Gene Delivery Using Chemical Methods - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 172

for dendrimer construction, poly(amidoamine) (PAMAM) dendrimers are the most
widely used and characterized family [290] .
4.6.1 Polyamidoamine Dendrimers
The PAMAM polymer (“starburst”) dendrimer is the first type of polycationic mol-
ecules described, with high transfection potential in the absence of endosomolytic
agents [291-293] . The dendrimer demonstrates high transfection potential in the
absence of any additional membrane-active agent. The dendrimer's amines (terminal
and internal) have a low p K a that provides buffering action, thereby preventing DNA
degradation within the endosomes/lysosomes. Their ease of synthesis, terminal group
modification, and commercialization has led to extensive investigation of the biologi-
cal properties of PAMAM dendrimers of various generations.
PAMAM dendrimers are synthesized by the divergent approach, which involves
in situ branch cell construction in stepwise, iterative stages around a desired core
to produce mathematically defined coreshell structures. Typically, ethylenediamine
( N c  4) or ammonia ( N c  3) are used as cores and allowed to undergo reiterative
two-step reaction sequences: (a) the Michael addition of a primary amine to two
molecules of methyl acrylate; and (b) amidation of the ester by ethylenediamine. The
branches of the dendrimer are built by a repetition of two reactions. The nature of
core, that is a trivalent initiator core or a tetravalent initiator core, determines several
structural characteristics of the molecule, including its overall shape, density, and
surface charge [294] .
At low dendrimer-DNA charge ratios (0-1), small changes to DNA conformation
occur, but the complex remains soluble and not compacted. At intermediate charge
ratios [1-100] , insoluble aggregates form due to DNA charge neutralization. Finally,
at high ratios (100), resolubilization occurs due to a salting-in effect [295] . The
dendrimer-DNA ratio for optimum transfection efficiency was 6:1; however, den-
drimers were found toxic in vitro at that ratio [296] . Alternatively, partially degraded
or fractured dendrimers were found to be more efficient in transfecting cells as com-
pared to intact dendrimers. The probable reason was that complexes are more soluble
and do not aggregate [291,297] .
DNA complexes with dendrimers are assumed to be uptake by multiple mech-
anisms. Though previously it was thought that dendrimers were internalized via
adsorptive endocytosis, raft-dependent pathway was suggested for dendrimer inter-
nalization [298] . It was concluded that lipid compositions of different membranes,
the pH, and intracellular trafficking between endosomes and Caveolae can affect
transfection efficiencies significantly [299,300] .
It was observed that the generational number determines gene transfection activity
and the cytotoxicity of PAMAM starburst dendrimers. As a general rule, the transfec-
tion activity of dendrimers with a high generational number is expected to be superior
to those with a low generational number [301] . PAMAM dendrimer displayed expo-
nential increase in transfection efficiency with an increase in generational number from
G5 to G8, when evaluated in vitro in the presence of DEAE-dextran. Also, plateau in
activity was observed after G8 generation [292] . Gene delivery to the rabbit carotid