Solid tumors often develop an acidic environment due to the Warburg effect. The effectiveness of diagnosis and therapy may therefore be enhanced by the design and use of pHsensitive agents that target acidic tumors. Recently, a novel technology was introduced to target acidic tumors using pH low insertion peptide (pHLIP), a peptide that inserts across cell membranes as an A-helix when the extracellular pH (pH e ) is acidic. In this study, we expanded the application of the pHLIP technology to include positron emission tomography imaging of the acidic environment in prostate tumors using 64 Cu conjugated to the pHLIP ( 64 Cu-DOTA-pHLIP). Studies showed that this construct avidly accumulated in LNCaP and PC-3 tumors, with higher uptake and retention in the LNCaP tumors. Uptake correlated with differences in the bulk pH e of PC-3 and LNCaP tumors measured in magnetic resonance spectroscopy experiments by the 31 P chemical shift of the pH e marker 3-aminopropylphosphonate. This article introduces a novel class of noninvasive pH-selective positron emission tomography imaging agents and opens new research directions in the diagnosis of acidic solid tumors. [Cancer Res 2009;69(10):4510-6]

We find that pH-(low)-insertion-peptide (pHLIP)-facilitated translocation of phalloidin, a cell-impermeable polar toxin, inhibits the proliferation of cancer cells in a pH-dependent fashion. The monomeric pHLIP inserts its C terminus across a membrane under slightly acidic conditions (pH 6-6.5), forming a transmembrane helix. The delivery construct carries phalloidin linked to its inserting C terminus via a disulfide bond that is cleaved inside cells, releasing the toxin. To facilitate delivery of the polar agent, a lipophilic rhodamine moiety is also attached to the inserting end of pHLIP. After a 3 h incubation at pH 6.1-6.2 with 2-4 μM concentrations of the construct, proliferation in cultures of HeLa, JC, and M4A4 cancer cells is severely disrupted (>90% inhibition of cell growth). Treated cells also show signs of cytoskeletal immobilization and multinucleation, consistent with the expected binding of phalloidin to F actin, stabilizing the filaments against depolymerization. The antiproliferative effect was not observed without the hydrophobic facilitator (rhodamine). The biologically active delivery construct inserts into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipid bilayers with an apparent pK a of ∼6.15, similar to that of the parent pHLIP peptide. Sedimentation velocity experiments show that the delivery construct is predominantly monomeric (>90%) in solution under the conditions employed to treat cells (pH 6.2, 4 μM). These results provide a lead for antitumor agents that would selectively destroy cells in acidic tumors. Such a targeted approach may reduce both the doses needed for cancer chemotherapy and the side effects in tissues with a normal pH. drug delivery | hydrophilic drugs | cytoplasmic delivery | targeting acidity | targeted chemotherapy

The membrane-associated folding/unfolding of pH (low) insertion peptide (pHLIP) provides an opportunity to study how sequence variations influence the kinetics and pathway of peptide insertion into bilayers. Here, we present the results of steady-state and kinetics investigations of several pHLIP variants with different numbers of charged residues, with attached polar cargoes at the peptide's membrane-inserting end, and with three single-Trp variants placed at the beginning, middle, and end of the transmembrane helix. Each pHLIP variant exhibits a pH-dependent interaction with a lipid bilayer. Although the number of protonatable residues at the inserting end does not affect the ultimate formation of helical structure across a membrane, it correlates with the time for peptide insertion, the number of intermediate states on the folding pathway, and the rates of unfolding and exit. The presence of polar cargoes at the peptide's inserting end leads to the appearance of intermediate states on the insertion pathway. Cargo polarity correlates with a decrease of the insertion rate. We conclude that the existence of intermediate states on the folding and unfolding pathways is not mandatory and, in the simple case of a polypeptide with a noncharged and nonpolar inserting end, the folding and unfolding appears as an all-or-none transition. We propose a model for membrane-associated insertion/folding and exit/unfolding and discuss the importance of these observations for the design of new delivery agents for direct translocation of polar therapeutic and diagnostic cargo molecules across cellular membranes.

We develop a method for pH-dependent fusion between liposomes and cellular membranes using pHLIP® (pH Low Insertion Peptide), which inserts into lipid bilayer of membrane only at low pH. Previously we establish the molecular mechanism of peptide action and show that pHLIP can target acidic diseased tissue. Here we investigate how coating of PEGylated liposomes with pHLIP might affect liposomal uptake by cells. The presence of pHLIP on the surface of PEGylated-liposomes enhanced membrane fusion and lipid exchange in a pH dependent fashion, leading to increase of cellular uptake and payload release, and inhibition of cell proliferation by liposomes containing ceramide. A novel type of pH-sensitive, “fusogenic” pHLIP-liposomes was developed, which could be used to selectively deliver various diagnostic and therapeutic agents to acidic diseased cells.

Drug molecules are typically hydrophobic and small in order to traverse membranes to reach cytoplasmic targets, but we have discovered that more polar molecules can be delivered across using water-soluble, moderately hydrophobic membrane peptides of the pHLIP® (pH Low Insertion Peptide) family. Delivery of polar cargo molecules could expand the chemical landscape for pharmacological agents that have useful activity but are too polar by normal drug criteria. The spontaneous insertion and folding of the pHLIP peptide across a lipid bilayer seeks a free energy minimum, and insertion is accompanied by a release of energy that can be used to translocate cell-impermeable cargo molecules. In this study we report our first attempt to tune the hydrophobicity of a polar cargo, phallacidin, in a systematic manner. We present the design, synthesis and characterization of three phallacidin cargoes, where the hydrophobicity of the cargo was tuned by the attachment of diamines of various lengths of hydrophobic chains. The phallacidin cargoes were conjugated to pHLIP, and shown to selectively inhibit the proliferation of cancer cells in a concentration-dependent manner at low pH.