The membrane peptide pH (low) insertion peptide (pHLIP) lives in three worlds, being soluble in aqueous solution at pH 7.4, binding to the surface of lipid bilayers, and inserting as a transbilayer helix at low pH. With low pH driving the process, pHLIP can translocate cargo molecules attached to its C-terminus via a disulfide and release them in the cytoplasm of a cell. Here we examine a key aspect of the mechanism, showing that pHLIP is monomeric in each of its three major states: soluble in water near neutral pH (state I), bound to the surface of a membrane near neutral pH (state II), and inserted across the membrane as an alpha-helix at low pH (state III). The peptide does not induce fusion or membrane leakage. The unique properties of pHLIP made it attractive for the biophysical investigation of membrane protein folding in vitro and for the development of a novel class of delivery peptides for the transport of therapeutic and diagnostic agents to acidic tissue sites associated with various pathological processes in vivo.

The pH-selective insertion and folding of a membrane peptide, pHLIP [pH (low) insertion peptide], can be used to target acidic tissue in vivo, including acidic foci in tumors, kidneys, and inflammatory sites. In a mouse breast adenocarcinoma model, fluorescently labeled pHLIP finds solid acidic tumors with high accuracy and accumulates in them even at a very early stage of tumor development. The fluorescence signal is stable for >4 days and is approximately five times higher in tumors than in healthy counterpart tissue. In a rat antigen-induced arthritis model, pHLIP preferentially accumulates in inflammatory foci. pHLIP also maps the renal cortical interstitium; however, kidney accumulation can be reduced significantly by providing mice with bicarbonatecontaining drinking water. The peptide has three states: soluble in water, bound to the surface of a membrane, and inserted across the membrane as an ␣-helix. At physiological pH, the equilibrium is toward water, which explains its low affinity for cells in healthy tissue; at acidic pH, titration of Asp residues shifts the equilibrium toward membrane insertion and tissue accumulation. The replacement of two key Asp residues located in the transmembrane part of pHLIP by Lys or Asn led to the loss of pH-sensitive insertion into membranes of liposomes, red blood cells, and cancer cells in vivo, as well as to the loss of specific accumulation in tumors. pHLIP nanotechnology introduces a new method of detecting, targeting, and possibly treating acidic diseased tissue by using the selective insertion and folding of membrane peptides.cancer targeting ͉ imaging ͉ peptide insertion M any pathological conditions such as cancer, ischemic stroke, inflammation, atherosclerotic plaques, and others are associated with increased metabolic activity and hypoxia resulting in an elevated extracellular acidity (1-6). Hypoxia and acidity have emerged as important factors in tumor biology and responses to cancer treatment. Imaging of hypoxic and acidic regions could provide new information about disease localization and progression and might enhance diagnosis and therapy. Here we describe the use of a peptide, pHLIP [pH (low) insertion peptide], to selectively accumulate in and label acidic tissues. We previously reported that the pHLIP bionanosyringe, a 36-aa peptide derived from the bacteriorhodopsin C helix, has three states: soluble in water, bound to the surface of a membrane, and inserted across the membrane as an ␣-helix. At physiological pH the water-soluble form is favored, whereas at acidic pH the transmembrane ␣-helix predominates (Fig. 1a) (7). We show that at low pH, pHLIP can translocate cell-impermeable cargo molecules that are disulfide-linked to the C terminus across a cell membrane, where they are released in the cytoplasm by reduction of the disulfide bond (8, 9). Among the successfully translocated molecules are organic dyes, phalloidin (a polar, cyclic peptide), and a 12-mer peptide nucleic acid.

. We now use a related peptide, pH (low) insertion peptide, to translocate cargo molecules attached to its C terminus across the plasma membranes of living cells. Translocation is selective for low pH, and various types of cargo molecules attached by disulfides can be released by reduction in the cytoplasm, including peptide nucleic acids, a cyclic peptide (phalloidin), and organic compounds. Because a high extracellular acidity is characteristic of a variety of pathological conditions (such as tumors, infarcts, stroke-afflicted tissue, atherosclerotic lesions, sites of inflammation or infection, or damaged tissue resulting from trauma) or might be created artificially, pH (low) insertion peptide may prove a useful tool for selective delivery of agents for drug therapy, diagnostic imaging, genetic control, or cell regulation.drug delivery ͉ peptide nucleic acid delivery ͉ tumors ͉ membrane transport ͉ helix formation W e suggest that spontaneous insertion and formation of transbilayer ␣-helices can be used as an approach to moving chemical reagents through the permeability barrier of biological membranes. Membrane proteins, which represent Ϸ30% of all ORFs in sequenced genomes (1, 2), play a key role in many fundamental cellular processes, such as signal and energy transduction, active transport, ion flow, cell-cell interactions, and nerve conduction. Based on the two-stage model of helical membrane protein folding, the formation of independently stable helices across the membrane lipid bilayer occurs first, and then the interaction of the helices with each other leads to the stabilization of higher-order structure (3, 4). Folding and insertion of large-membrane protein domains into targeted membranes require active participation of complex translocation machineries (5-7), whereas insertion of short (Ͻ50-to 60-residue) protein sequences can occur spontaneously (8-10). In recent work, some cases of apparent spontaneous insertion have been shown to require a membrane protein, YidC (11). We previously reported that a polypeptide derived from the bacteriorhodopsin C helix, consisting of the transmembrane sequence and two flanking sequences, is soluble in aqueous solution and spontaneously inserts across lipid bilayers in a pH-dependent manner (12, 13), with no requirement for additional proteins such as YidC. Here we report that a related peptide, pH (low) insertion peptide (pHLIP), which inserts across a cell membrane at low pH (Ͻ7.0), can translocate a drug or imaging molecule into the cell and release it in the cytoplasm (Fig. 1a). ResultsAt neutral pH, a polypeptide derived from the bacteriorhodopsin C helix binds weakly to the surface of a liposome, whereas at acidic pH (Ͻ7.0) it inserts across a membrane and forms a transmembrane ␣-helix (12, 13). Our strategy is to conjugate a cargo molecule by means of a disulfide bond to the end of the transmembrane peptide that inserts into the cell, where the reducing environment of the cytoplasm will break the disulfide and release the cargo. Our first task was to identi...