Biomedical Engineering Reference
In-Depth Information
pDNA/PEI polyplexes to the lung. Gene expression in the lungs of mice after
aerosol delivery of pDNA with iloprost-grafted PEI was 14-fold higher than for
plain PEI polyplexes.
The selection of the receptor ligand can have an impact both on cell binding and
intracellular uptake. “Dual targeting” options have been developed with the option
to combine two targeting ligands in an effective way; for example, combining
ligands with unique cell binding characteristics but moderate internalization with
co-ligands mediating efficient endocytosis. In transfections of prostate cancer cells,
synergistic dual targeting characteristics were observed with DNA polyplexes
containing two different peptide ligands. PEGylated PEI/DNA polyplexes were
decorated with RGD peptide ligands for integrin targeting and with peptide B6 for
TfR targeting. In a series of flow cytometer experiments, either cell association or
cell internalization with and without ligand competition were evaluated. RGD was
found to play a major role in cell surface binding, whereas B6 had a major role in
intracellular uptake [
150
].
2.3
Intracellular Transport
Several enveloped viruses, and some physical gene transfer techniques such as
electroporation, deliver the nucleic acid into the cell by direct crossing of the cell
membrane. Lipid-based, enveloped systems can do this by a physiological, self-
sealing membrane fusion process, avoiding physical damage of the cell membrane.
For cationic lipid-mediated delivery of siRNA, most material is taken up by endo-
cytotic processes. Recently, direct transfer into the cytosol has been demonstrated
to be the bioactive delivery principle for certain siRNA lipid formulations [
151
].
Other systems like electroporation have no lipids that might help in membrane
sealing or fusion; for direct transfer of the nucleic acid across membranes they have
to generate transient pores, a process where efficiency is usually directly correlated
with membrane destruction and cytotoxicity. Alternatively, like for the majority
of polymer-based polyplexes, cellular uptake proceeds by clathrin- or caveolin-
dependent and related endocytic pathways [
152
-
156
]. The polyplexes end up inside
endosomes, and the membrane disruption happens in intracellular vesicles. It is
noteworthy that several observed uptake processes may not be functional in deliv-
ery of bioactive material. Subsequent intracellular obstacles may render a specific
pathway into a dead end [
151
,
154
,
156
]. With time, endosomal vesicles become
slightly acidic (pH 5-6) and finally fuse with and mature into lysosomes. Therefore,
polyplexes have to escape into the cytosol to avoid the nucleic acid-degrading
lysosomal environment, and to deliver the therapeutic nucleic acid to the active site.
Either the carrier polymer or a conjugated endosomolytic domain has to mediate
this process [
157
], which involves local lipid membrane perturbation. Such a lipid
membrane interaction could be a toxic event if occurring at the cell surface or
mitochondrial membrane. Thus, polymers that show an endosome-specific mem-
brane activity are favorable.
