Yana K. Reshetnyak - US grants

DESCRIPTION (provided by applicant): 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.

DESCRIPTION (provided by applicant): It is a central idea of the NIH that basic research will lead to new approaches in medicine, and we believe that we have found one. As a result of earlier funding of this grant, we have discovered a peptide that (1) targets acidic tissues in vivo, including tumors, (2) can deliver polar molecules into cells, releasing them in the cytoplasm, and (3) gives an opportunity to better understand how peptides can insert across membranes. We now plan to explore both the basic and applied aspects of this discovery. The peptide, which we call pHLIP (for pH (Low) Insertion Peptide) is soluble as an unstructured monomer in aqueous solution, binds as an unstructured monomer to the surface of a bilayer or membrane, and inserts across the bilayer as a trans-membrane helix (TM) when the pH is lowered. We have established the basic energetics and kinetics of peptide insertion. We have shown that a labeled version of pHLIP targets and images tumors as small as 1 mm in mice, and that the imaging accurately identifies tumor borders. We have also established that large, polar cargo molecules (M ~ 1000 Da, log P ~ -2) attached to the inserting end of pHLIP by a disulfide are delivered across membranes and released in the cytoplasms of cultured tumor cells at low pH. By continuing our basic research we hope to frame the technology for use in the clinic. We will study the process of and sequence requirements for insertion of water soluble peptides into membranes, find improved ways to target tumors and other acidic tissues, and develop expanded ways to deliver polar molecules into cells, releasing them into the cytoplasm by disulfide or ester cleavage. Targeting imaging agents to tumors with pHLIP could aid in diagnosis or act as a guide for surgery, and delivering therapeutics could assist in treatment. Using biophysical, biochemical, and biological approaches, we will seek answers to the following questions: 1. What are the kinetic intermediates, energetics and structures of the bilayer and peptide during insertion? 2. Which sequence features allow a water-soluble peptide to insert spontaneously to form a TM? 3. What role(s) do lipids play in TM insertion? 4. Can pHLIP be used to image cargo delivery in vivo? 5. What is the range of polar molecules that can be delivered to cells?

DESCRIPTION (provided by applicant): The acidity is associated with development of various pathological states such as solid tumors, ischemic stroke, neurotrauma, epileptic seizure, inflammation, infection, wounds, cystic fibrosis and others. Normal cell could be distinguished from highly glycolytic cell (for example, metastatic cancer cell) by transmembrane pH gradient and value of pH at surface of plasma membrane. We propose to develop novel tool to map pH at the extracellular and intracellular surfaces of individual cell in highly heterogeneous environment of cells in vivo. The tool would allow opening an opportunity to contribute in understanding of diseases progression and development of approaches of pH-based image-guided intervention. We will employ optical spectroscopic and imaging approaches, which allow achieving cellular resolution. Our strategy is based on use of peptides of pHLIP (pH Low Insertion Peptide) family. pHLIPs are water-soluble membrane peptides, which insert and fold in lipid bilayer of membrane only at slightly acidic conditions. Since the equilibrium is strongly shifted toward membrane inserted form at low pH, pHLIP injected into blood, circulates in body and accumulates in acidic tissue of tumors, site of inflammatory arthritis and ischemic regions. At 24 h after i.p. or i.v. administration of pHLIP, it is washed out completely from the blood and stays in plasma membrane of cells with low extracellular pH. pHLIP labeled with optical, PET or SPECT probes is considered to be first acidity markers, which are currently under development for clinical uses. We plan to conjugate pHLIP peptides of different pKa of insertion into membrane ranging from 4.5 to 6.5 with pH-sensitive fluorophore, SNARF-1. The main goal of using pHLIPs is to deliver and tether optical probe to the outer or inner leaflet of bilayer of plasma membrane. The SNARF-1 was selected, since it demonstrates shift of the emission spectra in response to pH, which solves the problem of calibration for the probe concentration. The probe will be attached to the N- or C- terminus of pHLIPs. In first case, SNARF-1 will stay in the extracellular space being tethered to the cell surface. On the other hand, when SNARF-1 would be conjugated with the peptide inserting end (C-terminus), pHLIP would flip SNARF-1 across the bilayer and expose it to the intracellular space, while keeping it close to the inner leaflet of membrane. Thus, we propose to measure pH from the outer and inner leaflets of plasma membrane and identify transmembrane pH gradient. Experiments in solution, 2D and 3D cell culture, as well as on mouse cancer models will be performed. Our goals are: - to map pH at the surface of cancer cells in a process of cell division and migration in 3D culture; - to map pH on the surface of individual cells in tumors implanted into mice; - to monitor kinetics of pH changes at the surface of cancer cells in real time induced by the glucose infusion; - to establish the minimal size of metastatic and non-metastatic tumors, which can acidify microenvironment below pH 7.0. PUBLIC HEALTH RELEVANCE: The acidity is associated with development of various pathological states such as solid tumors, ischemia, stroke, inflammation, infection, wounds, cystic fibrosis and others. We propose to develop a novel tool to map pH at the extracellular and intracellular surfaces of individual cell.