Purpose Malignant cancer foci develop acidic extracellular environments. Mild acidic conditions trigger insertion and folding of the pH (low) insertion peptide (pHLIP™) across a cellular membrane, enabling targeting of such lesions. Procedures We employed optical imaging to follow targeting by fluorescent pHLIP given i.v. in mice. For visualization, Cy5.5 and Alexa750 were covalently attached to the N terminus of pHLIP, which stays outside of a cell membrane after transmembrane insertion. Results We demonstrate that pHLIP targets: (a) tumors of different origins established by subcutaneous injection of cancer cells, (b) spontaneous prostate tumors in TRAMP mice and (c) metastatic lesions in lung. pHLIP accumulation in tumors correlates with tumor aggressiveness. Within a tumor, it stains extracellular spaces and cellular membranes. Conclusions Tissue acidity can be detected by pHLIP peptide insertion and used to diagnose primary tumors, metastatic lesions, and lipid bodies in necrotic tissues. The ability of pHLIP to differentially bind metastatic and non-metastatic tumors may provide a new approach for evaluating cancer prognosis.

Progress in nanomedicine depends on the development of nanomaterials and targeted delivery methods. In this work, we describe a method for the preferential targeting of gold nanoparticles to a tumor in a mouse model. The method is based on the use of the pH Low Insertion Peptide (pHLIP), which targets various imaging agents to acidic tumors. We compare tumor targeting by nonfunctionalized nanogold particles with nanogold–pHLIP conjugates, where nanogold is covalently attached to the N terminus of pHLIP. Our most important finding is that both intratumoral and i.v. administration demonstrated a significant enhancement of tumor uptake of gold nanoparticles conjugated with pHLIP. Statistically significant reduction of gold accumulation was observed in acidic tumors and kidney when pH-insensitive K-pHLIP was used as a vehicle, suggesting an important role of pH in the pHLIP-mediated targeting of gold nanoparticles. The pHLIP technology can substantially improve the delivery of gold nanoparticles to tumors by providing specificity of targeting, enhancing local concentration in tumors, and distributing nanoparticles throughout the entire tumor mass where they remain for an extended period (several days), which is beneficial for radiation oncology and imaging.

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.

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.

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.

We have developed an improved tool for imaging acidic tumors by reporting the insertion of a transmembrane helix: the pHLIP-Fluorescence Insertion REporter (pHLIP-FIRE). In acidic tissues, such as tumors, peptides in the pHLIP family insert as α-helices across cell membranes. The cell-inserting end of the pHLIP-FIRE peptide has a fluorophore–fluorophore or fluorophore–quencher pair. A pair member is released by disulfide cleavage after insertion into the reducing environment inside a cell, resulting in dequenching of the probe. Thus, the fluorescence of the pHLIP-FIRE probe is enhanced upon cell-insertion in the targeted tissues but is suppressed elsewhere due to quenching. Targeting studies in mice bearing breast tumors show strong signaling by pHLIP-FIRE, with a contrast index of ∼17, demonstrating (i) direct imaging of pHLIP insertion and (ii) cargo translocation in vivo. Imaging and targeted cargo delivery should each have clinical applications.

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 pH 50 of 6.1 while most solid tumors have extracellular pH (pH e ) of 6.5-7.0. To close this gap, a ** We thank Prof. Dr. Donald M. Engelman (Yale) for discussion and support; Prof. Dr. Eriks Rozners and Prof. Dr. Susan Bane (both of SUNY-Binghamton) for frequent use of their fluorimeter and plate reader, respectively. We also thank Raemer J. Lapid (for assistance in processing data), Rebecca A. Chandler, Meghan M. Bell, Vladyslav Nazarenko, Ilana G. Bandler (for assistance in cell assays) and Emma A. Gordon (for assistance in synthesis). This work was supported by SUNY-Binghamton University (BU) (Start-up funds to M.A. and L.Y., various financial supports to J.O., L.K. and M.C., HHMI-BU undergraduate summer fellowships to C-H. E. and R. L.), NIH (R01-GM073857 for training of M.A. and initial work), and NSF grant CHE-0922815 for the Regional NMR Facility (600 MHz instrument) at SUNY-Binghamton.

The acid-labile behavior of di-substituted maleamic acid (DMA) and its equilibrium with di-substituted maleimide (DMI) are exploited to build an ultra acid-sensitive, small molecule prodrug that can be activated by tumor extracellular pH (pHe) in the range of 6.5-6.9. Such a DMA prodrug reversed the unfavorable pH-profile of doxorubicin (Dox), which may improve its therapeutic window.