Delivery to Intracellular Targets by Nanosized Particles - Intracellular Delivery: Fundamentals and Applications - page 82

barrier to nucleic acid delivery. Therapeutic genes and oligonucleotides which form
triple helices with DNA must be transported into the nucleus, while AS-ODN and
siRNA act in the cytoplasm at the level of protein synthesis on the ribosomes.
However, if the formulation containing the nucleic acid is internalized by phagocy-
tosis or endocytosis, it will be sequestered in a membrane-bound vacuole, which is
subsequently acidified and fused with a lysosome containing acid hydrolases.
Therefore, some mechanism must be included to allow the nucleic acid to escape
from the endosome before it is degraded.
Viral vectors have been developed for gene therapy, but have serious drawbacks
in terms of toxicity, immunogenicity and the size of the plasmid which can be
inserted. A number of other strategies have been adopted: physical methods such as
the “gene gun” and electroporation, complexes with cationic polymers such as
chitosan, poly (lysine) and poly (ethyleneimine) (PEI) and complexes with cationic
lipids, known as lipoplexes. This section will concentrate on recent developments
in intracellular delivery systems for AS-ODN and siRNA.

4.1 Lipid-Based Systems

The concept of “lipofection” was advanced by Felgner et al. ( 1987 ). Small lipo-
somes, formed from dioleoylphosphatidylethanolamine (DOPE) and a cationic
lipid, DOTMA, were mixed with DNA and the resulting “lipoplexes” were able to
introduce reporter genes into various cell lines. The cationic lipid is able to con-
dense the linear DNA into a complex, but the original lipid vesicle morphology is
not conserved. DOTMA is available commercially as Lipofectamine®. Many other
cationic lipids have been developed, including DOTAP, DOGS, DC-cholesterol and
SAINT-2. These lipids are usually mixed with a “helper” lipid, such as DOPE or
cholesterol which improves their stability and may aid cellular penetration.
However, these complexes show low transfection efficiency compared with viral
systems and in particular do not perform well in vivo. The net positive charge of
the complexes is probably responsible for their high toxicity and also promotes the
adsorption of plasma protein which leads to their rapid elimination. As a result,
attempts have been made to modify the surface of these complexes. The surface
charge can be modified by the addition of anionic lipids (Lee and Huang 1996 ) or
by the inclusion of PEGylated lipids (Fenske et al. 2001 ).
Li and Szoka ( 2007 ) have reviewed the development of lipid-based colloidal
particles with a diameter of less than 100 nm, which would be better adapted to
in-vivo nucleic acid delivery. In particular, they describe a detergent dialysis
method which allows the different components to be associated in a controlled
fashion. They also present a model for the interaction of these lipid articles with
cells. It was originally assumed that cationic lipoplexes were able to fuse directly
with the plasma membrane, which has a negative charge and deliver their cargo
directly to the cytoplasm. However, it is now accepted that the cationic complexes
are taken up by endocytosis after electrostatic interaction with the plasma mem-
brane. Within the endosome, endogenous lipids in the endosomal membrane are