Combinatorial-designed Hyaluronic Acid Nanoparticles for Tumour Targeted Drug and Small Interfering Ribonucleic Acid Delivery - Hyaluronic Acid for Biomedical and Pharmaceutical Applications - page 69

HA to block CD44 prior to Cy5.5-labelled HA-NP treatment,
they barely detected any fluorescent signal, suggesting a lack of
cellular uptake of HA-NP. Also, when they treated the fibroblast
cells (which do not express CD44) with these NP, at they saw a
very weak signal, suggesting that these particles in fact selectively
bind to CD44 and internalise into cells through a receptor-mediated
pathway. In addition, Jiang and co-workers demonstrated that their
anti-PGL3-luc siRNA encapsulated HA-PEI complexes appeared to
silence the PGL3-luc gene in the range of 50-85%. Similarly they also
demonstrated that this cell uptake is receptor-mediated by performing
competition assays [21].
As far as our system is concerned, we observed very similar findings
to those described by Choi and co-workers [7], and Jiang and
co-workers [21]. Since it is known that the CD44 receptors are
involved in the cellular entry of HA-based particles, we initially
determined the relative amounts of CD44 receptor expression on
the surface of different types of tumour cells by flow cytometry
( Figure 3.4a ). To confirm that these HA nanoparticles are entering
the cells by the CD44 receptor-mediated pathway, we tested the
Cy3-labelled siRNA loaded HA nanosystems in CD44 expressing
MDA-MB468 cells [19]. Similar to other findings, the confocal
microscopic evaluations revealed a bright fluorescence signal in the
cytoplasm of the cells due to the Cy3-siRNA localisation within the
cells that were treated with Cy3-siRNA loaded HA-PEI nanosystems
( Figure 3.4b ). Interestingly, Cy3-siRNA encapsulated HA-octylamine,
HA-stearylamine, HA-choline and HA-spermine also demonstrated
compelling results with high cellular uptake when tested in the same
cell lines. However, no detectable signal was observed with any of
those derivatives in Hep3B cells that do not, or minimally, express
CD44 receptors. These data clearly suggested that several of the
HA derivatives were able to internalise into cells that overexpress
CD44 receptors. We also demonstrated that these particles are
predominantly trafficked into the cells via a receptor-mediated
endocytosis pathway by performing a competition assay by pre-
treating the cells with free soluble HA containing serum-free medium
prior to incubating the cells with Cy3-labelled siRNA/HA-PEI