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. [Display omitted] •Ca2+ and Mg2+ promote the membrane insertion of pHLIP at neutral to basic pH.•Divalent cations modulate the insertion pKa of pHLIP.•Divalent cations promote pHLIP cellular insertion and drug delivery.•Effect of divalent cations is modulated by lipid composition.