Delivery to Intracellular Targets by Nanosized Particles - Intracellular Delivery: Fundamentals and Applications - page 83

transferred into the particle to form ion pairs with the cationic lipid. This destabilizes
the endosomal membrane and the nucleic acid is able to escape into the cytoplasm
(Xu and Szoka, 1996 ).
Another, similar, strategy for cytoplasmic delivery is pH-sensitive liposomes
(reviewed by Fattal et al. 2004 ). In this case the nucleic acid is encapsulated in
neutral or anionic liposomes including lipids in their composition which undergo a
change in organization at the pH of the endosomes, destabilize the endosomal
membrane and release the nucleic acid into the cytoplasm. DOPE is one of the
lipids used in this context, because it adopts a hexagonal phase at low pH. Oleic
acid or cholesteryl hemisuccinate (CHEMS) are often added to the formulation.
Some results have been obtained in vitro showing that these liposomes increase
nucleic acid delivery to cells; for example, the replication of Friend virus in NIH 3
T3 cells was inhibited by an AS-ODN in pH-sensitive cells (Ropert et al. 1996 ).
Interestingly, this work suggested that virally infected cells preferentially take up
particulate carrier systems by reflex endocytosis during virus budding. However,
observations in vivo with pH-sensitive liposomes have shown limited efficacy,
because interactions with plasma proteins reduce the pH-sensitivity (De Oliveira
et al. 2000 ). Recently, more sophisticated systems have been developed. Thus,
Mudhakir et al. ( 2008 ) coupled an arginine-rich peptide onto the surface of lipid
systems, to direct them to the macropinocytosis uptake pathway. When siRNA was
incorporated into this type of particle, which also contained a pH-sensitive lipid
combination DOPE/CHEMS, gene expression was effectively inhibited. Another
pH-sensitive system, developed by the laboratory of Robert Langer, used a coating
of PEG-polycation copolymer on pH-sensitive liposomes encapsulating siRNA
(Auguste et al. 2008 ). At the pH of the endosomes, the protective polymer is des-
orbed and the nucleic acid released. Efficient knockdown of GFP in transfected
HeLa cells and GAPDH in HUVEC cells by these particles has been demonstrated.
This is interesting in the light of results reported by Remaut et al., ( 2007 ) showing
that PEGylation of liposomes fails to protect the nucleic acid cargo from degraga-
tion in the lysosomes. On the other hand, an assembly of cationic lipid and
AS-ODN conjugated to PEG was able to promote rapid delivery to the nucleus in
KB cells (Jeong et al. 2006 ). Experimental results from Pakunlu et al. ( 2006 )
showed that PEGylated liposomes containing both AS-ODN to drug resistance
genes and doxorubicin could be taken up by cancer cells and in this way the
efficacy of the cytotoxic drug was enhanced.
A alternative strategy for intracellular delivery is the use of so-called “fusogenic”
liposomes in which a viral peptide included in the formulation allows cytoplasmic
delivery. An example is the promotion of cytoplasmic delivery of DNA oligonucle-
otides with a system using inactivated Sendai virus (Kunisawa et al. 2005 ).
The intracellular delivery of antisense oligonucleotides has been reported in a
large number of publications. Delivery has been evidenced by detection of fluores-
cent oligonucleotides within the cells and by an antisense effect on the target gene.
Thus, Ruozi et al. ( 2005 ) showed the role of DOTAP in liposomes for the intracel-
lular delivery of AS-ODN to COS 1 and HaCaT cells.
In the same way, uptake of siRNA loaded lipid complexes has been demon-
strated in many different cells types. For example, Santel et al. ( 2006 ) showed