Theoretical and in Vitro Understanding of the Effect of Ligand-Receptor Valency on Dual-Ligand Nanoparticle Gene Delivery to Cancer Cells

Cancer biomarker targeting has greatly improved cancer specific delivery of therapeutics, but its effectiveness is limited by on-target, off-tumor delivery. It was proposed that off-tumor delivery can be minimized with simultaneous targeting to two cancer specific receptors, requiring the high expression of both receptors uniquely present in cancer cells for efficient delivery. To test this hypothesis, we produced dual-ligand stealth liposomes functionalized with α₅β₁ and α₆β₄ integrin specific ligands. Binding affinity of multivalent nanoparticles is dependent on ligand valency, receptor valency and monovalent ligand-receptor affinity. Therefore, heteromultivalent nanoparticle binding is a complex interaction dependent upon valency and affinity of each ligand-receptor pair, as well as the ratios of these variables. A kinetic model was created to guide the design of this complex system. Dual-ligand liposomes were produced with varying ligand ratios and delivered to model cell lines with varying expression levels and ratios of the α₅β₁ and α₆β₄ integrins. Nanoparticle binding and internalization as well as integrin internalization rates were also 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 expressing cancer cells. With a quantitative understanding of dual ligand liposome binding, the insights gained from this study can inform future design of heteromultivalent nanoparticles for cancer therapy delivery.