142 Advances in Polymer Science Editorial Board: A. Abe. A.-C ...

Dendrimers and Dendrimer-Polymer Hybrids 221
mixture of hepta-and octapeptide is obtained. The method which allows the use
of unprotected polypeptides was expanded to other reactions producing oxime
and hydrazone dendrimer-peptide linkages [150, 151]. These are, however, less
stable than the thiazolidine ring at elevated pH.
5.2
Dendrimer-DNA Complexes
Dendrimers with primary amines form complexes with nucleotides by electrostatic
interaction between the cationic ammonium and anionic phosphate
groups [152–157]. Such interactions are also known to occur with polylysine
and other linear polycations. The first account of such interaction has been described
for the case of PAMAM dendrimers with plasmid DNA encoding for either
luciferase or bacterial b-galactosidase [156]. The complexation is monitored
by DNA immobilization in electrophoresis [152, 155, 156] or by enhanced
fluorescence anisotropy [153]. Complexes are completely immobile when the
terminal amine and nucleotide base stoichiometry is around 1:1 to 1.5:1 [152,
155, 156]. Excess nucleotide shows no retardation of mobility, while excess dendrimer
amine groups, i.e., 5:1 and higher, leads to positively charged complexes
which migrate to the cathode [152, 155, 156]. It is observed that complexes are
better prepared at low pH and low ionic strength [152]. Nevertheless, the dendrimer-DNA
complexes are quite stable against variations in pH between 3 and
10 [152, 155] with some loss of complexation above pH 10 [153]. Ionic strengths
between 50 mmol/l and 1.5 mol/l are tolerated. Disruption of the complex is only
effected with strong ionic detergents [155, 157]. An important advantage of the
complex is that it makes the DNA quite resistant to nuclease digestion [157].
The structure of the dendrimer-DNA complexes is not well characterized. It
has been estimated that under conditions of maximum transfection, i.e., the
process of bringing the DNA through the cell wall into the cell, the size of the luciferase
plasmid is such that about 320 PAMAM dendrimers are present, although
not all may be directly involved in the complexation [156]. Electron microscopy
suggests that the dendrimer-DNA complexes form condensed aggregates
[157]. Delong et al. have studied the complex formed between a third generation
PAMAM dendrimer and a small 15 base oligonucleotide [152]. They
found a broad distribution of MW by ultrafiltration with most material between
10 and 30 kD. Light scattering experiments, however, are obscured by the presence
of a small amount of very high MW material.
The PAMAM dendrimer-DNA complexes have been shown to promote transfection
[152–156]. The earlier studies used reporter luciferase which allows for
measurement of luciferase activity in host cells [155, 156]. Although there is
some dependence on the cell type being invaded [155], experiments have shown
that generations 6–8 dendrimers are most efficient in transfection [155, 156].
There is little transfection with generations 2–4 PAMAM dendrimers and no
further improved efficiency with generations 9 and 10 dendrimers. The preferred
dendrimer to DNA stoichiometry is 5:1 or higher [155, 156]. It has since