Orthogonal Conjugation of Anchoring-Dependent Membrane Active Peptides for Tuning of Liposome Permeability

Abstract

Liposomes are widely utilized in drug delivery systems to enhance drug pharmacokinetics and reduce side effects. Bioresponsive membrane active peptides (MAPs) can be employed to modulate the release of encapsulated drugs from liposomes to improve the therapeutic efficacy, but their selective and controlled conjugation to liposomes remains a challenge, which complicates translational efforts. Thiol-maleimide Michael addition is attractive for lipid conjugation of cysteine containing MAPs but suffers from issues with thiol oxidation. Strain-promoted azide-alkyne cycloaddition (SPAAC) is an alternative emerging conjugation strategy but the effects of SPAAC on liposome and peptide functionality are not fully understood. Here we demonstrate how these two different conjugation strategies for anchoring of MAPs to liposomes affect peptide-liposome interactions and lipid membrane permeability. Both conjugation strategies result in efficient accumulation of MAPs on the liposomes, but their impact on membrane integrity and release dynamics varies significantly. Notably, the presence of cholesterol in liposomes dramatically influences the conjugation reactions, leading to unexpected cross-reactivity and differences in lipid phase behaviors. Taking these effects into account enabled orthogonal MAP liposome functionalization and selective sequential release from mixed populations of liposomes using different MAPs. These findings underscore the critical role of conjugation chemistry and lipid composition in the design of MAP-based bioresponsive liposomal drug delivery systems. Understanding these interactions enables the fine-tuning of liposomal formulations for optimized drug release, offering new avenues for enhancing the efficacy of therapeutic agents.