Self-Assembling Peptide Amphiphile Contrast Agents As a Tumor Diagnostic Tool

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

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

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.

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Dedication

This document is dedicated to my family

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.

Vita

January 7, 1983…………………………...... BornBelleville, 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

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

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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

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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

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

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 sequence

in the H-Bonding region on self-assembly. Palmitoyl-AAXXEEEE-NH was used as (D) 2 xi a base molecule, where X is isoleucine, valine, tyrosine, and threonine. A. CD spectrum for 10 μΜ palmitoyl-AAIIEEEE-NH2. B. CD spectrum for 10 μΜ palmitoyl-AAVVEEEE. C. CD spectrum for 10 μΜ palmitoyl-AAYYEEEE. D.

CD spectrum for 10 μΜ palmitoyl-AATTEEEE. Note: all CD spectra were recorded in the 2.2 mM CaCl2 and 150 mM NaCl.……………………..………………….....37

Figure 3.10 TEM images of palmitoyl-AATTEEE-NH2 at pH 4……...……..…….39

Figure 3.11: Circular dichroism spectra of palmitoyl-AAYYEEEE-NH2, myristoyl-

AAYYEEEEE-NH2, and lauryl-AAYYEEEE-NH2, used for examining the impact of chain length of the lipophilic tail on self-assembly. A. 10 μM palmitoyl-

VVAAEEEE-NH2 in 2.2 mM CaCl2 and 150 mM NaCl. B. myristoyl-AAYYEEEE-

NH2 in 2.2 mM CaCl2 and 150 mM NaCl. C. lauryl-AAYYEEEE-NH2 in 2.2 mM

CaCl2 and 150 mM NaCl………..………………..…………………………………40

Figure 3.12: CD spectra investigating the role of the relative strength of the H-Bond forming region and its implication in self-assembly of peptide amphiphiles. A. 10

μΜ palmitoyl-VVAAEEEE-NH2 in 2.2 mM CaCl2 and 150 mM NaCl. B. 10 μΜ palmitoyl-VAAAEEEE-NH2 in 2.2 mM CaCl2 and 150 mM NaCl. C. 10 μΜ palmitoyl-VAAAEEEE-NH2 in 150 mM NaCl and 2.2 mM CaCl2 used to precisely define the pH required for self-assembly…..……………………….………………42

Figure 4.1: Fine scan CD spectra used to precisely determine the pH responsive self- assembly. A. Palmitoyl-VAAAEEEE-NH2 in 150 mM NaCl and 2.2 mM CaCl2. B.

Palmitoyl-VAAAEEEE-NH2 in 150 mM NaCl and 2.2 mM CaCl2. C. Palmitoyl-

xii

VAAAEEEE-NH2 in 150 mM NaCl and 2.2 mM CaCl2. D. Palmitoyl-VAAAEEEE-

NH2 in 150 mM NaCl and 2.2 mM CaCl2.…………………………………….……51

Figure 4.2: A. The structure of the molecule of interest, palmitoyl-IAAAEEEE, the molecule undergoes a pH responsive self-assembly near pH 6.6. B. TEM image of self-assembled palmitoyl-IAAAEEEE-NH2 in 150 mM NaCl and 2.2 mM CaCl2...53

Figure 4.3: CD spectra used to illustrate the reversibility of self-assembly by alternating the additions of NaOH and HCl. A. 10 uM palmitoyl-VAAAEEEE-NH2 is 150 mM NaCl and 2.2 mM CaCl2. B. 10 uM palmitoyl-IAAAEEEE-NH2 in 150 mM NaCl and 2.2 mM CaCl2.………………………………………………………54

Figure 4.4: Titration and 1st derivative plots of peptide amphiphiles used to determine the pKa at 10 μM PA, 150 mM NaCl and 2.2 mM CaCl2. A. Titration of

ST palmitoyl-VAAAEEEE-NH2. B. 1 derivative palmitoyl-VAAAEEEE-NH2

ST titration data. C. Titration of palmitoyl-FAAAEEEE-NH2. D. 1 derivative of palmitoyl-FAAAEEEE-NH2 titration data…………..…………...…………………55

Figure 4.5: Critical aggregation concentration determination using the pyrene 1:3 method. All fluorescence measurements were done at 150 mM NaCl, 2.2 mM

CaCl2, 0.3 μM pyrene with 1% MeOH. A. Palmitoyl-VAAAEEEE-NH2 B.

