Divalent Cations and Lipid Composition Modulate Membrane Insertion and Cancer-Targeting Action of pHLIP

The pH-Low Insertion Peptide (pHLIP) has emerged as an important tool for targeting cancer cells; it has been assumed that its targeting mechanism depends solely on the mild acidic environment surrounding tumors. Here, we examine the role of Ca²⁺ and Mg²⁺ on pHLIP's insertion, cellular targeting, and drug delivery. We demonstrate that physiologically relevant concentrations of either cation can shift the protonation-dependent transition by up to several pH units toward basic pH and induce substantial protonation-independent transmembrane insertion of pHLIP at pH as high as 10. Consistent with these results, the ability of pHLIP to deliver the cytotoxic compound monomethyl-auristatin-F to HeLa cells is increased several fold in presence of Ca²⁺. Complementary measurements with model membranes confirmed this Ca²⁺/Mg²⁺-dependent membrane-insertion mechanism. The magnitude of this alternative Ca²⁺/Mg²⁺-dependent effect is also modulated by lipid composition-specifically by the presence of phosphatidylserine-providing new clues to pHLIP's unique tumor-targeting ability in vivo. These results exemplify the complex coupling between protonation of anionic residues and lipid-selective targeting by divalent cations, which is relevant to the general signaling on membrane interfaces.