Identification and optimization of tunable endosomal escape parameters for enhanced efficacy in peptide-targeted...

Endosomal escape of nanoparticles (NPs) is a weighty consideration for engineering successful nanomedicines. Although it is well-established that incorporation of histidine (His) in particle design improves endosomal escape for NPs, our understanding of its effects for ligand-targeted nanoparticles (TNPs) remains incomplete. Here, we systematically evaluated the cooperativity between targeting ligands and endosomolytic elements using liposomal TNPs with precise stoichiometric control over functional moieties (>90% loading efficiency). We synthesized endosomolytic lipid conjugates consisting of 1 to 10 consecutive His residues presented at the end of linkers between 2 to 45 repeating units of ethylene glycol (His-EG). His-EG had minimal effect on NP size (∼115 nm) and had no significant effect on the receptor specificity of TNPs (>90% inhibition by competing peptide). We evaluated various formulations with 8 different targeting ligands relevant to two disease models. Incorporation of His₁-EG₈ resulted in up to ∼170- and ∼12.9-fold enhancement in intracellular accumulation relative to non-endosomolytic NP and TNP, respectively. These observations were time-dependent, targeted receptor-dependent, and showed different trends for NPs and TNPs. Further evaluation demonstrated short linkers (EG_2-4) significantly enhanced nanoparticle internalization compared to EG₈ or longer by up to ∼2.5-fold. Finally, rationally optimized formulation, His₁-EG₂-TNP, improved in vitro toxicity of a DM1 prodrug to SK-BR-3 cells by ∼4.2-fold, with IC₅₀ ∼8.5 nM compared to ∼36 nM for no-His TNP, and >100 nM for non-targeted/no-His NP. This study uncovers an intricate relationship between endosomal escape and ligand-targeted drug delivery, as well as tunable parameters. Furthermore, our findings highlight the value of rational design and systematic analysis for optimization of multifunctional NPs.