Versatile Click Linker Enabling Native Peptide Release from Nanocarriers upon Redox Trigger

Nanocarriers have shown their ability to extend the circulation time of drugs, enhance tumor uptake, and tune drug release. Therapeutic peptides are a class of drug compounds in which nanocarrier-mediated delivery can potentially improve their therapeutic index. To this end, there is an urgent need for orthogonal covalent linker chemistry facilitating the straightforward on-the-resin peptide generation, nanocarrier conjugation, as well as the triggered release of the peptide in its native state. Here, we present a copper-free clickable ring-strained alkyne linker conjugated to the N-terminus of oncolytic peptide LTX-315 via standard solid-phase peptide synthesis (SPPS). The linker contains (1) a recently developed seven-membered ring-strained alkyne, 3,3,6,6-tetramethylthiacycloheptyne sulfoximine (TMTHSI), (2) a disulfide bond, which is sensitive to the reducing cytosolic and tumor environment, and (3) a thiobenzyl carbamate spacer enabling release of the native peptide upon cleavage of the disulfide via 1,6-elimination. We demonstrate convenient "clicking" of the hydrophilic linker-peptide conjugate to preformed pegylated core-cross-linked polymeric micelles (CCPMs) of 50 nm containing azides in the hydrophobic core under aqueous conditions at room temperature resulting in a loading capacity of 8 mass % of peptide to polymer (56% loading efficiency). This entrapment of hydrophilic cargo into/to a cross-linked hydrophobic core is a new and counterintuitive approach for this class of nanocarriers. The release of LTX-315 from the CCPMs was investigated in vitro and rapid release upon exposure to glutathione (within minutes) followed by slower 1,6-elimination (within an hour) resulted in the formation of the native peptide. Finally, cytotoxicity of LTX CCPMs as well as uptake of sulfocyanine 5-loaded CCPMs was investigated by cell culture, demonstrating successful tumor cell killing at concentrations similar to that of the free peptide treatment.