similar. But, the polymer with the block structure, despite the low content of PEG
compared to some of the comb-shape polymers, protected DNA from enzymatic
degradation to the greatest extent, giving an indication that the structure in solution
might be different. Nevertheless, it should be mentioned that even at high polymer:
DNA ratio (4 and above), the charge of the polyplexes remained negative for all the
polymers, probably reflecting the low protonation degree of histidine at pH 7. At pH
5, the polyplexes had diameters between 100 and 300 nm and, most importantly,
were stable for over a week at room temperature. It could be observed that
increasing the PEG content also increased the diameter of the polyplexes. Once
transferred to pH 7, their stability was limited and aggregation occurred after 24 h,
showing a certain limitation of this approach. Moreover, the transfection efficiency
of all these polymers was poor in COS-7 cells (comparable to the tested PLL, but
three orders of magnitude lower than PEI).
In contrast to PLL, polyaspartamide modified with chosen oligoethyleneimine
side chains of various lengths had the advantage of possessing both primary and
secondary amines. PEG-b-PAsp derivatives modified with various amines
(Fig. 18d) were synthesized by the group of Kataoka [203]: the PAsp segment
was modified with ethylene diamine [PEG-b-P(Asp-EDA), Fig. 18d-R 1 ], diethylene
triamine [PEG-b-P(Asp-DETA), Fig. 18d-R 2 ], 4-methyldiethylene triamine
[PEG-b-P(Asp-MDETA), Fig. 18d-R 3 ], and N,N-diethyldiethylene triamine
[PEG-b-P(Asp-DEDETA), Fig. 18d-R 4 ]. The focus was on PEG-b-P(Asp-DETA),
which showed a two-step protonation process (pK a 6.0 and 9.5) due to the presence
of the ethylene diamine moiety, as illustrated in Fig. 19. At pH 7.4, this group is
in the mono-protonated state (gauche form) and is capable of exerting a substantial
buffering effect in the pH range down to 5. At pH 5, where 95% of the ethylene
diamine unit is protonated (~diprotonated) the fluorescence in the EtBr dyeexclusion assay leveled off at N:P ratio of 1, while at pH 7.4 where the monoprotonated form is present, a N:P ratio of 2 is necessary to obtain substantial
quenching.
It is interesting to note that the diameter of the polyplex micelles stayed
constant at around 70–90 nm throughout the range of N:P ratios (1–20), even at
neutral zeta potential, showing the efficiency of the hydrophilic shell to prevent
aggregation. The transfection efficiency of this polymer was compared to the other
PAsp derivatives [PEG-b-P(Asp-EDA), PEG-b-P(Asp-MDETA), PEG-b-P(AspDEDETA)], which all showed comparable sizes and zeta potential to PEG-bP(Asp-DETA). The polyplex based on the polymer modified with the 2-aminoethyl
group (pK a 9.4), PEG-b-P(Asp-EDA), was far less efficient than PEG-b-P
(Asp-DETA) (factor 10
3 at N:P ratio of 20), presumably because of the impaired
buffering capacity of the polymer but also because of its marginal internalization
by cells [203]. Concerning PEG-b-P(Asp-MDETA), which possesses a tertiary
amine instead the secondary amine of PEG-b-P(Asp-DETA) (primary amine unchanged), and PEG-b-P(Asp-DEDETA), which possesses a tertiary amine instead
of the primary amine of PEG-b-P(Asp-DETA) (secondary amine unchanged), they
both showed lower transfection efficiency compared to PEG-b-P(Asp-DETA),
166
A. Bertin
compared to some of the comb-shape polymers, protected DNA from enzymatic
degradation to the greatest extent, giving an indication that the structure in solution
might be different. Nevertheless, it should be mentioned that even at high polymer:
DNA ratio (4 and above), the charge of the polyplexes remained negative for all the
polymers, probably reflecting the low protonation degree of histidine at pH 7. At pH
5, the polyplexes had diameters between 100 and 300 nm and, most importantly,
were stable for over a week at room temperature. It could be observed that
increasing the PEG content also increased the diameter of the polyplexes. Once
transferred to pH 7, their stability was limited and aggregation occurred after 24 h,
showing a certain limitation of this approach. Moreover, the transfection efficiency
of all these polymers was poor in COS-7 cells (comparable to the tested PLL, but
three orders of magnitude lower than PEI).
In contrast to PLL, polyaspartamide modified with chosen oligoethyleneimine
side chains of various lengths had the advantage of possessing both primary and
secondary amines. PEG-b-PAsp derivatives modified with various amines
(Fig. 18d) were synthesized by the group of Kataoka [203]: the PAsp segment
was modified with ethylene diamine [PEG-b-P(Asp-EDA), Fig. 18d-R 1 ], diethylene
triamine [PEG-b-P(Asp-DETA), Fig. 18d-R 2 ], 4-methyldiethylene triamine
[PEG-b-P(Asp-MDETA), Fig. 18d-R 3 ], and N,N-diethyldiethylene triamine
[PEG-b-P(Asp-DEDETA), Fig. 18d-R 4 ]. The focus was on PEG-b-P(Asp-DETA),
which showed a two-step protonation process (pK a 6.0 and 9.5) due to the presence
of the ethylene diamine moiety, as illustrated in Fig. 19. At pH 7.4, this group is
in the mono-protonated state (gauche form) and is capable of exerting a substantial
buffering effect in the pH range down to 5. At pH 5, where 95% of the ethylene
diamine unit is protonated (~diprotonated) the fluorescence in the EtBr dyeexclusion assay leveled off at N:P ratio of 1, while at pH 7.4 where the monoprotonated form is present, a N:P ratio of 2 is necessary to obtain substantial
quenching.
It is interesting to note that the diameter of the polyplex micelles stayed
constant at around 70–90 nm throughout the range of N:P ratios (1–20), even at
neutral zeta potential, showing the efficiency of the hydrophilic shell to prevent
aggregation. The transfection efficiency of this polymer was compared to the other
PAsp derivatives [PEG-b-P(Asp-EDA), PEG-b-P(Asp-MDETA), PEG-b-P(AspDEDETA)], which all showed comparable sizes and zeta potential to PEG-bP(Asp-DETA). The polyplex based on the polymer modified with the 2-aminoethyl
group (pK a 9.4), PEG-b-P(Asp-EDA), was far less efficient than PEG-b-P
(Asp-DETA) (factor 10
3 at N:P ratio of 20), presumably because of the impaired
buffering capacity of the polymer but also because of its marginal internalization
by cells [203]. Concerning PEG-b-P(Asp-MDETA), which possesses a tertiary
amine instead the secondary amine of PEG-b-P(Asp-DETA) (primary amine unchanged), and PEG-b-P(Asp-DEDETA), which possesses a tertiary amine instead
of the primary amine of PEG-b-P(Asp-DETA) (secondary amine unchanged), they
both showed lower transfection efficiency compared to PEG-b-P(Asp-DETA),
166
A. Bertin