Dual-receptor targeted strategy in nanoparticle design achieves tumor cell selectivity through cooperativity

Targeted liposomal nanoparticles are commonly used drug delivery vehicles for targeting cancer cells that overexpress a particular cell surface receptor. However, typical target receptors are also expressed at variable levels in healthy tissue, leading to non-selective targeting and systemic toxicity. Here, we demonstrated that the selectivity of peptide-targeted liposomes for their target cells can be significantly enhanced by employing a dual-receptor targeted approach to simultaneously target multiple tumor cell surface receptors. The dual-receptor targeted approach can be tuned to create cooperativity in binding only for the cancer cells, therefore leaving the healthy cells and tissue unharmed. We evaluated this strategy in a multiple myeloma disease model where the liposomes were functionalized with two distinct peptide antagonists to target VLA-4 and LPAM-1, two receptors with increasing relevance in multiple myeloma. By employing a multifaceted strategy to synthesize dual-receptor targeted liposomes with high purity, reproducibility, and precisely controlled stoichiometry of functionalities, we identified optimal design parameters for enhanced selectivity via systematic analysis. Through control of the liposomal formulation and valency of each targeting peptide, we identified that the optimal dual-receptor targeted liposome consisted of a peptide density of 0.75% VLA4pep and 1% LPAM1pep, resulting in an 8-fold and 12-fold increased cellular uptake over VLA-4 and LPAM-1 single targeted liposomes respectively. This formulation resulted in a cooperative ratio of 4.3 and enhanced uptake for myeloma cells that simultaneously express both VLA-4 and LPAM-1 receptors, but displayed no increase in uptake for cells that express only one or neither of the receptors, resulting in a 28-fold selectivity of the dual-targeted liposomes for cells displaying both targeted receptors over cells displaying neither receptor. These results demonstrated that through refined design and well-characterized nanoparticle formulations, dual-receptor targeted liposomes have the potential to improve cancer therapy by providing enhanced selectivity over conventional single-receptor targeted approaches.