Antisense Oligonucleotides and RNA Interference - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 336

larger than in any other body tissue, allowing access of even large oligonucleotides.
AS ODNs also traverse across these endothelial cells transcellularly via claveolin-
based transcytosis [50] . This transcellular transport is size independent, allowing pas-
sage of both small and large oligonucleotide molecules. Cell-penetrating peptides,
targeting ligands, or molecular conjugates, can be used to facilitate passage of AS
ODNs across endothelial lining [18] .
7.4.2 Cellular Barrier
To exert its action, an antisense agent needs to enter the cell and then reach the actual
target. To do so, it faces some of the following challenges.
7.4.2.1 Cell Entry
Nonviral vector-AS ODN complexes, by being highly cationic, bind nonspecifically
to negatively charged cell membranes and are easily taken up by cells of the RES by
endocytosis or membrane fusion [51] . This nonspecific cellular uptake by nontarget
tissues results in severe toxic manifestations due to unwanted protein expression. This
nonspecific cellular uptake can be reduced by coating the nonviral vector-AS ODN
complexes with PEG and by attaching cell-specific targeting ligands such as transferrin
[52] , folate [53] , surface receptor-specific antibodies [54] , and so on. Coupling with
membrane-permeable peptides like transportan and penetratin also enhances the cel-
lular internalization [55] . Coating with PEG not only reduces uptake by the RES but
also reduces uptake by target cells. Hence, it is more rational to use cell-specific tar-
geting ligands along with PEG coating. However, use of receptor-specific antibodies
can evoke immunogenic manifestations. Therefore, these antibodies must be suitably
tailored before being used as targeting ligands [56] .
7.4.2.2 Endosomal Release
Once an AS ODN reaches the target cells, escape from pericellular vesicles (endo-
somes) or lysosomes is required for transfection [57] . Hence, the transfection efficiency
of nonviral vectors depends on cellular internalization as well as the endosomal escape
of the active moiety to reach the actual target [57,58] . Two strategies are widely used
to enhance the endosomal escape. The first uses fusogenic lipids or peptides to rupture
lysosomal membranes, by forming pores in membranes [59,60] . A pH-sensitive lipo-
some system, such as Lipofectin, composed of cationic lipids along with a fusogenic
helper lipid such as DOPE (1,2-dioleoyl- sn -glycero-3-phosphoethanolamine), has been
reported, which readily releases the entrapped oligonucleotides at low-pH environ-
ments [61] . Interaction of the cationic lipids of these liposomes with the anionic lipids
of the cell membranes (endosomal membrane) results in phase separation, thereby cre-
ating DOPE-rich regions that form pores in the membranes, causing membrane desta-
bilization [62,63] . The second strategy involves using delivery systems that possess
high buffering capacity. This prevents acidification of the endosomes, resulting in dis-
ruption of the endosomal membrane [64,65] . Polyethylenimine is used as a buffering