Date of Award

2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Electrical Engineering

First Advisor

Ying Sun

Abstract

The design and development of pH-sensitive peptides for cancer diagnostics provides an opportunity to study and address fundamental questions of in vivo bio-distribution of pH-sensitive peptides, as well as developing new targeting imaging and therapeutic agents for acidic diseased tissue such as cancer, infections, ischemia, stroke and others.

The main goal of the work presented here is to investigate the following problems:

- Targeting of highly metastatic mammary tumors and spontaneous breast tumors in transgenic mice with fluorescently labeled pHLIPs® (pH (Low) Insertion Peptides) pHLIP variants;

- Direct imaging of pHLIP insertion and cargo translocation in vivo;

- Targeting of mammary tumors using cyclic pH-sensitive peptides;

- Biodistribution of different pHLIP variants conjugated with various fluorescent dyes with the main purpose to identify the best pHLIP-based constructs for clinical applications.

It has been shown that extracellular acidity is associated with tumor progression. Elevated glycolysis and acidosis promote the appearance of aggressive malignant cells with enhanced multidrug resistance. The most effective pH-sensitive tumor targeting agents should sense pH at the surface of cancer cells, where it is expected to be the lowest. Thus, targeting tumor acidity might represent a novel approach for the prediction of tumor aggressiveness and delivery of therapeutic agents to tumor cells with the greatest metastatic potential. pHLIP belongs to the class of pH-sensitive agents capable of delivering imaging and/or therapeutic agents to cancer cells within tumors. Also, cyclic pH-sensitive peptides containing a number of Trp and Glu residues can be developed for imaging of acidity in tumors.

pHLIP insertion is associated with the protonation of Asp/Glu residues in the transmembrane sequence and its inserting end. Carboxyl group protonation leads to an increase in the hydrophobicity that further triggers transmembrane formation and insertion of the peptide across a lipid bilayer pHLIP insertion is predominantly uni-directional. In contrast to cell-penetrating peptides, pHLIP remains in the cellular membrane after insertion, translocating one end across the bilayer and comes out in the cytoplasm, while leaving the other end in the extracellular space. Therefore, pHLIP possesses dual delivery capabilities: it can translocate cell-impermeable cargo molecules into the cell cytoplasm or it can tether cargo molecules to the cell surface. Also, the process of peptide folding into a membrane ensures a much higher co-operativity of the transition compared to the pH-diffusion across a membrane. A family of novel pHLIP variants were introduced and demonstrated that tumor targeting, blood clearance and biodistribution of this peptides can be modulated by tuning their sequence and, as a result, their physical and chemical properties and their interaction with the cell membrane.

For demonstrating direct imaging of pHLIP insertion and cargo translocation in vivo, the cell-inserting end of the pHLIP-FIRE peptide has a fluorophore-fluorophore or fluorophore-quencher pair. A pair member is released by disulfide cleavage after insertion into the reducing environment inside a cell, resulting in de-quenching of the probe. Thus, the fluorescence of the pHLIP-FIRE probe is enhanced upon cell-insertion in the targeted tissues, but is suppressed elsewhere due to quenching.

The cyclic pH-sensitive peptides used in this work, contain: i) single cysteine (Cys, C) residue for conjugation purposes, ii) at least one tryptophan (Trp, W) for ability to record fluorescence signal, iii) 3-5 protonatable glutamic acid (Glu) residues to trigger pH-dependent interaction with membrane. As in the case of pHLIP peptides, the presence of low pH leads to the protonation of carboxyl groups of Glu residues, which increases peptides hydrophobicity and promotes partition of peptides in bilayer.

Thus, there is an opportunity to develop new imaging and/or therapeutic agents, which are based on the use of pH-sensitive peptides. We have successfully established the bio-distribution of both pHLIP and cyclic pH-sensitive peptides attached to different fluorescent dyes and identified the best candidate for imaging and therapeutic applications.

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