Palmitoyl-IAAAEEEE-NH2………………..……..………………..………....…….57

Figure 4.6: CD fines scans of A. palmitoyl-IAAAEEEE-NH2 , self assembly occurs at pH 6.6 in simulated physiological salts. B. palmitoyl-IAAAEEEEK-

DOTA(Gd3+), assembly occurs at pH 5.7.……………………………….……...….58

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List of Abbreviations

ATP- Adenosine triphosphate CD- Circular dichroism DCM- Dichloromethane DIPEA- N,N-Diisopropylethylamine DMF- N,N-Dimethylformamide DOTA- 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri t-butyl ester ECM- Extracellular matrix EDTA- Ethylenediaminetetraaceticacid EPR- Enhanced permeability and retention effect ESI-MS- Electrospray ionization mass spectrometry FMOC- Fluorenylmethyloxycarbonyl chloride HBTU- O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate HATU- O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate HOAt- 1-Hydroxy-7-azabenzotriazole HOBt-1-Hydroxybenzotriazole hydrate HPLC- High performance liquid chromatography MALDI- Matrix assisted laser desorption ionization MeCN- Acetonitrile MeOH- Methanol MRI- Magnetic Resonance Imaging Mtt- Methyl trityl NADH- Nicotinamide adenine dinucleotide NMR- Nuclear Magnetic Resonance PA- Peptide amphiphile PEG- Polyethylene glycol PET- Positron Emission Tomography SAXS- Small angle x-ray scattering TEM- Transmission electron microscopy TCA- Tricarboxylic acid TFA- Trifluoracetic acid TIS- Triisopropylsilane TLC- Thin layer chromatography TOF- Time of flight

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Chapter 1

Introduction

Cancer is an ever-growing problem in our society; currently 1 in 4 deaths1 in the

United States is attributed to a form of cancer. With the current state of treatment, this figure is only expected to grow. According to the American Cancer Society, early detection is the most effective way to treat cancer2. Annual checkups are effective for a patient showing symptoms of cancer, however, these symptoms often only manifest during later stages of cancer. Some screening efforts designed to produce early detection, such as colonoscopies for colon cancer, pap-smears for cervical cancer, and mammograms for breast cancer, have been shown to result in curative treatments.

Despite all these advances, current diagnostic tests for many cancer types yield numerous false positives requiring subsequent imaging and biopsies for verification. For example, screening for prostate cancer requires either monitoring for elevated levels of a prostate- specific antigen3 in the blood, or detecting an enlarged prostate via digital rectal examination. Both tests have a 70% false positive4 rate since infection or non-cancerous inflammation of the prostate can exhibit these symptoms. Thus, subsequent surgically invasive and expensive biopsies of the prostate are required. A direct impact on decreasing the number of deaths due to cancer is possible in the long term by developing 1 early screening materials and methods that can easily and non-invasively image cancerous tissue.

The mechanism by which normal proliferating cells transform into tumor cells has been extensively studied over the last 30 years. For this transformation to occur, cells must adapt and tend to exhibit the six “Hallmarks of Cancer5”. The ability to resist cell death, induce angiogenesis, enable replicative immortality, activate invasion and metastasis, evade growth suppressors, and sustain proliferative signaling comprises the six hallmarks of cancer. The third hallmark, constant replication of tumor cells, causes an increased demand for energy6 (schematic shown in Figure 1.1), specifically in the form of Adenosine Triphosphate (ATP). Most tumor cells use glycolysis as the main form of energy production7,8 with the net products of pyruvic acid, ATP, NADH, and H+.

