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

222 J. Roovers, B. Comanita
become clear that perfect generation 6 PAMAM-DNA complexes are not effectively
transfected [158]. Experiments with solvolytically degraded PAMAM-
DNA complexes suggest that partially degraded high MW complexes are the
true transfection agents. This has been confirmed by the synthesis of defective
PAMAM dendrimers which on complexation with DNA showed high transfection
activity [158]. In the case of oligonucleotides, generations 4 and 5 are about
equally efficient [153].
The uptake of the dendrimer-DNA is an active energy dependent endocytosis
in living cells [155]. Flow cytometry has shown that the complex is located in the
cell [152, 153] and microscopy shows cytoplasmic as well as nuclear uptake
[152]. The cell uptake of DNA is also measured by its biological consequences.
The biological activity of the DNA in the DNA-dendrimer complex seems to be
reduced as less transcription to m-RNA is observed [157]. However, in cases
where antisense oligonucleotides are transfected, the activity of a luciferase carrying
cell is downgraded by the introduction of an antisense oligonucleotide
specific to a portion of the luciferase RNA.
An important question in cell uptake of DNA is the potential toxicity of the
dendrimer nucleotide complexes. In general, the toxicity has been found to be
lower for dendrimer-nucleotides than for other transfection systems including
polylysine [154–156].
5.3
Dendrimer-Antibody Conjugates
Antibodies are ideal for targeting specific cells due to their specific interaction
with antigens on the cell surface. They can therefore be used as carriers of radioisotopes,
toxins, and cytotoxins to the selected cells. This method requires the
linkage of the agent to the antibody without affecting the immunoreactivity of the
antibody. Direct linkage of the agent to the antibody is known to be limited and
to decrease the immunoreactivity. Roberts et al. coupled porphyrin molecules
with a third (24 NH 2 groups) or fourth (48 NH 2 groups) generation PAMAM dendrimer
and then linked rabbit immunoglobin by means of remaining amino
groups [159]. The porphyrin rings have then been used to complex with radioactive
67 CuCl 2 . They established that the dendrimer is attached to the carbohydrate
region of the glycoprotein and that the immune response is retained.
Gansow and his coworkers developed conjugates of monoclonal antibodies
and complexing agent modified PAMAM dendrimers [160] (Scheme 21). All reactions
are performed in water. After purification and creation of the sulfhydryl
group these molecules are linked to monoclonal antibodies that have been premodified
with maleimide groups. The dendrimer-antibody ratio is always near
unity. The uptake of 90 Y by the complexing sites in the conjugate takes about
30 min at 35 ˚C and about 3 h at room temperature. The amount of 90 Y labeling
and the resulting radioactivity exceed by far that obtainable by direct linkage of
the complexing agent to the antibodies. This result is obtained with loss of immunoreactivity.