Intracellular Delivery: A Multifunctional and Modular Approach - Intracellular Delivery: Fundamentals and Applications - page 203

de-stabilizing effect of the LL component on the endosomal membranes. After 4 h
incubation, larger, fused fluorescent endosomal structures became apparent in the
case of LL-free micelles, whereas cells incubated with PEG-PE/LL micelles had
smaller punctuate fluorescent structures in the cytoplasm. Increased cytoplasmic
delivery of paclitaxel was confirmed by the results of in vitro cytotoxicity studies
against BT-20 cells (human breast carcinoma) and A2780 cells (human ovarian
carcinoma). The paclitaxel-loaded PEG-PE/LL micelles were significantly more
cytotoxic compared to that of free paclitaxel or paclitaxel delivered using nonca-
tionic LL-free PEG-PE micelles: in A2780 cancer cells, the IC50 values for free
paclitaxel, paclitaxel in PEG-PE micelles, and paclitaxel in PEG-PE/LL micelles
were 22.5, 5.8 and 1.2 mM, respectively. In BT-20 cancer cells, the IC50 values of
the same preparations were 24.3, 9.5 and 6.4 mM, respectively.
However, use of cationic lipids has sometimes been associated with cytotoxicity,
especially when used in the high amounts usually used for gene delivery (Torchilin
et al. 2003a ). Therefore, it is necessary to find a novel ligand that enhances both the
cellular uptake and the escape from lysosomal degradation without cytotoxicity or
immunogenicity.

3 Intracellular Delivery of Nanocarriers Using Cell

Penetrating Peptides (CPPs)

A promising approach for the intracellular delivery that has emerged over the last
decade is the use of CPPs (Schwarze et al. 1999 ). Many different short peptide
sequences have been identified that promote transport of a variety of cargoes across
the plasma membrane and deliver their payload intracellularly. This process is
termed “protein transduction”. Such proteins or peptides contain domains of less
than 20 amino acids and are referred to as Protein Transduction Domains (PTDs) or
CPPs, which are highly enriched with basic residues (Schwarze and Dowdy 2000 ).
TATp, the most frequently used CPP, is derived from the transcriptional activator
protein encoded by human immunodeficiency virus type 1 (HIV-1) (Jeang et al.
1999 ). Authors Green (Green and Loewenstein 1988 ) and Frankel (Frankel and
Pabo 1988 ) demonstrated that the 86-mer trans-activating transcriptional activator,
Tat, protein encoded by HIV-1, was efficiently internalized by cells in vitro when
introduced in the surrounding media. Later it was shown that residues 49-57 were
responsible for membrane translocation, with the positive charge contributing
largely to the transduction ability of TAT (Park et al. 2002 ).
TATp-mediated cytoplasmic uptake of plasmid DNA (Astriab-Fisher et al. 2002 ;
Nguyen et al. 2008 ), nanoparticles (Zhao et al. 2002 ; Lewin et al. 2000 ; Rao et al.
2008 ), liposomes (Torchilin et al. 2001b ; Fretz et al. 2004 ; Levchenko et al. 2003 ;
Torchilin 2001 ; Zhao et al.) and micelles (Sethuraman and Bae 2007 ; Sawant et al.
2008 ) has been reported. A variety of uptake mechanisms appear to be involved in
different systems, and in some cases, the mechanism is cell-type or cargo-specific
(Zorko and Langel 2005 ). Smaller molecules attached to TATp seem to transduce