2.3 Brush Polymer Synthesis and DNA Complexation
The PLL-brush polymers were synthesized by grafting Z-protected lysine by ringopening polymerization from a macroinitiator, as described elsewhere [87]. The
total weight-average molar mass (M w ) of the deprotected PLL brush was determined in aqueous 5 mM LiBr by light scattering to be 1.03Â10
7 g/mol, yielding an
average of 55 lysine units per side chain [87]. The overall brush dimensions were
R g ¼ 84.4 nm and R h ¼ 56.7 nm. The degree of polymerization of the
macroinitiator was evaluated by light scattering to be P w ¼ 900. The polydispersity
measured by gel permeation chromatography in dimethyl formamide (DMF) was
M w /M n ¼ 1.5 (polystyrene calibration). Linear PLL with M w ¼ 326,000 g/mol,
R g ¼ 27 nm, and R h ¼ 21 nm was also utilized for complex formation and transfection studies. The DNA/polycation complexes were prepared by adding DNA
(pUC19) solution dropwise to a polycation solution.
Complexes can be formed by dropwise addition of DNA solution to the
polycation solution. At low excess of polycation, the radii of the complex mixture
remain small, in the size regime of either the pure polycation or the DNA. In this
part of the phase diagram, complexes are known to coexist with excess polycation
[89–91]. When approaching the point where the molar mass and the radii of the
complexes start to diverge, the excess component becomes fully incorporated into
the complexes. Upon further addition of DNA, the solution becomes unstable due to
bridging of the primary complexes by additional DNA. These experiments were
performed with pUC19 DNA but similar results are to be expected for the larger
GFP-DNA (Fig. 8). For linear PLL, this diverging point lies in the equimolar charge
regime, whereas it is shifted to a bigger excess of polycation for brush molecules in
qualitative agreement with our former results on similar systems [89]. Above
z
+
/z
À
¼ 1.5 for complexes with linear PLL and z
+
/z
À
¼ 2.2 with PLL brushes,
no free DNA could be detected.
For transfection experiments, these critical DNA weight fractions (w DNA ) limit
the choice of mixing ratios to 0.15 < w DNA < 0.2, because w DNA should be well
below the diverging point and the solution for transfection should not contain
too-large an excess of uncomplexed polycation.
Typically, one complex contains 20 DNA molecules and 10 cylindrical brush
molecules, whereas more than 100 linear PLL chains are involved in a complex
containing similar numbers of DNA molecules.
For a successful transfection, DNA must be released from complexes. One
prominent hypothesis postulates that anionic competitors, most likely polyions,
could replace DNA in the complex [92, 93]. Hence, lower molar mass heparin has
been established as a model for DNA release experiments, although heparin is not
abundant in cells. In addition, the serum protein albumin (which is also not abundant
in the cell cytoplasm) as representative of proteins with a small anionic charge, NaCl,
and RNA were tested for their ability to release DNA from brush polymers and linear
Polymer Complexes in Biological Applications
227
The PLL-brush polymers were synthesized by grafting Z-protected lysine by ringopening polymerization from a macroinitiator, as described elsewhere [87]. The
total weight-average molar mass (M w ) of the deprotected PLL brush was determined in aqueous 5 mM LiBr by light scattering to be 1.03Â10
7 g/mol, yielding an
average of 55 lysine units per side chain [87]. The overall brush dimensions were
R g ¼ 84.4 nm and R h ¼ 56.7 nm. The degree of polymerization of the
macroinitiator was evaluated by light scattering to be P w ¼ 900. The polydispersity
measured by gel permeation chromatography in dimethyl formamide (DMF) was
M w /M n ¼ 1.5 (polystyrene calibration). Linear PLL with M w ¼ 326,000 g/mol,
R g ¼ 27 nm, and R h ¼ 21 nm was also utilized for complex formation and transfection studies. The DNA/polycation complexes were prepared by adding DNA
(pUC19) solution dropwise to a polycation solution.
Complexes can be formed by dropwise addition of DNA solution to the
polycation solution. At low excess of polycation, the radii of the complex mixture
remain small, in the size regime of either the pure polycation or the DNA. In this
part of the phase diagram, complexes are known to coexist with excess polycation
[89–91]. When approaching the point where the molar mass and the radii of the
complexes start to diverge, the excess component becomes fully incorporated into
the complexes. Upon further addition of DNA, the solution becomes unstable due to
bridging of the primary complexes by additional DNA. These experiments were
performed with pUC19 DNA but similar results are to be expected for the larger
GFP-DNA (Fig. 8). For linear PLL, this diverging point lies in the equimolar charge
regime, whereas it is shifted to a bigger excess of polycation for brush molecules in
qualitative agreement with our former results on similar systems [89]. Above
z
+
/z
À
¼ 1.5 for complexes with linear PLL and z
+
/z
À
¼ 2.2 with PLL brushes,
no free DNA could be detected.
For transfection experiments, these critical DNA weight fractions (w DNA ) limit
the choice of mixing ratios to 0.15 < w DNA < 0.2, because w DNA should be well
below the diverging point and the solution for transfection should not contain
too-large an excess of uncomplexed polycation.
Typically, one complex contains 20 DNA molecules and 10 cylindrical brush
molecules, whereas more than 100 linear PLL chains are involved in a complex
containing similar numbers of DNA molecules.
For a successful transfection, DNA must be released from complexes. One
prominent hypothesis postulates that anionic competitors, most likely polyions,
could replace DNA in the complex [92, 93]. Hence, lower molar mass heparin has
been established as a model for DNA release experiments, although heparin is not
abundant in cells. In addition, the serum protein albumin (which is also not abundant
in the cell cytoplasm) as representative of proteins with a small anionic charge, NaCl,
and RNA were tested for their ability to release DNA from brush polymers and linear
Polymer Complexes in Biological Applications
227