Decoration of nanoparticle surfaces with receptor-specific ligands can improve functional drug
delivery to cancer cells. Our ligand of interest is a short peptide, U11, sequenced to show
high affinity for the Urokinase Plasminogen Activator receptor (uPAR), a receptor
overexpressed on the surface of many tumours. We prove that the presentation of the
peptide ligand, along with the nature of the liposomal platform, play synergic roles in the
exhibition of the nanoparticle’s targeting effect.
U11 peptides were loaded onto PEGylated liposomal nanoparticle surfaces by covalent
conjugation. In vitro pDNA transfection and uptake studies established the viability of the U11
peptide as a ligand for cancer cell targeting. Circular dichroism and fluorescence
spectroscopy methods were used to examine the secondary and tertiary conformations of the
surface peptides. siRNA encapsulation assays, nanoparticle turbidity and in vitro gene
silencing experiments were used to optimise the targeted nanoparticle for in vivo delivery. In
vivo siRNA (luciferase) experiments were performed on mice carrying uPAR-positive (DU145-
luc and MDA-MB-231-luc) and uPAR-negative (PC3-luc) cancer xenografts. Biodistribution
properties of the nanoparticles were examined by administrating fluorescently-labelled or
microRNA-encapsulated nanoparticles.
U11-targeted nanoparticles were able to enhance gene delivery (pDNA and siRNA) to uPARpositive
cells. Nanoparticle platforms of lower zetapotential reduced non-specific electrostatic
interactions with cell membranes, hence allowed enhanced exhibition of the ligand’s targeting
effect. Adding further percentages of PEG increased nanoparticle stability, and maximised
ligand exposure by forcing the U11-associated PEG chains into a brush conformation. In vivo,
U11-nanoparticles indicated enhanced delivery efficacies of siRNA to models bearing small,
uPAR-positive tumours. In larger, uPAR-negative xenografts, the accumulation of both
targeted and non-targeted nanoparticles (by the enhanced permeation and retention effect)
into tumours was equal, and indicated reduced protein expression to the same extent. 24-
hours post-administration, accumulation into the liver and the spleen of targeted nanoparticles
was higher compared to non-targeted nanoparticles, most likely a result of increased
recognition of the nanoparticles by organ-specific macrophages.
Without doubt, the introduction of surface ligands can enhance nanoparticle deliver to cancer
cells, although careful organisation is required to maximise their effect. Low ligand densities,
ligand extension via a PEG spacer, high platform PEGylation and low zetapotentials can aid
the maximum exhibition of the ligand’s targeting effect. Such a presentation of surfaceligands
can increase nanoparticle interaction with cancer cell receptors, although it can also
lead to greater accumulation into the liver and spleen.