Discovery of a Macropinocytosis‐Inducing Peptide Potentiated by Medium‐Mediated Intramolecular Disulfide Formation

Macropinocytosis is a ubiquitous cellular uptake mechanism of peptide‐based intracellular delivery. This entry pathway shows promise as a route for the intracellular uptake of biomacromolecules and nanoparticles. In this work, we obtained the 8‐residue analogue P4A bearing higher macropinocytosis induction ability. P4A contains vital cysteine residues in its sequence, which immediately reacts with cystine in culture medium to convert into its oxidized forms, including the intramolecularly oxidized form (oxP4A) as the dominant and active species. The conjugate of oxP4A and the membrane lytic peptide LK15 delivered bioactive proteins into cells; notably, this peptide delivered functional proteins fused with a negatively charged protein tag at a significantly reduced amount (up to nanomolar range) without compromising the delivery efficiency and the cellular activities of delivered proteins. Macropinocytosis is as a ubiquitous cellular delivery route. A short macropinocytosis‐inducing peptide P4A is identified that undergoes thiol‐disulfide exchange reaction, yielding a potent intramolecular oxidized analogue (oxP4A). The conjugate of oxP4A and the membrane lytic peptide LK15 delivered functional proteins. This peptide delivers proteins fused with a supernegatively charged protein tag at nanomolar concentrations.