Gene Delivery Using Chemical Methods - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 170

TAT-DNA complexes demonstrated caveolae-dependent cellular uptake in both
HepG2 and CHO1 cell types, transfection of the BGM cell line exhibited an alterna-
tive route of endocytosis [272] . Hence, cellular uptake of free TAT-peptides occurred
by an energy-independent pathway, and transfection of TAT-DNA complexes pro-
ceeds by endocytosis. Anti-MDR antisense ODN conjugated with TAT-peptide, which
displayed significant inhibition of P-glycoprotein expression in vitro even in the pres-
ence of serum, which could lead to a two- to threefold increase in drug uptake [273] .
Various modifications were explored to improve gene transfer after the initial suc-
cess of TAT-based ODN transfection. Ternary complexes of TAT-peptide, pDNA, and
PEI were prepared by TAT-peptide conjugation with DNA, followed by complexation
with the cationic polymer. These ternary complexes show higher gene transfer effi-
ciency compared to either PEI or Superfect complexes. This increase in gene transfer
was assumed to be because of substantial cellular uptake and endosome lysis medi-
ated by the PEI/Superfect moieties followed by translocation into the cell nucleus
mediated by the TAT-peptide [274] . TAT-liposomes prepared by covalently attaching
the TAT-peptide to p -nitrophenylcarbonyl-PEG-phosphatidyl ethanolamine liposomes
displayed significantly higher gene expression as compared to TAT-free liposome
plasmid complexes when injected directly into tumor tissue. In vitro studies of TAT
liposomes showed comparable gene transfection to Lipofectin TM despite higher cyto-
toxicity [275] .
TAT-polylysine conjugates exhibited moderate gene transfer activity. However,
highest efficiency was achieved only in the presence of chloroquine. This suggests
inefficiency of TAT-peptides in promoting endosomal escape despite having signifi-
cant plasma and nuclear membrane-fusion capabilities [276] . The above drawback
was overcome by covalently attaching 10 histidine residues to the C-terminus of the
TAT-peptide. The modified complex exhibited up to 7000-fold higher gene transfec-
tion efficiency as compared to the unmodified TAT-peptide, with lower cytotoxicity
[277] . TAT-peptide was conjugated with cationic peptide  (mu), a 19-amino acid
sequence peptide (Met-Arg-Arg-Ala-His-His-Arg-Arg-Arg-Arg-Ala-Ser-His-Arg-Arg-
Met-Arg-Gly-Gly) that exhibited the highest transfection efficiency in the presence of
the commercially available cationic lipids Lipofectamine TM and DC-Chol [278] .
The antennapedia homeodomain is a 60-amino acid polypeptide correspond-
ing to the Drosophila melanogaster antennapedia homeobox sequence synthesized
first in the early 1990s by Prochiantz et al. [279] . When the 60-amino acid struc-
ture was reduced to a 16-mer peptide (pAntp, residues 43-58, Arg-Gln-Ile-Lys-Ile-
Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys) without affecting cellular effect,
it was concluded that the R-helix of the antennapedia homeodomain is involved in
promoting translocation. Cellular uptake of pAntp followed a nonendocytic path-
way [280] . It was observed that the replacement of several amino acids with proline
disrupted the R-helical structure of pAntp but did not hinder cellular uptake. This
finding indicated that the R-helical conformation is not required for cellular uptake
[281] . Further, translocation of the reverse helix of pAntp and a helix composed of
D-enantiomers indicates that internalization of pAntp is not receptor mediated. Based
on the earlier findings, Prochiantz et al. have suggested that internalization of pAntp
proceeds via electrostatic interactions between the basic amino acid residues of the