Applications of pHLIP Technology for Cancer Imaging and Therapy

Abstract

Acidity is a biomarker of cancer that is not subject to the blunting clonal selection effects that reduce the efficacy of other biomarker technologies, such as antibody targeting. The pH (low) insertion peptides (pHLIP^®s) provide new opportunities for targeting acidic tissues. Through the physical mechanism of membrane-associated folding, pHLIPs are triggered by the acidic microenvironment to insert and span the membranes of tumor cells. The pHLIP platform can be applied to imaging acidic tissues, delivering cell-permeable and impermeable molecules to the cytoplasm, and promoting the cellular uptake of nanoparticles. Since acidosis is a hallmark of tumor development, progression, and aggressiveness, the pHLIP technology may prove useful in targeting cancer cells and metastases for tumor diagnosis, imaging, and therapy.

Keywords: acidity; drug delivery; imaging; nanotechnology; tumor targeting.

Figure I. The main features of sequences of the pHLIP peptide family

Members of the peptide of pHLIP family follow a similar pattern in terms of peptide sequence: an N-terminal flanking region (yellow) consists mainly of polar residues; a transmembrane region (red) consists mainly of hydrophobic residues, but also includes protonatable residues which prevent insertion at physiological pH; and a C-terminal flanking region (green), which may not present in all peptides, contains a few additional protonatable residues. Cargo can be conjugated either to the C- or N-terminus via single cysteine or lysine residues.

Figure I. pHLIP delivery capability

pHLIPs are used to increase the tumor targeting and delivery of various types of cargoes, such as imaging agents and therapeutics, by conjugating cargo to pHLIP in a one-to-one ratio (A). pHLIPs can also be used to decorate nanoparticles (B), to increase the biocompatibility, tumor targeting and promote cellular uptake of nanoparticles by cancer cells.

Figure 1. pHLIP membrane interaction and insertion

The peptides of the pHLIP family (blue) exist in equilibrium between solvated and membrane-adsorbed conformation at the normal extracellular pH found in healthy tissue, whereas peptides insert across cellular membrane at the low extracellular pH found in acidic, diseased tissue.

Figure 2. Mechanism of pHLIP insertion into the cellular membrane

When the pHLIP (blue) encounters healthy tissue where the extracellular pH is around pH 7.4, the protonatable residues of the pHLIP (red circles) remain deprotonated and negatively charged, and the peptide resides at or near the hydrophilic surface of the cellular membrane. Weakly bound to the membrane, the pHLIP is washed from the membrane via normal perfusion and continues to circulate through the body. Cancer cells, however, produce excess acidity as a consequence of their malfunctioning metabolisms and overexpression of certain surface proteins, and pump these acidic byproducts out of the cell interior in order to maintain comfortable conditions inside the cell, resulting in the acidification of tumor tissue. When the pHLIP encounters tumor tissue, it senses the low extracellular pH at the cancer cell surface (i.e., the concentration of protons (cyan circles) at the surface of the cellular membrane is high), and the protonatable residues and negatively charged C-terminal carboxyl group of the pHLIP become neutrally charged (green circles). The protonation leads to an increase in the overall hydrophobicity of the pHLIP, increasing the affinity of the peptide to the hydrophobic core of the cellular membrane and triggering the pHLIP to spontaneously fold into a helix and insert across the membrane, resulting in the formation of a transmembrane helix. When the C-terminal protonatable residue and carboxyl group are then exposed to the normal intracellular pH of the cell, they are deprotonated, again becoming negatively charged, and anchor the pHLIP in the membrane.

Figure 3. Two uses for pHLIPs: tethering cargo to the cell surface or translocating cargo across the membrane into the cytoplasm

A pHLIP (blue) can be used to target and tether cargo molecules to the surfaces of cells in low pH environments (A). The cargo could be an optical marker, a PET or SPECT imaging agent, or an antigen or protein delivered to induce certain cellular processes. A pHLIP can also be used for the intracellular delivery of payloads, translocating cargo (green) across the membranes of cells with low extracellular pH, such as those cells found in acidic, diseased tissue (B). These payloads are conjugated to the membrane-inserting end of the pHLIP, typically via a cleavable link (magenta), and could include toxins, chemotherapeutic agents, or agents to alter gene expression.

Figure 4. A postulated pathway for fusion and cellular uptake of pHLIP-coated liposomes

pHLIP-coated liposomes interact with the membrane and promote either direct fusion with the lipid bilayer of the membrane (A), or uptake via the endocytotic pathway (B), followed by fusion with the endosomal membrane. Both uptake mechanisms result in the cytoplasmic delivery of encapsulated polar cargo (green), or delivery of lipid membrane-incorporated hydrophobic payloads to the membrane (red). Schematic is not to scale.