pH-Low Insertion Peptides (pHLIPs) as Conjugates, for Imaging and Therapeutics

pH-Low Insertion Peptides (pHLIPs) are water-soluble peptides derived from the transmembrane C-helix of bacteriorhodopsin. These peptides selectively target, stain, and deliver therapeutic or diagnostic agents to acidic microenvironments (pH), including solid tumors, ischemic tissue, and sites of chronic inflammation.

John Hunt, during his Ph.D. work with Engelman in 1993-1997, studying membrane protein folding, discovered the first pH-sensitive peptide of the pHLIP family. During his studies, he synthesized seven polypeptides corresponding to the α-helices of the membrane protein bacteriorhodopsin (BR). He studied the structure of each polypeptide in phospholipid vesicles to test the then-current idea that they would be independently stable in a membrane (Hunt et al., 1997). Six of these seven peptides were found to be stable and irreversibly associated with reconstituted vesicles near neutral pH. However, one peptide corresponding to the C-helix in the primary sequence of BR was only weakly associated with lipid vesicles. It could be recovered at significant concentrations in a lipid-free supernatant following sedimentation of reconstituted vesicles.

Hunter et al. found that the BR-C peptide is soluble at neutral pH in aqueous buffers free of both detergents and chaotropic agents. The peptide's solubility allows it to interact with phospholipid membranes. Membrane insertion of the peptide is pH-dependent and occurs rapidly at room temperature with a pK of 6.0, rapid and fully reversible.

pHLIPs respond to environmental pH variations by forming transmembrane α-helices. Localized extracellular acidity in solid tumors can be exploited for cancer diagnosis and treatment. To this end, the 36-residue pH-low insertion peptide has been developed as an imaging tool and a carrier of therapeutic agents, and as an important model system for studying α-helix formation in the membrane environment and α-helix insertion into the lipid bilayer. This is an example how basic scientific studies, in this case the study or peptide folding in membranes, allow the development of commercial products.

Anatomy of a pHLIP Peptide

A pHLIP peptide consists of an N-terminal flanking region followed by the membrane insertion α-helical peptide sequence and a C-terminal flanking region.

Luecke et al. (1999) solved the crystal structures of the Asp96 to Asn mutant of the light-driven proton pump bacteriorhodopsin and its M photointermediate produced by illumination at ambient temperature to 1.8 and 2.0 angstrom resolution ( 1C8S). As of July 2026, over 300 structures of bacteriorhodopsin have been solved.

pHLIP peptides originate from one bacteriorhodopsin peptide.

Reshetnyak et al. (2007) studied the transition states of pHLIP peptides. These peptides transition between three states state, (I), the solution state, at physiological pH (7.4) in which pHLIP exists as a soluble, unstructured monomer in the aqueous environment, (II), pHLIP attached to the membrane bilayer, here the peptide adsorb to the surface of a lipid bilayer while maintaining its unstructured, monomeric form, and (III) pHLIP inserted across the bilayer, in acidic environments at pH 6.5 or lower, in which specific aspartic and glutamic acid residues become protonated, increasing the peptide's hydrophobicity and forcing it to fold into an α-helix that inserts entirely across the lipid membrane.

This study used the following pHLIP variants:

pHLIP-1: GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT,

pHLIP-2: AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG,

pHLIP-3: ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG.

Andreev et al. (2014) observed that members of the pHLIP family of peptides bind to cell surfaces at all pH values and therefore sense pH at the surfaces of cancer cells, where pH is the lowest, further accentuating the pH sensitivity of the peptides making them useful tools for delivering cargo molecules inside cancer cells, for example, by attaching the cargo to the inserting end of the peptides, or for tethering cargo to the cell membrane, for example, by conjugating to the non-inserting end of the peptides.

Adochite et al. (2014) utilized the pHLIPs to target breast cancer for monitoring metabolic changes and to deliver imaging and therapeutic agents to tumors selectively. The pHLIPs WT, Var3, and Var7 were used for this study.

Shu et al. (2015) showed that pHLIP binds to a membrane at pH 7.4 in an unstructured conformation but folds across the bilayer as a transmembrane helix at pH∼6. The researchers utilized NMR spectroscopy to study residue-specific conformation and membrane locations of membrane-associated pHLIP at pH 7.4, 6.4, and 5.3. They found that the critical membrane-adsorbed state is more complex than previously envisioned. At pH 6.4, for the major unstructured population, the peptide sinks deeper into the membrane in a state II′ that is distinct from the adsorbed state II observed at pH 7.4, which may enable pHLIP to sense slight change in acidity even before insertion.

Wei et al. (2017) investigated superparamagnetic iron oxide nanoparticles (SPION) as contrast agents for noninvasive tumor magnetic resonance imaging (MRI). SPIONs are contrast-enhancing agents used in noninvasive magnetic resonance imaging for tumors. In this study, Wei et al. tested the effectiveness of pH-responsive SPIONs via pHLIP to target acidic tumor microenvironments, using Polylysine polymers to modify the SPIONs with pHLIP to achieve pH-responsive capability. SPION pHLIP nanoclusters of 64, 82, 103, and 121 nm size were then assembled by the pH-responsive polymers in a size-controlled manner. The pH-responsive SPION nanoclusters (64 nm) showed the most effective pH-responsive retention in cells and tumor-selective imaging in MRI, demonstrating that pH-responsive pHLIP nanoclusters can target different tumors and facilitate noninvasive tumor diagnosis using MRI.

