Abstract 5137: Utility of pH (low) insertion peptide (pHLIP® peptide) variants in drug delivery
Authors: Wyatt Linden C. , Moshnikova Anna , Crawford Troy , Andreev Oleg A. , Reshetnyak Yana K. 
Published: July 2017

Introduction: The pH (Low) Insertion Peptides (pHLIP® peptides) show utility in a wide variety of medical applications due to their ability to target cancer cells by exploiting the acidity that is ubiquitous to tumor tissue. Targeting tumor acidity offers many advantages over traditional biomarker targeting and has allowed pHLIP® peptides to target primary tumor tissue as well as sub-millimeter metastases. Previously, pHLIP®s conjugated to PET/SPECT tracers or fluorescent labels have found applications in diagnostic nuclear imaging and fluorescence-guided surgery. The purpose of this study was to evaluate a diverse collection of pHLIP® variants, including those incorporating non-standard amino acids, consisting of two or four pHLIP®s linked together, and one of de novo design, in order to determine the pHLIP® peptide best suited for the targeting and intracellular delivery of therapeutic cargo molecules to primary tumors and metastases.

Methods: Biophysical experiments, namely fluorescence and circular dichroism spectroscopy, using liposomes as model membranes, were used to study the localization of pHLIP® variants at the bilayer surface at high pH, the tertiary structure and orientation within the membrane of the folded variants at low pH, the parameters of pH-dependent peptide insertion, and the rate at which insertion occurs. In vitro experiments, wherein the polar toxin amanitin was conjugated to the membrane-inserting end of the pHLIP® variants via a cleavable disulfide bond, were used to study intracellular cargo delivery and the resulting inhibition of cancer cell proliferation. In vivo experiments, wherein the fluorescent dye Alexa Fluor 546 was conjugated to the non-inserting end of the pHLIP® variants via a non-cleavable bond, were used to study biodistribution and tumor uptake.

Results: All investigated pHLIP® variants demonstrated the behavior characteristic of wild-type pHLIP® with some differences in membrane affinity and membrane insertion profiles, namely pKa of insertion and cooperativity. Inhibition of cancer cell proliferation was induced by the pH–dependent translocation of amanitin across the plasma membrane by the pHLIP® variants. Biophysical measurements, values of EC50 obtained at pH 6.0 and pH 7.4, and fluorescence imaging of triple-negative breast tumors in mice using pHLIP® peptides were analyzed collectively.

Conclusions: Different pHLIP® variants are best suited for different purposes, such as the intracellular delivery of highly or moderately toxic polar or hydrophobic payloads to primary tumor tissue as well as metastases. pHLIP® Variant 3 (Var3), exhibited the most favorable properties for the targeted delivery of amanitin to tumor tissue.

Acknowledgement: This research was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R01GM073857 to OAA and YKR.

Citation Format: Linden C. Wyatt, Anna Moshnikova, Troy Crawford, Oleg A. Andreev, Yana K. Reshetnyak. Utility of pH (low) insertion peptide (pHLIP® peptide) variants in drug delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5137. doi:10.1158/1538-7445.AM2017-5137