Oleg A. Andreev

Mechanism and uses of a membrane peptide that targets tumors and other acidic tissues 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 bicarbonate-containing drinking water. The peptide has three states: soluble in water, bound to the surface of a membrane, and inserted across the membrane as an α-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.

A novel technology for the imaging of acidic prostate tumors by positron emission tomography.

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 pH-sensitive 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 alpha-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.

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

The pH low-insertion peptide (pHLIP) serves as a model system for peptide insertion and folding across a lipid bilayer. It has three general states: (I) soluble in water or (II) bound to the surface of a lipid bilayer as an unstructured monomer, and (III) inserted across the bilayer as a monomeric α-helix. We used fluorescence spectroscopy and isothermal titration calorimetry to study the interactions of pHLIP with a palmitoyloleoylphosphatidylcholine (POPC) lipid bilayer and to calculate the transition energies between states. We found that the Gibbs free energy of binding to a POPC surface at low pHLIP concentration (state I–state II transition) at 37°C is approximately −7 kcal/mol near neutral pH and that the free energy of insertion and folding across a lipid bilayer at low pH (state II–state III transition) is nearly −2 kcal/mol. We discuss a number of related thermodynamic parameters from our measurements. Besides its fundamental interest as a model system for the study of membrane protein folding, pHLIP has utility as an agent to target diseased tissues and translocate molecules through the membrane into the cytoplasm of cells in environments with elevated levels of extracellular acidity, as in cancer and inflammation. The results give the amount of energy that might be used to move cargo molecules across a membrane.

pHLIP peptide targets nanogold particles to tumors

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.

Family of pH (low) insertion peptides for tumor targeting

Cancer is a complex disease with a range of genetic and biochemical markers within and among tumors, but a general tumor characteristic is extracellular acidity, which is associated with tumor growth and development. Acidosis could be a universal marker for cancer imaging and the delivery of therapeutic molecules, but its promise as a cancer biomarker has not been fully realized in the clinic. We have discovered a unique approach for the targeting of acidic tissue using the pH-sensitive folding and transmembrane insertion of pH (low) insertion peptide (pHLIP). The essence of the molecular mechanism has been elucidated, but the principles of design need to be understood for optimal clinical applications. Here, we report on a library of 16 rationally designed pHLIP variants. We show how the tuning of the biophysical properties of peptide–lipid bilayer interactions alters tumor targeting, distribution in organs, and blood clearance. Lead compounds for PET/single photon emission computed tomography and fluorescence imaging/MRI were identified, and targeting specificity was shown by use of noninserting variants. Finally, we present our current understanding of the main principles of pHLIP design.

pH-(low)-insertion-peptide (pHLIP) translocation of membrane impermeable phalloidin toxin inhibits cancer cell proliferation.

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 pKa 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.

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

What are the molecular events that occur when a peptide inserts across a membrane or exits from it? Using the pH-triggered insertion of the pH low insertion peptide to enable kinetic analysis, we show that insertion occurs in several steps, with rapid (0.1 sec) interfacial helix formation, followed by a much slower (100 sec) insertion pathway to give a transmembrane helix. The reverse process of unfolding and peptide exit from the bilayer core, which can be induced by a rapid rise of the pH from acidic to basic, proceeds approximately 400 times faster than folding/insertion and through different intermediate states. In the exit pathway, the helix–coil transition is initiated while the polypeptide is still inside the membrane. The peptide starts to exit when about 30% of the helix is unfolded, and continues a rapid exit as it unfolds inside the membrane. These insights may guide understanding of membrane protein folding/unfolding and the design of medically useful peptides for imaging and drug delivery.

pH-sensitive membrane peptides (pHLIPs) as a novel class of delivery agents

Abstract Here we review a novel class of delivery vehicles based on pH-sensitive, moderately polar membrane peptides, which we call pH (Low) Insertion Peptides (pHLIPs), that target cells located in the acidic environment found in many diseased tissues, including tumours. Acidity targeting by pHLIPs is achieved as a result of helix formation and transmembrane insertion. In contrast to the earlier technologies based on cell-penetrating peptides, pHLIPs act as monomeric membrane-inserting peptides that translocate one terminus across a membrane into the cytoplasm, while the other terminus remains in the extracellular space, locating the peptide in the membrane lipid bilayer. Therefore pHLIP has a dual delivery capability: it can tether cargo molecules or nanoparticles to the surfaces of cells in diseased tissues and/or it can move a cell-impermeable cargo molecule across the membrane into the cytoplasm. The source of energy for moving polar molecules attached to pHLIP through the hydrophobic layer of a membrane bilayer is the membrane-associated folding of the polypeptide. A drop in pH leads to the protonation of negatively charged residues (Asp or Glu), which enhances peptide hydrophobicity, increasing the affinity of the peptide for the lipid bilayer and triggering peptide folding and subsequent membrane insertion. The process is accompanied by the release of energy that can be utilized to move cell-impermeable cargo across a membrane. That the mechanism is now understood, and that targeting of tumours in mice has been shown, suggest a number of future applications of the pHLIP technology in the diagnosis and treatment of disease.

Measuring Tumor Aggressiveness and Targeting Metastatic Lesions with Fluorescent pHLIP

PurposeMalignant cancer foci develop acidic extracellular environments. Mild acidic conditions trigger insertion and folding of the pH (low) insertion peptide (pHLIPTM) across a cellular membrane, enabling targeting of such lesions.ProceduresWe 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.ResultsWe 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.ConclusionsTissue 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.

Tuning the Insertion Properties of pHLIP

The pH (low) insertion peptide (pHLIP) has exceptional characteristics: at neutral pH it is an unstructured monomer in solution or when bound to lipid bilayer surfaces, and it inserts across a lipid bilayer as a monomeric alpha-helix at acidic pH. The peptide targets acidic tissues in vivo and may be useful in cancer biology for delivery of imaging or therapeutic molecules to acidic tumors. To find ways to vary its useful properties, we have designed and analyzed pHLIP sequence variants. We find that each of the Asp residues in the transmembrane segment is critical for solubility and pH-dependent membrane insertion of the peptide. Changing both of the Asp residues in the transmembrane segment to Glu, inserting an additional Asp into the transmembrane segment, or replacing either of the Asp residues with Ala leads to aggregation and/or loss of pH-dependent membrane insertion of the peptide. However, variants with either of the Asp residues changed to Glu remained soluble in an aqueous environment and inserted into the membrane at acidic pH with a higher pK(app) of membrane insertion.