Summary: | In this work, different targeted formulations have been designed and
evaluated in order to improve gene delivery to cancer cells by non-viral vectors
in vitro and in vivo. All the nanosystems are based on the dendrimeric carrier
PAMAM, to which four different ligands (hyaluronic acid, transferrin, B6 and
GE11 peptides) have been attached, in order to evaluate the targeting capacity
of each nanocarrier. First, a novel PAMAM-hyaluronic acid conjugate has been
synthetized by an amine bond formation between PAMAM and oxydized
hyaluronic acid. This conjugate was able to form nanoparticles in the presence
of pDNA and exhibited excellent capacity to effectively bind pDNA and
protect it from enzymatic degradation by nucleases. In vitro evaluation of
PAMAM-hyaluronic acid dendriplexes showed an increase in transfection
activity in MDA-MB231 and B16F10 cells compared to non-targeted
complexes. A competition study with an excess of free HA confirmed the
uptake via specific receptor-mediated mechanism. Toxicity studies showed a
good cell viability and lower toxicity than the highly used PEI-polyplexes. In vivo
results showed an increase in luciferase expression in the liver and heart of
Balb-C mice compared to non-targeted complexes. These systems were also
able to transfect efficiently B16F10 tumors in C57BL/6 tumor-bearing mice,
although no significant differences compared to non-targeted ones were
detected. Secondly, PAMAM-Transferrin conjugates were prepared and
evaluated. In vitro evaluation of this new PAMAM-Transferrin conjugate
demonstrated increased gene delivery to cancer cells (HeLa, HepG2 and CT26)
when complexes were formulated at N/P ratio of 6. Toxicity was lower than
PEI-polyplexes. The uptake via receptor-mediated endocytosis was ensured by
a competition assay. Finally, B6 and GE11 peptides were studied as targeting
ligands in these systems. Small interfering RNA (siRNA) was formulated in targeted complexes containing each peptide in the presence of PEG (2 kDa).
PAMAM-PEG-B6 and PAMAM-PEG-GE11 dendriplexes formed stable
nanoparticles, able to condense siRNA effectively. In vitro evaluation of the
gene silencing capacity of siRNA encapsulated into PAMAM-PEG-B6 or
PAMAM-PEG-GE11 complexes showed that specific siRNA-Luc was able to
reduce luciferase expression in HeLa and LS174 cells, without leading to
toxicity effects.
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