Pyruvic acid is then shuttled through The Citric Acid (TCA) cycle, where it is converted into cellular energy. The constant glycolytic cycle causes an increased production of H+, which is further released into the extracellular environment of tumor tissue and cells. The constant flux of protons into the extracellular matrix (ECM) causes it to be slightly more acidic than normal tissue and blood. The extracellular pH of “normal” blood and tissue is

7.4 and is typically maintained constant throughout the body9. However, tumor cells exhibit an abnormally acidic extracellular environment (shown in Figure 1.2) with the pH ranging from 6.8-7.410,11. The extracellular pH of noncancerous inflammation does not result in an acidic tissue environment. Consequently, designing imaging probes that

2 target the acidic extracellular pH may eventually lead to cancer diagnoses with fewer false positives and less invasive diagnostic tests.

A crucial step to increase the detection of tumors is designing molecules capable of targeting tumors. Two approaches currently used to increase tumor detection are active and passive targeting. Active targeting12 involves the conjugation of Magnetic Resonance

Imaging13 (MRI) and Positron Emission Tomography14 (PET) imaging agents to ligands capable of binding to the receptors, over-expressed on the tumor cell surface.

Conjugation of contrast agents to folic acid15 is one such example currently used to target tumors. Various types of tumors have been shown to over-express folic acid16 receptors on the cell membrane surface. Thus, conjugation of diagnostic imaging reporter molecules to folic acid exhibits a considerable increase in efficacy for imaging of the reporter molecules like Gd in MRI or 18F in PET. Conversely, passive targeting17 is premised on designing molecules capable of persisting in the bloodstream by eluding macrophages and renal clearance, making them more effective at reaching tumor sites. A common method of passive targeting is the attachment of polyethyleneglycol (PEG) to an albumin conjugated liposome18,19 increasing the delivery of liposome encapsulated doxorubicin (a cancer therapeutic) in vivo. Passive targeting approaches are so far only modestly effective for tumor detection. In this work, we describe a hybrid (active/passive targeting) approach, by designing a dynamic system that is capable of sensing and transforming its molecular structure in response to minute changes in pH found only at the tumor. The formation of long nanofibers at the tumor traps diagnostic molecules at

3 the tumor site due to the formation of nanofibers in the acidic tumor environment, making them too large to escape the tumor tissue.

A well-studied approach to tumor targeting is the Enhanced Permeability and

Retention (EPR) effect. The EPR effect20 exhibited by many tumor cells is partly due to the production of many vascular permeability factors such as bradykinin21 and vascular endolithelal growth factors22 (VEGF) needed for tumor metastasis. Increased production of VEGF and other growth factors cause tumor tissue to show drastic increases in permeability, allowing larger than usual molecules to penetrate the tumor vasculature.

Inflamed tissue and tumor tissue exhibit similar permeabilites to larger molecules, but vary in their rate of clearance23 from the body. Macromolecules can be cleared from tumor tissue over the course of days whereas inflamed or normal tissue can take minutes to hours24 indicating large molecules enter the tumor vasculature, but cannot escape easily by the same route. Much of the work on the EPR effect has used macromolecules conjugated to a reporter for tumor detection, as well as its efforts in drug delivery.

Several groups have shown that the conjugation of polymers25,26 to current marketed drugs has increased the distribution of drugs to the liver, the kidney and more importantly, tumor sites. Doxorubicin conjugated polymers are an example of anticancer drugs exhibiting such a bio-distribution characteristic in mice27,28. The enhanced biodistribution for this class of drugs is likely due to size increases from conjugation to polymers that help slow down the pharmacokinetics of the molecules and lead to increased exposure of the tumor to the drug.

Liposomes are artificial phospholipid bilayers that can be specifically designed to encapsulate many drugs and release their contents in response to external stimuli.

