DNA was more easily
released from the cylindrical brush complexes than from linear PLL complexes,
experiments with ethidiumbromide revealed that all DNA phosphate groups were
complexed by the cationic charges for both linear and cylindrical brush PLLs. Thus,
subtle topology-dependent enthalpic and entropic effects may play a deciding role
in the DNA release out of the complexes.
2.4 Transfection with Brush Polymers
Prior to transfection, the question of cell cytotoxicity needs to be addressed.
Porcine microvascular endothelial cell (PBMEC) viability was affected in a
concentration-dependent manner and was similar for linear PLL and cylindrical
PLL brushes, with a 50% cell survival concentration of 0.5 mg/mL. For comparison, the cell cytotoxicity of linear PLLs and stiff cylindrical PLL-brush polymers
on fibroblasts (NIH/3T3) was also tested, which is known to be very high for
linear PLL [99]. These results could be confirmed because linear as well as brush
PLL polymers were found to be very toxic for fibroblasts (50% cell survival
concentration 0.01 mg/mL). The differences in cytotoxicity between the two cell
lines may be explained by cell type-dependent differences. Brain endothelial cells
possess transport systems (efflux transporter of ABC family) characteristic for
barrier endothelial cells, which constitute a physical barrier to the brain
[100]. The PLL-brush polymers showed a 40% reduction in cell viability at a
concentration of 0.1 mg/mL and no effect at a concentration of 0.01 mg/mL. PLL
brush concentrations used for transfection were <0.1 mg/mL; hence, only moderate effects on cell viability are to be expected during transfection. Compared to
the well-known cytotoxic side effects of other transfection reagents (e.g.,
lipofectins [101]), the moderate effects on cell viability observed here are judged
to be of only minor importance.
For in vitro transfection studies, complexes of DNA and cylindrical brush PLL with
PLL side chains were formed at a polycation-to-DNA charge ratio of z
+
/z
À
¼ 10.6
and z
+
/z
À
¼ 6.3. [94]. Transfection was controlled by confocal microscopy and
quantified by cell cytometry. Only complexes with brush PLL provided significant
cell transfection of brain capillary endothelial cells (PBMEC), whereas complexes
with linear PLL were not effective, as shown in Fig. 9.
It was demonstrated that cylindrical brush/DNA complexes showed the highest
transfection efficiencies, even higher than for Lipofectamine controls. Quantitative
analysis revealed that cylindrical brush/DNA complexes formed at an excess of 6.3
and 10.6 positive charges per negative charge (z
+
/z
À ) evoked an increase in
Polymer Complexes in Biological Applications
229
not allow a detailed discussion on the importance of DNase protection on the
transfection efficiency.
Whereas the release experiments clearly showed that DNA was more easily
released from the cylindrical brush complexes than from linear PLL complexes,
experiments with ethidiumbromide revealed that all DNA phosphate groups were
complexed by the cationic charges for both linear and cylindrical brush PLLs. Thus,
subtle topology-dependent enthalpic and entropic effects may play a deciding role
in the DNA release out of the complexes.
2.4 Transfection with Brush Polymers
Prior to transfection, the question of cell cytotoxicity needs to be addressed.
Porcine microvascular endothelial cell (PBMEC) viability was affected in a
concentration-dependent manner and was similar for linear PLL and cylindrical
PLL brushes, with a 50% cell survival concentration of 0.5 mg/mL. For comparison, the cell cytotoxicity of linear PLLs and stiff cylindrical PLL-brush polymers
on fibroblasts (NIH/3T3) was also tested, which is known to be very high for
linear PLL [99]. These results could be confirmed because linear as well as brush
PLL polymers were found to be very toxic for fibroblasts (50% cell survival
concentration 0.01 mg/mL). The differences in cytotoxicity between the two cell
lines may be explained by cell type-dependent differences. Brain endothelial cells
possess transport systems (efflux transporter of ABC family) characteristic for
barrier endothelial cells, which constitute a physical barrier to the brain
[100]. The PLL-brush polymers showed a 40% reduction in cell viability at a
concentration of 0.1 mg/mL and no effect at a concentration of 0.01 mg/mL. PLL
brush concentrations used for transfection were <0.1 mg/mL; hence, only moderate effects on cell viability are to be expected during transfection. Compared to
the well-known cytotoxic side effects of other transfection reagents (e.g.,
lipofectins [101]), the moderate effects on cell viability observed here are judged
to be of only minor importance.
For in vitro transfection studies, complexes of DNA and cylindrical brush PLL with
PLL side chains were formed at a polycation-to-DNA charge ratio of z
+
/z
À
¼ 10.6
and z
+
/z
À
¼ 6.3. [94]. Transfection was controlled by confocal microscopy and
quantified by cell cytometry. Only complexes with brush PLL provided significant
cell transfection of brain capillary endothelial cells (PBMEC), whereas complexes
with linear PLL were not effective, as shown in Fig. 9.
It was demonstrated that cylindrical brush/DNA complexes showed the highest
transfection efficiencies, even higher than for Lipofectamine controls. Quantitative
analysis revealed that cylindrical brush/DNA complexes formed at an excess of 6.3
and 10.6 positive charges per negative charge (z
+
/z
À ) evoked an increase in
Polymer Complexes in Biological Applications
229