Delivery of Single siRNA Molecules - Nanotechnology for the Delivery of Therapeutic Nucleic Acids - page 97

(TAT) protein was able to enter the cytoplasm of cells without any
transporter (Frankel and Pabo, 1988). At the same time, in another
laboratory, chemically synthesized TAT was also shown to enter cells
rapidly (Green and Loewenstein, 1988). Some years later, it was
found that a 60-amino-acids-long peptide from another transcription
factor, corresponding to the homeobox of the Antennadapedia
protein in 1991).

This initiated a new field of delivery vehicles named PTDs or CPPs
that has evolved and expanded greatly over the last decades. The
PTDs are short cationic peptides approximately 10-30 amino acids
long and can deliver different cargoes into cells, such as drugs and
oligonucleotides, both

Drosophila , entered cells efficiently (Joliot et al.,

in vitro and in vivo (Snyder and Dowdy, 2004,

Eguchi 2009). Moreover, several PTDs are now being analyzed

in clinical trials, as reviewed elsewhere (van den Berg and Dowdy,
2011).

et al.,

4.2.2 Delivery of siRNA-PTD Nanoparticles

The uptake mechanism of PTDs has been studied intensively and
several pathways of endocytosis have been suggested as well as
direct penetration through the membrane (Kaplan et al., 2005;

Palm-Apergi 2005). It

has also been shown that different forms of endocytosis can function
simultaneously, and by blocking one pathway, other pathways may
become more active. Furthermore, the uptake is dependent on
both concentration and cargo. Several studies have utilized PTDs
to deliver siRNA into the cytoplasm, both as covalently linked
conjugates and electrostatically bound nanoparticles. However, the
efficiency of covalently conjugated PTD-siRNA has been low due
to the precipitation and aggregation that occur when the cationic
residues of the PTD interact with the anionic phosphate backbone
of the siRNAs (Moschos

et al. ; 2009, Ferrari et al. ; 2003, Richard et al.,

2007). In contrast, some successful
studies have taken advantage of the negative charges of the siRNA
and the positively charged PTD to form electrostatically bound PTD-
siRNA nanoparticles to induce knockdown of target mRNA both

et al.,

in

vitro .

One of the first studies to utilize the electrostatic interaction
between the siRNA and the PTD synthesized a peptide named MPG
that consisted of two domains. The first domain was hydrophilic
and cationic in order to bind the anionic siRNA, whereas the second
domain contained hydrophobic amino acids that engaged the lipids

and in vivo