Biomedical Engineering Reference
In-Depth Information
11.2.4.2 Cationic polymers
Cationic polymers for gene delivery may be synthetic or naturally occurring. The most effec-
tive and extensively researched is the biodegradable peptide poly(L-lysine), first reported as
an effective gene delivery agent by Wu and Wu [82] in 1988. Following its success there
have been numerous adaptations and manipulations of the polylysine peptide, as well as a
huge number of different cationic polymers in linear or branched configurations explored as
DNA delivery vehicles. These include cationic proteins (e.g. histones, protamines, spermine)
and peptides, polyethyleneimine (PEI), dendrimers, polyaminoesters, cationic dextran and
chitosans.
Polylysine has not only been shown to be a good DNA condensing agent, shielding it from
degradation, but also has been suggested to possess some nuclear trafficking properties. The
length and type of positively charged amino acid chain influences the stability and efficacy
of vector/DNA particles [83].
Alone, it is capable of gene delivery; however, polylysine chains of varying length are
most effective when linked with a targeting ligand, forming a receptor-mediated gene deliv-
ery approach. A wide range of peptide vectors have been explored [84]. The ligand takes
advantage of the ability of receptors on a cell's surface to bind and internalize recognized
molecules, adding specificity to the DNA delivery system. Targeting ligands include natu-
rally occurring proteins (such as transferrin [85], insulin [86] or asialorosomucoid [82, 87]),
structural motifs from natural receptor binding ligands (e.g. sugar residues [88] or syn-
thetic peptides [89-91]) or antibodies against an epitope on the extracellular portion of the
receptor (e.g. polymeric immunoglobulin receptor [92]).
There is some evidence that polylysine can induce an inflammatory response follow-
ing direct administration
in vivo
[93]; however, the same is not seen with DNA-ligand-
polylysine complexes [94].
One drawback to polylysine-based vectors is the requirement for assistance with endo-
somolysis. These delivery systems lack any buffering capacity and, therefore, the DNA is
readily degraded following the drop in pH within the endosomal-lysosomal pathway. The
simple addition of the organic lysosomotropic agent chloroquine reduces enzyme degrada-
tion and allows escape of the DNA [95]. Its widely reported toxicity
in vitro
, however,
limits its clinical applications.
A more sophisticated method of preventing endosomal-lysosomal degradation has been
adapted from the many destabilizing proteins found in viruses. The best studied is a
20-amino-acid peptide from the hemagglutinin protein of the influenza virus [96]. The
peptide gains fusogenic activity once in the acidic environment of the lysosome, resulting
in disruption of the membrane, releasing the DNA. This has been successfully incorporated
into receptor-mediated gene delivery systems [97].
PEI is a widely used polymer in the manufacturing industry, but has more recently been
exploited as a vehicle for gene delivery [98]. PEI is an efficient and economic gene transfer
agent available in both linear and branched forms in a variety of molecular weights (see
Figure 11.3, reviewed in Kircheis
et al
. [99]). Its highly positive charge enables effective
DNA condensation to form nuclease-protected stable particles, allowing electrostatic binding
to the cell surface and subsequent endocytic internalization. Once inside the cell, the high
number of protonable nitrogen groups results in a high buffering capacity, called the 'proton
sponge' hypothesis. This buffers the endosomal compartment, leading to rupture of the
membrane, allowing the DNA increased access to the nucleus, without the addition of any
