Self-Assembling Peptide Amphiphile Contrast Agents As a Tumor Diagnostic Tool
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Self-Assembling Peptide Amphiphile Contrast Agents as a Tumor Diagnostic Tool Thesis Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Mark A. Haverick B.S Graduate Program in Chemistry The Ohio State University 2011 Master’s Examination Committee: Professor Joshua Goldberger, Advisor Professor Michael Tweedle, Advisor Professor Thomas Magliery Copyright by Mark A. Haverick 2011 Abstract Cancer is a disease that affects millions of people each year, with early detection often enabling the most effective treatment. A characteristic shared amongst tumors is an acidic extracellular matrix, resulting from the constant glycolytic cycle required to produce energy for uncontrolled replication. The Enhanced Permeablity and Retention (EPR) effect describes the ability of macromolecules to enter tumor tissue through “leaky” vasculature and temporarily evade clearance from the body. Combining these ideas, it may be possible to increase tumor detection through an active and passive targeting approach with creative molecular design. Biomaterials have been developed for use in many biological applications such as tissue engineering, cellular signaling, and tumor imaging. Specifically, peptide amphiphiles are a class of biocompatible molecules comprised of amino acids and lipids known to self-assemble into ordered structures including spherical micelles, cylindrical micelles, and ribbons. The work presented herein describes the development of a self- assembling peptide amphiphile (PA), capable of dynamically transitioning into nanofibers in a pH range corresponding to the extracellular vasculature of tumor tissue (pH 6.4-7.4). We have explored the role of molecular design on the pH dependent self- assembly behavior through a combination of techniques: circular dichroism (CD), ii transmission electron microscopy (TEM), cryo-TEM, critical aggregation concentration (CAC) measurements, and pKa titrations. This work has produced a series of self- assembling PA molecules that assemble into nanofibers when the pH is reduced from 7.4 to 6.6, in isotonic salt solutions simulating the acidic extracellular environment of cancer cells. This transition is rapid and reversible, indicating the system to be under thermodynamic equilibrium. By fine-tuning the attractive hydrophobic and hydrogen bonding forces with repulsive electrostatic forces, the single molecule to nanofiber transition pH can be systematically shifted. This transition occurs despite incorporating MRI imaging moieties onto these molecules, making them prospective candidates as cancer imaging agents. iii Dedication This document is dedicated to my family . iv Acknowledgments There are many people that played a crucial role in my success with this thesis. I would like to wholeheartedly thank my advisors Dr. Joshua Goldberger and Dr. Michael Tweedle, for their continued support and always being available, whether it was to brainstorm ideas or to give me the motivation needed to succeed during desperate times. All the members of the Goldberger Lab played an instrumental role in the success of this thesis, especially Arijit Ghosh and Keith Stump for all their efforts to move this project along. A special thanks to all the members of the Tweedle lab especially, Dr. Natarajan Raju, Dr. Shankaran Kothandaraman, and Dr. Krishan Kumar for their daily guidance in the lab, our lengthy discussions in times of frustrations have helped me grow not only as a scientist but also as a person. I would like to thank Xiangyu Yang and Rick Layman for their help in collecting MRI data. To my family, thank you for believing in me and giving me the support I needed to complete this difficult task. Finally, I would like to thank OSU Research Funding for their support on this project, without it this work would not have been possible. v Vita January 7, 1983…………………………....... BornBelleville, New Jersey 2001……………………………………….... Belleville High School, New Jersey 2009……………………………………….... B.S. Biochemistry, Montclair State University, New Jersey 2009 - 2010…………………………............. Graduate Teaching Assistant, Montclair State University, New Jersey 2010 - 2011………………………………..... Graduate Teaching Assistant, The Ohio State University, Ohio Fields of Study Major Field: Chemistry vi Table of Contents Abstract…………………………………………………………………………….ii Dedication………………………………………………………………………....iv Acknowledgements……………………………………..……………..…………...v Vita………………………………………………………………………………...vi List of Tables……………..……………………………………………………….ix List of Figures……………..……………………………………………………….x List of Abbreviations………...…………………………………………………..xiv Chapter 1: Introduction….…………………………………………………………1 Chapter 2: Materials and Methods…………...……………………...