Liposome delivery can be useful for the integration of material into the leaky vasculature of tumor tissue, when liposomes29 are less than 200 nm in diameter. Discher et al. has shown the incorporation of filo-micelles to anticancer agents leads to longer blood circulation of materials up to 8 μm in length, allowing the drug to remain uncleared by phagocytes over ten times longer than similarly prepared spherical micelles30. The morphology of a molecule directly impacts its ability to be cleared from the body due to the EPR effect. One goal of the present work is to create a material that is capable of dynamically transitioning from spherical micelles or isolated molecules to nanofibers in response to pH changes, serving as a sensor for acidic tumor environments while expanding upon the EPR effect with the production of nanofibers. Incorporating imaging agents to such a morphologically dynamic material could possibly increase the detection of many cancers using this hybrid targeting approach.

MRI is a noninvasive diagnostic tool commonly used to detect tumor tissue by measuring the relaxation of the protons of water molecules in response to an externally administered magnetic field. The relaxation of the protons depend on the surrounding environment31. For example, the environments of tumor tissue and areas of inflammation are vastly different from extracellular matrix of normal tissue. The addition of a contrast

3+ agent, such as the paramagnetic ion Gd , increases the spin-lattice relaxation time (T1) of water, therefore increasing the signal intensity. MRI resolution can be dramatically

5 enhanced by the addition of Gd3+, however free (i.e. unchelated) Gd3+ in the blood is highly toxic32. Clinically available contrast agents are chelated to macrocylic chelators33 neutralizing any toxic effects. The commonly used macrocyclic chelator, DOTA is effective at binding Gd3+, having a binding constant34 of 1.0x1024. Conjugation of a

DO3A chelate to a molecule targeted for cancer is an approach used to increase detection of tumors that has not generally succeeded because biochemical tumor targets (e.g. cell surface proteins) are only available to nM concentrations, while Gd in µM concentrations is required to visualize the Gd in an MRI scan35. The approach taken here could localize

Gd in the µM range. This work utilizes a DOTA derivative, known as DO3A (Figure

1.3) in its molecular design to create dynamic pH sensitive materials that should be capable of tumor detection.

The focus of this thesis is to design materials that reversibly change size and shape in response to the extracellular pH of tumor tissue. A dynamic and reversible material can be created in a system whose assembly behavior is dictated via supramolecular forces. Supramolecular chemistry36 focuses on the use of weak forces

(hydrogen bonding, hydrophobic interactions, and electrostatic interactions) to hold molecules together in lieu of traditional covalent and ionic bonds. Self-assembly is the organization of molecules into larger structures, such as micelles, cylindrical micelles, fibers and ribbons through a spontaneous yet controllable process through changes in ionic strength37, temperature38, and pH39. This self-organization creates a system using weak forces to form ordered molecular structures dynamic in character. The morphological thermodynamic equilibrium structures created through weak forces are

6 structurally different in various extracellular conditions. Through judicious design of sequence and structure, it is possible to fine-tune the environmental dependence of these self-assembled structures.

Self-assembly has successfully been used to enhance a number of biological processes, using both polymer- and peptide-based materials. Many peptide-based monomers can self-assemble into fibrous structures typically occurring through the formation of β-sheet like structures. Bowerman et al. studied amyloid structures, naturally occurring proteins, favoring the aggregation of monomers through noncovalent weak interactions that form β-sheet like structures40. They determined that hydrophobic and aromatic amino acids found in amyloid structures, favor β-sheet formation through the synthesis and characterization of a library of peptides containing these amino acids.

Additionally, Stupp et al, have developed amphiphilic peptide nanomaterials to mimic biological processes and enhance tissue regeneration41.

