Mechanism and uses of a membrane peptide that targets tumors and other acidic tissues in vivo

Abstract

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.

Energetics of peptide (pHLIP) binding to and folding across a lipid bilayer membrane

“…In contrast to previously studied amphipathic peptides, it does not aggregate, form helical structure on the surface of a membrane, or induce pore formation (6)(7)(8)(9)12). Folding (formation of a transmembrane helix) accompanies insertion of the peptide into the lipid bilayer.…”

“…pHLIP contains two tryptophan residues having fluorescence that reports the attachment of the peptide to the membrane surface and the insertion of the peptide across the lipid bilayer as an ␣-helix (6,8,9). We chose 100-nm large unilamellar vesicles (LUV) containing palmitoyloleoylphosphatidylcholine (POPC) lipids with zwitterionic head groups to minimize any electrostatic component of the pHLIP interaction and to study peptide-membrane interactions over a wider range of temperatures than was possible in earlier work with dimyristoylphosphatidylcholine (DMPC) (6).…”

“…The number of protons involved in the process of protonation of pHLIP is Ϸ1.5-1.8. Previously, we demonstrated by mutation that the two Asp residues located in the transmembrane part of pHLIP are involved in protonation and peptide insertion/folding (8). Recognizing the nature of the hydrophobic gradient, partial titration of one of the Asp groups could account for the fractional number.…”

pH (low) insertion peptide (pHLIP) inserts across a lipid bilayer as a helix and exits by a different path

“…The pKa of the transition from state II to state III is 6.0 (6,8). The insertion is driven by the protonation of two Asp residues in the transmembrane region, leading to an increase of hydrophobicity that results in the folding of the peptide across a membrane (9). Increasing the pH promotes the unfolding and exit of the peptide from the core of the lipid bilayer.…”

Mixed-surface, lipid-tethered quantum dots for targeting cells and tissues

“…Second, the charge and hydrophobicity of surface ligands covering the QD can tune the degree of cell and tissue binding. We expect this phenomenon to regulate other targeting approaches using either hydrophobic alpha-helix forming peptides [34] or specific targeting peptides. [35] Finally, QDs lacking specific targeting mechanisms such as antibody-antigen or ligandreceptor interactions are able to spatially localize to both subcellular and anatomical structures.…”