Orthogonal conjugation of anchoring-dependent membrane active peptides for tuning of liposome permeability.

Liposomes are widely utilized in drug delivery systems to enhance pharmacokinetics and reduce side effects. Bioresponsive membrane-active peptides (MAPs) can modulate the release of encapsulated drugs from liposomes, improving therapeutic efficacy. However, achieving efficient and specific conjugation of MAPs to liposomes remains challenging, complicating translational efforts. Thiol-maleimide Michael addition is an attractive strategy for lipid conjugation of cysteine-containing MAPs but is hindered by thiol oxidation. Strain-promoted azide-alkyne cycloaddition (SPAAC) presents an alternative conjugation approach, yet its effects on liposome and peptide functionality are not fully understood. Here, we demonstrate how these two conjugation strategies influence peptide-liposome interactions and lipid membrane permeability using a designed bioresponsive MAP. Both strategies result in MAP accumulation on liposomes, but their effects on membrane integrity and release dynamics differ significantly. SPAAC-based conjugation is generally much slower than the corresponding thiol-maleimide reaction. The inclusion of cholesterol in liposomes has a pronounced impact on both conjugation reactions, leading to phase separation and unexpected cross-reactivity. Accounting for these effects enabled orthogonal MAP-liposome functionalization and selective, sequential release from mixed liposome populations using different MAPs. These findings highlight the critical role of conjugation chemistry and lipid composition in designing MAP-based bioresponsive liposomal drug delivery systems. Understanding these interactions allows for the fine-tuning of liposomal formulations to optimize drug release, opening new avenues for enhancing the efficacy of liposomal therapeutics.