Amphiphilic molecules are composed of a region of hydrophobic residues and a region containing charged (hydrophilic) residues. The attractive hydrophobic and repulsive hydrophilic forces must be balanced for self-assembly to occur42. The hydrophobic region is a critical component for self-assembly as it causes hydrophobic collapse in aqueous solutions. In the hydrophilic region, counter-ions screen the charge created by the charged amino acids43. Interactions between the counter-ions and charged residues decrease the charge distribution in aqueous solution. Therefore, charge screening

7 is crucial for self-assembly since it is capable of decreasing the electrostatic repulsion between two PA monomers. Moreover, the charged region increases the solubility of the lipophilic peptide in aqueous solution. Amino acids containing ionizable side chains have electrostatic forces between neighboring molecules, making PAs containing them responsive to changes in pH. The balance of electrostatic charge through protonation and deprotonation or ionic screening allows amphiphilic molecules to self-assemble.

Recently, Goldberger et al. 44 have shown that the peptide amphiphile, palmitoyl-

VVAAEEEEGIKVAV-COOH self-assembles into spherical micelles in the absence of

Ca2+, and cylindrical nanofibers in the presence of Ca2+.

The work presented here describes our efforts in designing a pH sensitive self- assembling MRI contrast agent capable of transitioning from isolated molecules (or spherical micelles) to nanofibers in response to the acidic extracellular tumor environment. Chapter 2 discusses the methods for synthesizing and purifying peptide amphiphiles, conjugation to DO3A, and chelation to gadolinium. Chapter 3 probes the role of the 3 regions of this class of peptide amphiphiles and investigates the relationship between peptide sequence and self-assembly behavior. The role of ionic strength will be investigated, as it is critical in developing pH dependent self-assembled molecules for in vivo applications. Chapter 4 focuses on the development of biomaterial to function as a contrast agent that is capable of reversibly transitioning from isolated molecules (or spherical micelles) to cylindrical micelles in response to small changes in pH. Chapter 5 provides a summary as well as planned future work.

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)

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.

Chapter 2

Materials and Methods

Synthesis of Peptides Amphiphiles

All amino acids were purchased from AnaSpec Inc. unless otherwise specified.

Peptides were synthesized using two different methods depending on the quantity required. For PAs containing lysine, a Sieber resin (AAPPTEC) was used; all other peptides were synthesized with a Rink Amide Resin (AAPPTEC) in a linear fashion from the C-terminus to N-terminus direction using FMOC chemistry45. Peptides that were made on 0.25 mmol-scale, were synthesized on an automated peptide synthesizer

(Applied Biosystems Model No. 433A) with Applied Biosystem cartridges for all but palmitic acid (Sigma Aldrich). For peptides above 0.25 mmol, the following procedures were followed:

The resin was swelled in a shaker vessel with dichloromethane (DCM) for 30 minutes, the DCM was removed and dimethyl formamide (DMF) was added to the vessel and further shaken for 30 minutes. After the liquid was removed, 10% N,N-

Diisopropylethylamine (DIPEA) in DMF was added to the vessel and mixed for 10 11 minutes. For deprotection, 20% piperidine in DMF was used to remove the FMOC protecting group on the resin. A Kaiser test protocol confirmed removal of the FMOC protecting group. Coupling of the acid to the amine end of resin was done through activation using O-Benzotriazole N,N,N’,N’-tetramethyluronium hexafluorophosphate

(HBTU) or 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU). The coupling solution contained 4.0 Eq. of amino acid,

3.96 Eq. of HBTU/HATU, 4 Eq. of N-Hydroxybenzotriazole (HOBt) or1-Hydroxy-7- azabenzotriazole (HOAt), and 8 Eq. of DIPEA with respect to peptide allowing at least 3 hours of coupling per amino acid. The surfactant Triton X-100 was added to the coupling solution and to the latter amino acids to aid in coupling efficiency. Resin cleavage of the peptide was done by addition of the following solutions: For the Rink Amide resin, a solution of 95% Trifluoroacetic acid (TFA), 2% Anisole, 2% water was used and for

Sieber Resin cleavage, a solution of 1% TIS and 1% TFA, 2% Anisole, 1% Triisopropyl silane (TIS) and 94% water was used; shaken for at least 2 hours. The TFA was removed under vacuo and the PA was precipitated using two 20 mL portions of cold diethyl ether.

