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Cell-Penetrating Peptides as Vectors for Delivery of Nucleic Acids 357
from physiological to 5 pH, shown by CD spectra analysis. The ability of penetration
through membranes was shown at neutral and negatively charged liposomes at pH
range of 4 to 8.5, in which the KALA peptide induced 100% leakage of liposomeentrapped
dyes. A similar mechanism is suggested by the authors to account for
KALA peptide’s ability to exit from endosomes and transfer ONs or DNA. The
KALA peptide was also shown to be efficient in mediating transfection of various
cells with plasmid DNA. Transferrin addition did not influence transfection efficiency,
thus eliminating this type of uptake.
Fominaya et al. 65 demonstrated the use of the KALA peptide analog in gene
delivery. This peptide delivered noncovalent complexes of peptide–psVLUC plasmid
to various cell lines as detected by expressed luciferase activity measurements.
A peptide with similar amphiphilic α-helical properties was designed by Niidome
et al. 66 They demonstrated that COS-7 cells efficiently take up noncovalently
conjugated peptide–plasmid complexes and that these plasmids retain functionality
and express luciferase. The treatment of cells with chloroquine, an inhibitor of
lysosomal hydrolases, increased the expression of luciferase, pointing to endosomal
type of uptake. Later works by this group show the importance of hydrophobic
moieties in cellular uptake of these peptides. 67
Very recently Good et al. 68 used a peptide KFFKFFKFFK to target antisense
peptide nucleic acids to bacterial cells. This peptide, developed by Vaara and Porro, 69
does not exhibit antibacterial activity and is capable of penetrating bacterial cells.
PNA–peptide conjugates specifically inhibit Escherichia coli gene expression and
growth. PNA conjugates targeted to rRNA and mRNA encoding the essential fatty
acid biosynthesis protein Acp inhibited cell growth. The anti-acpP PNA at 2 µM
concentration cured HeLa cells from K12 E. coli infection.
16.4.7 POLYLYSINE AND LOLIGOMERS
Polylysine (PLL) has been used successfully as a DNA carrier in receptor–mediated
gene transfer; in such cases, PLL is conjugated to the ligand for cell surface receptors
like glycoproteins, 70 insulin, 71 transferrin, 72 lectins, 73 etc. These conjugates are
directed to specific cell surface receptors and their uptake is of a lysosomal type.
Some reports suggest possible involvement of PLL in nonendocytotic mechanism.
Polylysine alone usually possesses low uptake rates of ONs. 74 A recent report
demonstrates the uptake of plasmid DNA, conjugated to poly-(L)-Lys (PLL), poly-
(D)-Lys, or poly-Orn, into alveolar adenocarcinoma cells, A549. 75 Several other
reports assert that PLL uptake is nonspecific adsorptive endocytosis. 76,77
A recent article by Sazani et al. 78 demonstates the uptake of antisense PNA
molecules with 4-mer Lys moieties in C terminus. These constructs are efficiently
taken up by HeLa S3 cells; they blocked aberrant and restored correct splicing of
modified EGFP precursor mRNA, thus generating properly translated EGFP. The
authors suggest that the PNA–(Lys) 4 construct is taken up by a mechanism similar
to that of cell-penetrating homeodomain proteins, as incubation at decreased temperatures
retained the same number of fluorescent cells.
Another group of vector peptides, called loligomers, consists of branched “squidlike”
polylysine sequences. Design of loligomers is based on a concept that multi-