…..………..11 2.1: Synthesis of Peptide Amphiphiles.…...…………………………….….11 2.2: Purification of Peptide Amphiphiles…………..……………..…...……13 2.3: Synthesis and Purification of DOTA….………………………..…...…13 2.4: Chelation of PA with Gd3+……….…………….…………………...….15 2.5: Circular Dichroism…………..……………….…………………..…….16 2.6: Determination of Critical Aggreagation Concentration (CAC).….……16 Chapter 3: Development of a pH responsive Peptide Amphiphile……………….18 3.1: Molecular Tunablity of PAs……...…………………………………….20 3.2: Library Screening………………………………………………………21 vii 3.3: Influence of the Hydrogen Bond Forming Region……….…………….22 3.4: Screening of Electrostatic Repulsive Forces……………………………23 3.5: Effect of Electrostatic Repulsion.…………….…………………….…..24 3.6: Role of Amino Sequence of H-Bond Region………………………..…24 3.7: Effect of Hydrophobic Tail Length……………………………….….…27 3.8: Decreasing the Strength of β-sheet Propensity of the H-Bond region….27 Chapter 4: Examining a Series of Potential Peptide Amphiphile Contrast Agents…………………………………………………………………………….44 4.1: Discovery of a Possible pH Dependent PA Contrast Agent………...….45 4.2: Dynamic Reversibility of Self-Assembly………………………………46 4.3: Correlation of Peptide Sequence to Self-Assembly………….…………46 4.4: Determination of the Effect of Protonation of Glutamic Acid and its pKa..…….………………………………………………………………47 4.5: Critical Aggregation Concentration of PAs………………………….…48 4.6: Incorporation of an MRI Imaging Moiety……………………………...49 Chapter 5: Conclusion and Future Work…………………………………………59 References…………………………………………………………………...……61 viii List of Tables Table 3.1: A list of Gibbs free energy and probability values of amino acids…….30 Table 3.2: Complete list of all synthesized molecules.……..……………….…….31 Table 4.1: List of synthesized molecules with self-assembly pH and pKa values………………………………………...………………………………….…50 ix List of Figures Figure 1.1: Schematic of the cellular uptake and metabolism of glucose. As glucose is aerobically and anaerobically metabolized, a high concentration of [H+] is generated causing the extracellular matrix to have a slightly acidic pH (Figure taken from Reference 6)…………..…………………..……………..……...………….….9 Figure 1.2: In vivo image of a MDA-MB-435 breast tumor, displaying the heterogeneity of the acidic pH of the tumor cells. Image created by 1H NMR spectroscopy (Figure taken from Reference 6)…..……..…...………..…………….10 Figure 1.3: The structure of a DO3A (1,4,7,10-tetraazacyclododecane-1,4,7,10- tetraacetic acid) derivative of the macrocylic metal chelator DOTA.………......….10 Figure 2.1: Synthetic scheme for the unsymmetrical macrocyclic metal chelator, DO3A-tri- tert-butyl ester.…….............................................................................................……17 Figure 3.1: The structure of a Peptide Amphiphile, illustrating the 3 regions contributing to the molecules unique, self-assembling properties are shown; the hydrophobic region (Black), Hydrogen Bond forming region (Blue), and the hydrophilic region (Red). Note: PA shown is palmitoyl-VAAEEEE-NH2..……......30 Figure 3.2: Schematic of a pH responsive self-assembly PA in the unassembled and the self-assembled nanofiber states.……………………....………………...………31 Figure 3.3: SAXS, CD, and TEM data taken from Goldberger et. al. indicating the morphology of the PA, palmitoyl-VVAAEEEEIKVAV-NH2. A. SAXS data used to x determine the size and shape of molecules in solution. B. The CD spectra shown to emphasize the response to Ca2+. C. TEM image of the PA in a salt solution showing spherical micelles. D. TEM image of the PA showing cylindrical fibers formed (Figure taken from reference 32)….………………………………………………..32 Figure 3.4: A. Circular Dichroism spectra of palmitoyl-EEEE, peptide amphiphile shown lacks the hydrogen bond forming region. 10 μΜ palmitoyl-EEEE in the (D) presence of 150 mM NaCl and 2.2 mM CaCl2. B. The CAC determination of palmitoyl-EEEE-NH2 in physiological salt conditions. ……………………………34 Figure 3.5: Circular Dichroism spectra of palmitoyl-VVAAEEEE-NH2, utilized for the determination of the Ca2+ concentration dependence on self-assembly. A. 10 μΜ palmitoyl-VVAAEEEE-NH2 in the presence of 2.2 mM CaCl2 and 150 mM NaCl. B. 10 μΜ palmitoyl-VVAAEEEE-NH in the presence of 1.1 mM CaCl2 and 150 mM NaCl……………………….…………………………….……………………..34 Figure 3.6: Comparison of palmitoyl-VVAAEEEE-NH2 and palmitoyl- VVAAEEEEE-NH2 in 150 mM NaCl and 2.2 mM CaCl2. Dashed lines correspond to PA containing 5 glutamic acids and solid lines for PA with 4 glutamic acids…..35 Figure 3.7: CD spectrum for 10 μΜ palmitoyl-AAVVEEEE-NH2 in 2.2 mM CaCl2 and 150 mM NaCl.……………………………………………………………….…35 Figure 3.8: The peptide backbone used to show the 3 types (ω,,) of dihedrals seen in a peptide…………………………………………………………………………36 Figure 3.9 Circular Dichroism spectra for examining the effect of peptide