The crude peptide was filtered and washed with cold diethyl ether.

To attach the DO3A, the lysine used during peptide synthesis was Methyl trityl protected46, so that it could be removed by the mild cleavage conditions as the peptide was cleaved from the Sieber resin. The DO3A was coupled in the solution phase, using the aforementioned ratio of reagents with the exception of 1 Eq. of DO3A.

Purification of Peptide Αmphiphiles

The crude peptide amphiphile (PA) was dissolved in 0.1% NH4OH at approximately 10 mg/mL by vigorously shaking and sonicating until the solution turned clear. To aid in dissolution, an additional drop of concentrated NH4OH was added to the solution. The PA solution was filtered first using a 0.45 μm syringe filter (Whatman), followed by a 0.2 μm syringe filter. The sample was purified on a Shimadzu preparative

HPLC (dual pump system controlled by LC-MS solution software) with an Agilent

PLRP-S polymer column (Model No. PL1212-3100 150 mm x 25 mm) under basic conditions. The product was eluted with a linear gradient of 10% Acetonitrile to 100% over 30 minutes containing 0.1% NH4OH (v/v). The purity of the collected fraction was verified using an electrospray ionization time-of-flight mass spectrometer (Bruker)

Fractions greater than 90% pure were combined; the Acetonitrile (MeCN) was removed by vacuum before freeze-drying.

Synthesis and Purification of DO3A

The synthesis scheme is shown in Figure 2.1. All reagents were purchased from

Sigma Aldrich and used without further purification unless specified. 30 g of cyclen

(1,4,7,10-tetraazacyclododecane) was combined with powdered, dry 42.9 g of sodium acetate in 400 mL of N,N-dimethylacetamide in a round bottom flask and stirred for 30 minutes with an overhead glass rod stirrer. The round bottom flask containing the slurry

13 was placed in an ice bath until it reached 0C. 77.1 mL of tert-butyl bromoacetate was dissolved in 150 mL of N, N,-dimethylacetamide and added drop-wise to the slurry at

0C over a period of 25 minutes. The slurry equilibrated back to room temperature and was stirred for 5 days. A separate solution was made by dissolving 30 g of potassium bromide (KBr) in 2 L of deionized water (Millipore) followed by stirring and heating to a temperature of 50C. After the KBr solution reached 50C, it was added to the slurry forming a yellow colored solution. The pH was adjusted to 9.0 by the addition of powdered sodium bicarbonate and checked via litmus paper. Precipitation of the desired product, acetic acid tert-butyl ester hydrobromide (4,7-bis-tert-butoxycarbonylmethyl-

1,4,7,10-tetraaza-cyclododec-1-yl), settled for 4 hours without stirring, followed by vacuum filtering and drying, yielding a white powder. 10.0 g of acetic acid tert-butyl ester hydrobromide was dissolved in 50 mL of MeCN and combined with 5.1077 g (2.2 eq.) of finely powdered, dry potassium carbonate and stirred for 30 minutes. Benzyl bromo acetate 2.927 mL (1.1 eq.) was added drop-wise to the solution and stirred overnight at 50C-60C. The mixture was cooled to room temperature and vacuum filtered, with the desired product in the filtrate. The solid was washed twice with MeCN.

The combined MeCN was removed by evaporation under vacuum, yielding the tri-tert- butyl ester form of DO3A, a viscous yellow gel. NMR and ESI-MS confirmed the presence of the desired product. The crude product was purified using flash-column chromatography using 50.0 g of silica gel for every 1.0 g of tri-tert-butyl ester form of

DO3A using DCM as the mobile phase. The product was eluted from the column using a gradient elution, starting with 2% MeOH in DCM to 6% of MeOH in DCM. The elution

14 of the desired product was followed by TLC, using 10% MeOH in DCM as the mobile phase. Pure fractions were combined and the solvents evaporated under vacuum. The residue was then dissolved in approximately 50 mL of MeOH in deionized water

