The early detection of cancer is recognized by the American Cancer Society as the most effective way to improve survival outcomes, but can only be accomplished by developing diagnostic agents that can target smaller, earlier stage tumors. For example, a state-of-the-art cancer-specific imaging technique is 18F-Fluorodeoxyglucose Positron Emission Tomography (FDG-PET), which can locate tumors in vivo with a spatial resolution of ~1 cm. Magnetic Resonance Imaging (MRI) has far greater tissue contrast resolution than PET and spatial resolution of <1 mm, but lacks an imaging agent that can target a cancer hallmark, glycolytic metabolism. Developing stimuli-responsive imaging pharmaceuticals that localize passively in vivo via one of cancer’s generic hallmarks rather than specific biomarkers can prove effective in developing an MRI agent that can specifically image cancer.
One attractive hallmark target is the acidic extracellular microenvironment of tumor tissue (pHe 6.6-7.4) that arises due to the enhanced rate of glycolysis in cancer cells. Creating a material that is nano-sized in blood, but upon reaching the acidic extracellular tumor environment, transforms into a larger, more slowly diffusing object could serve as a novel mechanism for achieving high accumulation of imaging agents at the tumor site compared to the bloodstream.
Work in this dissertation includes the design, synthesis and characterization of self-assembling peptide amphiphile (PA) MRI contrast agents that reversibly transform from spherical micelles into nanofibers (microns in length) when entering the acidic tumor vasculature. The PA molecules consist of four segments: a hydrophobic alkyl tail, a ß-sheet-forming peptide sequence, a charged amino acid sequence and a DO3A-Gd MRI moiety, where DO3A=1,4,7-tris (carboxymethylaza) cyclododecane-10-azaacetyl amide. The PAs were synthesized via the solid phase technique, purified by High Performance Liquid Chromatography and characterized by mass spectrometry, analytical HPLC and peptide content analyses. Circular dichroism spectroscopy, critical aggregation concentration measurements and transmission electron microscopy were used to characterize the transitions and create concentration-pH phase diagrams for selected PAs. Finally, fluorescence anisotropy (FA) was used to probe self assembly in blood serum.
After extensive assessment of structure-property relationships in a series (>40) of rationally designed PAs, we developed the chemical insight for how this transitional pH can be systematically tuned with alkyl chain length, ß-sheet sequence, and number of charged residues. A ratio of one hydrophobic to three charged amino acids was necessary to enable this transition in the desired pH range. Finally, we successfully created a vehicle that transitions in blood serum at pH 7.0 using FA with 1.5% of the PA labeled with a Ru(bipy)3 2+ fluorophore. Surprisingly, albumin does not bind to these anionic PAs as it does to cationic and hydrophobic surfactants, but instead promotes nanofiber formation due to a molecular crowding effect. We also established that 150 mM NaCl, 2.2 mM CaCl2, and 1.8 mM of 20 kDa PEG replicates the ionic strength and crowding of pure serum. MRI relaxivity values of water protons in presence of the PA were found to be higher than that for a commercially available Magnevist control, providing a secondary mechanism for enhanced tumor detection.