According to DuPont et al. (2023), targeted antigen delivery activates the immune system to kill cancer cells. DuPont et al. reported the targeted delivery of several epitopes, including a peptide, a small molecule, and a sugar, to tumors by pHLIPs. Epitopes linked to the extracellular ends of pH Low Insertion Peptide were positioned at the surfaces of tumor cells and were recognized by corresponding anti-epitope antibodies. DuPont et al. utilized pHLIP Var3 and 2,4-dinitrophenyl (DNP) and alpha-galactose (Gal) as tri-Gal for the synthesis of peptide conjugates. Conjugating DNP-maleimide, DNP-Peg4-NHS, DNP-Peg12-NHS, and GAL-maleimide to pHLIP allows imaging of cell membranes. Conjugating specific epitopes to the extracellular end of pHLIP enables targeted extracellular delivery to tumors to induce an immune response.

A study by Dhuri et al. (2023) compared two pHLIP gamma-peptide nucleic acids (γPNA) conjugates targeting the seed region of microRNA‑155 (miR‑155), an oncogenic miRNA upregulated in multiple cancers. The pHLIP‑serine γPNA and pHLIP‑diethylene glycol γPNA conjugates utilized pHLIP to selectively insert the conjugates into acidic tumor microenvironments without binding to normal tissue. The study used a U2932‑derived xenograft mouse model to assess biodistribution, tumor accumulation, and miR‑155 silencing effects, and all‑atom molecular dynamics simulations to evaluate lipid bilayer insertion and interaction energetics. This study found that pHLIP‑serine γPNAs outperformed pHLIP‑diethylene glycol γPNAs in both in vitro uptake and in vivo tumor targeting, with computational modeling supporting the serine variant’s optimal membrane insertion. Computational results for pHLIP‑serine γPNA showed the most favorable transmembrane insertion energetics. In vitro, the serine‑based construct exhibited higher cellular uptake and stronger RNA binding than the diethylene glycol variant. In vivo, the pHLIP‑serine γPNA achieved superior tumor accumulation and miR‑155 knockdown compared to the diethylene glycol version. Both constructs targeted tumor tissue via pH‑dependent insertion, but the serine variant’s performance was consistently better. These results support pHLIP‑serine γPNA as a promising platform for tumor‑specific RNA silencing, with potential for further optimization in preclinical and clinical contexts. The combination of pH‑targeted membrane insertion and RNA‑targeting specificity may improve therapeutic index and reduce off‑target effects.

Reshetnyak et al. (2024) reviewed the targeting characteristics of pHLIP peptides. This review showed that rather than targeting a single cell-surface receptor protein, which often varies between tumor types and clones, pHLIP targets the low extracellular pH (pHe 6.0 to 6.8) characteristic of the tumor microenvironment. Tumor acidity results from elevated glycolysis (the Warburg effect) and oxidative phosphorylation, coupled with carbonic anhydrase activity across cancer cells, stromal cells, and immune cells. Because acidosis is a hallmark of solid tumors regardless of specific receptor expression, pHLIP acts as a broad, tumor-agnostic targeting platform. pHLIPs derived from the transmembrane helix of bacteriorhodopsin undergo a pH-triggered conformational transition: In states I & II at physiological pH (~7.4), key acidic residues, aspartic and glutamic acid, are negatively charged. The peptide remains unstructured and soluble or loosely associated with the cell surface, allowing it to be cleared from healthy tissues without toxic systemic accumulation. In state III at low pH (<7.0), the acidic environment induces protonation of carboxyl side chains, reducing the net negative charge and increasing overall hydrophobicity. Protonation triggers a coil-to-helix transition that drives the peptide to insert unidirectionally across the lipid bilayer as a transmembrane α-helix.

Ruchi et al. (2025) reviewed the progression of antisense oligonucleotides (ASOs) from basic proof-of-concept to targeted genetic therapies, highlighting how chemical modifications and advanced delivery systems such as bioconjugates and nanocarriers can overcome delivery challenges like nuclease degradation and extrahepatic tissue targeting.

The introduction of phosphorothioate (PS) linkages and other sugar modifications provides crucial resistance to nuclease degradation, greatly increasing metabolic stability. Conjugating ASO with ligands such as GalNAc has been a breakthrough, enabling highly specific delivery to hepatocytes in the liver. However, to expand the utility of ASOs beyond the liver, researchers are exploring non-viral vectors and conjugates, including fatty acids and peptides.

Table: List of Bacteriorhodopsin and pHLIP peptides

Please note: This article is for educational purpose only. The pHLIP Technology can be licensed from Yale, the University of Rhode Island, and the Memorial Sloan Kettering Cancer Center (https://physics.uri.edu/biophysics/phlip-technology/). The licenses include exclusive Worldwide commercial rights to the linear and cyclic pH peptides patent families and pHLIP® trademark. https://phlipinc.com/about/, PHLIP.INC 2026. https://phlipinc.com/news-2/

References

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