Visualizing pHLIP Insertion in Plasmamembrane and Endosomal Membrane

The pH-(Low) Insertion Peptide (pHLIP) has potential as a tumor-targeting drug carrier. At neutral pH, pHLIP has affinity for the surface of a lipid bilayer, whereas under slightly acidic conditions (e.g. pH ∼ 6) pHLIP inserts into the membrane, forming a transmembrane helix. Since many solid tumors are more acidic than healthy tissues, pHLIP may be used to translocate chemotherapeutic agents selectively into cancer cells. Knowledge of the exact location of pHLIP insertion in cells can guide the rational design of delivery constructs. We envision two scenarios for pHLIP insertion in cells: First, pHLIP may directly insert into the plasmamembrane; alternatively, cells may internalize pHLIP molecules via endocytosis, and subsequently pHLIP may insert into the endosomal membrane. In this study, several fluorescently self-quenched pHLIP constructs were synthesized to visualize to what extend these two scenarios are occurring in cells at pH 7.3 and 6.2. In these self-quenched pHLIP constructs, a rhodamine dye (TAMRA or Alexa Fluor 568) is attached to a C-terminal Lys residue, with the quencher QSY-9 conjugated to an adjacent Cys via a cleavable disulfide linker (or a stable thio-ether bond). Upon insertion, pHLIP would translocate its C-terminus into the cell cytoplasm, where cleavage of the disulfide linkage and release of the quencher QSY9 can take place. In turn, the pHLIP construct would become more fluorescent.