Unlocking pH-responsive dual payload release through hydrazone linkage chemistry

The capacity for simultaneous intracellular delivery of two payloads to various organelles, in a targeted and programmable manner, would represent a powerful tool to probe cellular function and responses or deliver synergistic combination therapies. While only single pathways are currently probed, this work introduces an acid-labile trifunctional hydrazone linker that releases two types of payloads, which we term payload W and payload Z. As a key and controlling feature, initial acid-mediated release of payload W triggers release of payload Z with 1:1 stoichiometry via intramolecular cyclization. An azide group is also built into the linker structure to allow optional conjugation to nanoparticles (NPs). Through overcoming significant synthetic challenges, we have prepared six target acylhydrazone linkers and evaluated their stability over a range of pH values. An acyl acetophenone hydrazone linker (linker 3) displays a particularly promising release profile, supporting the feasibility of the novel dual-release concept through high stability at physiological pH but rapid release of both payloads under pH conditions similar to those in late endosomal and lysosomal compartments (pH 4.5-5.5) or tumor sites (pH 6.5). Therefore, linker 3 holds the potential as an ideal candidate carrier for future nanoparticle conjugation, offering a mechanism for dual drug release after endosomal entrapment. A particularly promising application would be in combination therapy for controlled intracellular delivery of doxorubicin (DOX) and a nitric oxide (NO) donor, or proteins and/or siRNA and small molecules, to enable diverse synergistic treatment strategies.