Delivery to Intracellular Targets by Nanosized Particles - Intracellular Delivery: Fundamentals and Applications - page 84

uptake of lipid complexes containing siRNA against CD31 in mouse vascular
endothelial cells and also observed inhibition of tumor growth by an antiangiogenic
effect. Lavigne and Thierry ( 2007 ) were able to quantify the delivery of siRNA
directed against cyclin D1 associated with a lipoplex to specific cell compartments
in MCF-7 cells. Yadava et al. ( 2007 ) compared the performance of lipoplexes with
poly(ethyleneimine) (PEI, see below) and saw similar cellular uptake of a model
siRNA from the two systems, but a better silencing activity from the lipoplexes,
which they attributed to a more rapid dissociation of the lipid complexes.
Recent developments include the use of specific targeting ligands to promote
uptake of the nucleic acid complexes by specific cells types. Cardoso et al. ( 2007 )
used lipoplexes targeted by transferrin to deliver siRNA silencing marker genes to
glioma, hepatocarcinoma and HT-22 cells. Transferrin was also used as a ligand to
prepare “Trojan horse” liposomes to carry nucleic acids and other therapeutic
agents across the blood-brain barrier, taking advantage of the presence of transfer-
rin receptors promoting transcytosis across endothelial cells (Pardridge 2010a ). The
insulin receptor has been used for the same purpose (Pardridge 2010b ). The folate
receptor, overexpressed on many tumor cells, is another target which has been
exploited for intracellular nucleic acid delivery (Yu et al. 2009 ). For example, Bcl2
downregulation in KB cells was shown to be greatly enhanced by the presence of
folate acid on the surface of cationic liposomes (Chiu et al. 2006 ). The ligand is
attached to the extremity of PEG chains themselves coupled to phosphatidyletha-
nolamine in the liposome membrane. Delivery of anti HER-2 AS-ODN to head and
neck cancer cells was also improved by folate-liposomes and increased the sensitivity
of the cells to conventional chemotherapy (Rait et al. 2003 ).

4.2 Polymer-Based Systems

As far as polymer-based systems are concerned, three strategies have been adopted
for the delivery of nucleic acids: complexation with cationic molecules, adsorption
onto preformed nanoparticles and encapsulation within nanoparticles.
The most commonly used macromolecule for complexation of nucleic acids is
PEI, which provides a high density of positive charge. Complexes can be formed
easily by mixing the polymer and the nucleic acid; the resulting assemblies retain
a net positive charge, which leads to electrostatic interactions with the cell mem-
brane and uptake by endocytosis. Within the acidic endosome, the amino groups on
the polymer are protonated, leading to osmotic swelling and rupture of the endo-
some. This so-called proton sponge effect ensures the release of the nucleic acid
into the cytoplasm. However, a simple PEI/nucleic acid formulation presents draw-
backs in terms of toxicity and an unfavorable biodistribution. PEI, particularly
branched or high-molecular weight chains is toxic because of its ability to bind to
cell membranes and cause necrotic cell death (Roques et al. 2007 ). The net positive
charge of the complexes also provokes rapid adsorption of plasma proteins after
intravenous administration, leading to particle aggregation. These aggregates are