Our project is based on the use of a water-soluble membrane peptide, pHLIP, which we have shown, by whole-body fluorescence and PET imaging, to selectively target acidic solid tumors in vivo and to translocate polar cargo molecules into the cytoplasms of cultured cancer cells. pHLIP inserts unidirectionally across the lipid bilayer of a cell membrane as a monomer under mildly acidic conditions, as are found in tumors and forms a transmembrane alpha helix, whereas there is practically no insertion across the membranes of cells with the normal extracellular pH of healthy tissue. To date, no toxic effects of pHLIP exposure have been observed either for cells in culture or for mice. Here we propose to develop a nanotechnology platform for selective delivery of imaging and therapeutic agents to tumors based on the use of the pHLIP-bionanosyringe. By attaching cargo molecules to the end of pHLIP that stays outside of the membrane, we can anchor imaging or therapeutic probes to the surfaces of cancer cells, facilitating diagnosis, treatment and therapeutic monitoring. By attaching cargo to its inserted end via cleavable links, pHLIP can be used for the selective translocation of polar, cell-impermeable molecules into cancer cells. By combining the efforts of three laboratories, a broad development of this promising technology will be possible. We will use pHLIP targeting to test cancer models and establish how tumor growth and development correlate with tumor acidity. To improve pHLIP technology, we will design, synthesize and test various dendrimeric-pHLIP constructs to enable delivery of multiple therapeutic and/or imaging probes to tumors. We will introduce a synthetic scheme of simultaneous conjugation of cargo molecules and fluorescent dyes to the C-terminus of pHLIP via a cleavable S-S bond and establish the properties (polarity, shape, charge and size) of cargo molecules that pHLIP can translocate through the lipid bilayer of a membrane, defining a new, polar class of therapeutic molecules that can be delivered for tumor treatment. We will test pHLIP for the intracellular delivery of two functional cell-impermeable molecules in vivo: a toxin (phalloidin) and a gene regulation agent (Peptide Nucleic Acid). Importantly, we will attempt the simultaneous detection and treatment of tumors by labeled pHLIP-phalloidin, which is our first lead for a potential antimetastatic drug. Further, we will develop a two-step delivery scheme for the specific tethering and assembly of nanoparticles at the surfaces of cancer cells in vivo: 1) targeting tumors using pHLIP with a binding domain, which will be tethered to the surface of cancer cells and 2) targeting the pHLIP with liposomes containing therapeutic and/or imaging payloads and having a surface-exposed complementary binding domain. Inspired by the properties of pHLIP in its current version, we will further evaluate the effect of pHLIP sequence variation on peptide insertion into a membrane, enabling the design of a second generation of the nanosyringe with a range of useful properties. pHLIP nanotechnology offers a new approach for the disease-specific imaging and treatment of cancers. Our ultimate goal is to improve the diagnosis and treatment of cancer, which is responsible for about 25% of all deaths in the USA and other developed countries. There are several aspects of the problem where our technology development could be useful, but the major concept is the selective delivery of therapeutic and imaging agents to cells in tumors. Another aspect of the technology is that it permits the use of a new class of therapeutic agents: cell-impermeable molecules that would be translocated into cells only in diseased tissue while not affecting healthy cells. A therapy based on these concepts would exhibit much higher efficacy and/or significantly reduced side effects. Such improvements are especially important for cancer treatment, since the majority of anti-cancer drugs are poisons that damage normal cells. ? ? ?
| Antosh, Michael P; Wijesinghe, Dayanjali D; Shrestha, Samana et al. (2015) Enhancement of radiation effect on cancer cells by gold-pHLIP. Proc Natl Acad Sci U S A 112:5372-6 |
| Pereira, Mohan C; Arachchige, Mohan C M; Reshetnyak, Yana K et al. (2015) Advanced targeted nanomedicine. J Biotechnol 202:88-97 |
| Tapmeier, Thomas T; Moshnikova, Anna; Beech, John et al. (2015) The pH low insertion peptide pHLIP Variant 3 as a novel marker of acidic malignant lesions. Proc Natl Acad Sci U S A 112:9710-5 |
| Adochite, Ramona-Cosmina; Moshnikova, Anna; Carlin, Sean D et al. (2014) Targeting breast tumors with pH (low) insertion peptides. Mol Pharm 11:2896-905 |
| Cruz-Monserrate, Zobeida; Roland, Christina L; Deng, Defeng et al. (2014) Targeting pancreatic ductal adenocarcinoma acidic microenvironment. Sci Rep 4:4410 |
| Andreev, Oleg A; Engelman, Donald M; Reshetnyak, Yana K (2014) Targeting diseased tissues by pHLIP insertion at low cell surface pH. Front Physiol 5:97 |
| Karabadzhak, Alexander G; An, Ming; Yao, Lan et al. (2014) pHLIP-FIRE, a cell insertion-triggered fluorescent probe for imaging tumors demonstrates targeted cargo delivery in vivo. ACS Chem Biol 9:2545-53 |
| Fendos, Justin; Barrera, Francisco N; Engelman, Donald M (2013) Aspartate embedding depth affects pHLIP's insertion pKa. Biochemistry 52:4595-604 |
| Yao, Lan; Daniels, Jennifer; Danniels, Jennifer et al. (2013) pHLIP peptide targets nanogold particles to tumors. Proc Natl Acad Sci U S A 110:465-70 |
| Moshnikova, Anna; Moshnikova, Valentina; Andreev, Oleg A et al. (2013) Antiproliferative effect of pHLIP-amanitin. Biochemistry 52:1171-8 |
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