pHLIP® is a platform technology of pH-sensitive peptides: pH (Low) Insertion Peptides that exploit pH differences between healthy and diseased cells as a biomarker for targeting therapeutic and imaging/diagnostic agents to cells in diseased tissue.

pHLIP® peptide interactions with membrane

Molecular Mechanism

A pHLIP® peptide’s targeting responds to the pH at cell surfaces, where the pH is the lowest, thus providing high sensitivity. pHLIP® peptides exploit folding and insertion across the cell membrane, a cooperative process that gives high specificity.

Molecular mechanism of pHLIP® peptides insertion into lipid bilayer of membrane (Wyatt et al., Trends Biotechnol. 2017, 35, 653-664)

When the pHLIP® peptide (blue) encounters healthy tissue where the extracellular pH is around pH 7.4, the protonatable residues of pHLIP® peptide (red circles) remain deprotonated and negatively charged, and the peptide is located at or near the hydrophilic outside surface of the cellular membrane. Weakly bound to the membrane, pHLIP® is washed from the membrane via normal perfusion. When pHLIP® peptide encounters acidic, diseased tissue, it senses the low extracellular pH at cell surfaces (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 pHLIP® peptide have no net charge (green circles). The protonation leads to an increase in the overall hydrophobicity of the pHLIP® peptide, increasing the affinity of the peptide to the hydrophobic core of the cellular membrane and triggering pHLIP® to spontaneously fold into a helix and insert its C-terminus across the membrane, resulting in the formation of a stable 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 help to anchor pHLIP® peptide in the membrane.


Dual Delivery

pHLIP® peptides can be used to target and tether cargo molecules to the surfaces of cells in low pH environments of acidic diseased tissues. The cargo can be an optical marker, a PET, a SPECT, or a MR imaging agent, or an antigen or a protein delivered to induce certain cellular or immune responses.

Tethering Cargo to a Cell Surface

pHLIP® peptides can also be used for the intracellular delivery of payloads, facilitating the translocation of therapeutic cargoes across the membranes of cells with low extracellular pH, such as those cells found in acidic diseased tissue. These payloads are conjugated to the membrane-inserting end of pHLIP® peptide, typically via a link that is unstable inside the cell. Thus, pHLIP® peptide targets a payload to acidic diseased cells and flips the cargo across the plasma membrane, releasing it in the cytoplasm. Therapeutic cargoes may include toxins, metabolic activators/inhibitors, chemotherapeutic agents, or agents to alter gene expression.

Intracellular Delivery of Cargo

Tumor Acidity

Cancerous tumors are acidic (from glucose metabolism). The pH inside a normal cell and a cancer cell is the same (pHi=7.2). The pH outside of a normal cell is neutral (pHe=7.4), but the pH near a cancer cell is acidic (pHe=6.0-6.8). The pH is lowest at the surfaces of tumor cells (~pHs=6.0) compared to the bulk extracellular pH (pHe=6.8), and pH increases with distance from the cell membrane.

pH inside and outside of a normal cell and a cancer cell in tumors


pHLIP® peptides allows intracellular delivery of therapeutic cargo molecules for treatment of primary tumors as well as metastases. pH-selective pHLIP® peptide mediated inhibition of cancer cell proliferation has been shown in vitro and in vivo using a range of agents:

Cell-impermeable polar payloads: Polar drug molecules; immuno-simulators; gene-regulation agents, peptide-nucleic acids (PNA), which bind mRNA, miRNA, lncRNA; mushroom toxins; functional peptides; proteins

Cell-permeable drug payloads: DNA damaging agents; DNA repair inhibitors; Tubulin-binding agents; Kinase inhibitors.

Another use of pHLIP® technology is for the targeting of therapeutic agents (peptide, protein or immune cells) to the extracellular surfaces of cancer cells for selective activation of cellular signaling pathways, coagulation or immune responses.



Nuclear imaging methods, such as Positron Emission Tomography (PET) and Single-Photon Emission Computed Tomography (SPECT), rely on the delivery of radioactive imaging agents to diseased tissue. One of the main challenges in PET/SPECT imaging is to obtain images of targeted tissue that contrast highly with the image background. When a radionuclide is conjugated to the non-inserting terminus of a pHLIP® peptide, the resulting construct not only targets tumor tissue very well, but also remains securely in the tumor long enough to allow the excess, non-inserted construct to clear from normal tissues, resulting in higher contrast images. Success has been demonstrated using various radioisotopes conjugated to pHLIP® peptides. A 18F pHLIP® construct is in the process of clinical translation as novel nuclear imaging marker of diseased tissue acidity.

Fluorescence-guided surgery utilizing fluorescent molecules targeted by pHLIP® peptide could ease the challenges of tumor resection, such as visualization of all parts of cancerous lesions, ultimately reducing the number of surgeries that result in positive margins, thus, improving surgical outcomes and reducing tumor recurrences. Excellent targeting and labeling of primary tumors, lymph nodes, and sub-millimeter-sized metastatic lesions has been demonstrated by fluorescent pHLIP® peptides in various human and murine tumor models, transgenic mouse cancer models, and various human tissue specimens. Fluorescence Angiography (FA) allows visualization of blood flow during various surgical procedures. Currently, the main issue is that the agent is cleared very rapidly (minutes), which limits the observation time and requires multiple injections (up to 10 times) of fluorescent dyes during a single procedure. By conjugating pHLIP® peptide to an FDA approved near infrared fluorescent dye, indocyanine green (ICG), the time of circulation in blood is dramatically enhanced. ICG pHLIP® imaging agent provides outstanding contrast and extends observation time from a few minutes to a few hours in animal models, and is currently in clinical translation as a novel marker of blood flow and for visualization of cancerous lesions in primary tumors and lymph nodes.

Optoacoustic imaging is an emerging new technology with the potential to increase sensitivity and improve spatial resolution. It is a hybrid technique that incorporates advantageous properties of both light (for excitation) and sound (for detection), resulting in high-resolution of lesions deeply located in organs or tissues. Fluorescent pHLIP® peptides have been shown to be excellent probes for multispectral optoacoustic imaging. This new direction is rapidly developing and progressing toward clinical use.



pHLIP® peptides find a variety of uses in nanotechnology applications. Multiple pHLIP® peptides can be used to decorate a single nanoparticle, which can range in size from a few to hundreds of nanometers. Nanocarriers decorated with pHLIP® peptides are biocompatible, can target tumors, and demonstrate enhanced cellular uptake by cancer cells. Among the pHLIP® peptide-coated nanoparticles that have been investigated are lipid, polymer, and metal-based nanomaterials.

pHLIP® peptide-coated liposome for cytoplasmic delivery of polar and hydrophobic payloads (Pereira et al., J Biotechnol. 2015, 20, 88-97).