Dual-ligand α5β1 and α6β4 integrin targeting enhances gene delivery and selectivity to cancer cells

Nanoparticles functionalizedwith cancer-targeting ligands have shown promise but are still limited by off-tumor binding to healthy tissues that express lowlevels of the molecular target. Targeting two cancer biomarkers using dual-targeted heteromultivalent nanoparticles presents a possible solution to this challenge by requiring overexpression of two separate ligands for localization. In order to guide experimental design, a kinetic model was built to explore how the affinity and valency of dual-ligand liposomes affect the binding and selectivity of delivery to cells with various receptor expression. alpha(5)beta(1) and alpha(6)beta(4) integrin expression levels were quantified on 20 different cell lines to identify appropriatemodel cells for in vitro investigation. Dual-targeting heteromultivalent liposomes coveredwith polyethylene glycol (PEG) were synthesized using the PR_b peptide that binds to the alpha(5)beta(1) integrin and the AG86 peptide that binds to the alpha(6)beta(4) integrin. PEGylated liposomes with varying ratios of the targeting peptides were delivered to cells with different integrin concentrations. Nanoparticle binding and internalization as well as integrin internalization as a function of time were evaluated to understand the effect of valency and avidity on delivery. Results showed that of all formulations and cells tested, dual-ligand liposomes with equal ligand valencies achieved enhanced binding and selectivity for cancer cells expressing equal and high levels of receptor expression. These trends were consistent between theoretical and experimental results. The optimized liposomes were further used to achieve efficient and selective transfection in dual-receptor expressing cancer cells. With a quantitative understanding of dual-ligand liposome binding, the insights gained from this study can inform rational design of modular heteromultivalent nanoparticles for enhanced specificity to target tissue for the creation of more effective cancer treatments. (C) 2017 Elsevier B.V. All rights reserved.