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 pH-Low Insertion Peptide (pHLIP) offers the potential to deliver drugs selectively to the cytoplasm of cancer cells based on tumor acidosis. The WT pHLIP inserts into membrane with a pH50 of 6.1 while most solid tumors have extracellular pH (pHe) of 6.5-7.0. To close this gap, a SAR study was carried out to search for pHLIP variants with improved pH-response. We learned that (a) replacing Asp25 with α-aminoadipic acid (Aad) adjusts the pH50 to 6.74, matching average tumor acidity, and (b) replacing Asp14 with γ-carboxyglutamic acid (Gla) increases the sharpness of pH-response (i.e. transition over 0.5 instead of 1 pH unit). These effects are additive — the Asp14Gla/Asp25Aad double variant shows a pH50 of 6.79, with sharper transition than Asp25Aad. Further, the advantage of the double variant over WT pHLIP in terms of cargo delivery was demonstrated in turn-on fluorescence assays and anti-proliferation studies (using paclitaxel as cargo) in A549 lung cancer cells at pH 6.6.

SUMMARY Our goal is to define the properties of cell-impermeable cargo molecules that can be delivered into cells by pHLIP (pH (Low) Insertion Peptide), which can selectively target tumors in vivo based on their acidity. Using biophysical methods and fluorescence microscopy, we show that pHLIP can successfully deliver polar and membrane-impermeable cyclic peptides linked to its C-terminus through the membranes of lipid vesicles and cancer cells. Our results also indicate that the translocation of these cargo molecules is pH-dependent and mediated by transmembrane helix formation. Since a broad range of cell-impermeable molecules is excluded from discovery efforts because they cannot traverse membranes on their own, we believe that pHLIP has the potential to expand therapeutic options for acidic tissues such as tumors and sites of inflammation.

The pH-low insertion peptide (pHLIP) binds to a membrane at pH 7.4 unstructured but folds across the bilayer as a transmembrane helix at pH∼6. Despite their promising applications as imaging probes and drug carriers that target cancer cells for cytoplasmic cargo delivery, the mechanism of pH modulation on pHLIP-membrane interactions has not been completely understood. Here, we show the first study on membrane-associated pHLIP using solid-state NMR spectroscopy. Data on residue-specific conformation and membrane location describe pHLIP in various surface-bound and membrane-inserted states at pH 7.4, 6.4 and 5.3. The critical membrane-adsorbed state is more complex than previously envisioned. At pH 6.4, for the major unstructured population, the peptide sinks deeper into the membrane in a state II′ that is distinct from the adsorbed state II observed at pH 7.4, which may enable pHLIP to sense slight change in acidity even before insertion.

The pH-low insertion peptide (pHLIP) inserts into membranes and forms a transmembrane (TM) α-helix in response to slight acidity, and has shown great potential for cancer diagnosis and treatment. As a lead, pHLIP is challenging to optimize because the mechanism of its pH-dependent membrane interactions is not completely understood. Within pHLIP there are multiple D/E residues which could sense the pH change, the particular role played by each of them in the protonation-driven insertion process is not clear. The precise location of the TM helix within the pHLIP sequence is also unknown. In this work, solid-state NMR spectroscopy is used to address these central questions. Tracing backbone conformations revealed that the TM helix spans from A10 to D33 with a break at T19 to P20. Residue-specific pKa values of D31, D33, D25, and D14 were determined to be 6.5, 6.3, 6.1, and 5.8, respectively, and define the sequence of protonations which lead to insertion. Furthermore, possible intermediate states which disrupt membranes at pH 6.4 were proposed based on tryptophan fluorescence quenching and NMR data.