(Millipore) at a ration of 9:1. Palladium on carbon catalyst was added to the solution in

20% by weight with respect to tri-tert-butyl ester form of DO3A. The sample was hydrogenated under 50-psi hydrogen pressure overnight followed by filtration of the solid catalyst. The filtrate containing DO3A was evaporated under vacuum to remove the methanol then 100 mL of deionized water was added to the solution. Diethyl ether (50 mL) was added 3 times to the solution in a separatory funnel to extract the non- hydrogenated product. Solvent was removed by evaporation and the solution was freeze- dried to remove remaining deionized water, yielding a yellowish powder. NMR and ESI-

MS were used to confirm the presence of DO3A and check purity.

Chelation of PA with Gd3+

2.27 mg of previously prepared PA-DO3A was dissolved in 1.0 mL of water and combined with 2 eq. of GdCl3 in 0.01 M HCl. The reaction was set to stir in an oil bath at 60C for 30 min. The pH of the solution was gradually adjusted from approximately pH 2 to 4-5 (Litmus paper) using small amounts of 0.050 M NaOH. The resultant solution was stirred for 24 hours at 60C. A small sample was removed and analyzed by

MALDI to determine the extent of reaction completion. The pH was then raised over a period of an hour using ammonium hydroxide followed by the addition of EDTA and

15 filtered using a 0.2μm syringe filter. The solution was dialyzed against Millipore water to remove NaCl, free Gd3+, and EDTA-Gd3+. The buffer water for dialysis was changed 4 times over a period of 24 hours. The PA-DO3A-Gd3+solution was freeze-dried to recover a white fluffy powder.

Circular Dichroism

Measurements were done on a circular dichroism spectrometer (Jasco-815). The collected spectra were averaged over 3 accumulations and were baseline subtracted by the use of aqueous solutions containing only salts. Peptide amphiphiles stock solutions were prepared in water at pH 9 by the addition of NaOH. The stock solution was diluted to 10 μM to prepare solutions containing 150 mM NaCl and 2.2 mM CaCl2 and allowed to heat at 80C for 30 min. The solution was allowed to naturally cool to room temperature followed by the adjustment of the pH to the desired value.

Determination of Critical Aggregation Concentration (CAC)

Fluorescence measurements were performed on a fluorometer (BioTek Synergy

H4) at room temperature. A series of dilutions were performed for each PA from 245 μΜ to 95 nM in simulated physiological salts containing 0.3 μM pyrene. The pyrene was excited at 335 nm and the emission spectra recorded. The pyrene 1:3 method was used for determination of the CAC.

Figure 2.1: Synthetic scheme for the unsymmetrical macrocyclic metal chelator, DO3A- tri-tert-butyl ester.

Chapter 3

Development of a pH-responsive Peptide Amphiphile

This chapter describes the investigation of a class of molecules capable of self- assembly and examines various iterations of peptide design leading to the successful creation of a molecule that undergoes a pH-dependent self-assembly at physiologically relevant conditions. Numerous peptide amphiphile (PAs) molecules were synthesized, and their pH-dependent self-assembly behavior was characterized using circular dichroism (CD), conventional transmission electron microscopy (TEM) measurements, and critical aggregation concentration (CAC) experiments.

Recently, the development of self-assembling biomaterials for use in many biological applications including cell signaling47, tissue engineering48, and drug delivery49 has gained interest. PAs have generated much interest as biomaterials due to their biological compatibility and low risk of toxicity50. PAs are typically short peptide sequences, amphiphilic in nature, and composed of three main regions; 1) a lipophilic chain, 2) a hydrogen-bonding, β-sheet forming region located next to the hydrophobic region, and 3) a hydrophilic region containing charged amino acids (Figure 3.1). The hydrophilic region allows the self-assembly to be pH-responsive depending upon the 18 state of the charged residues, either protonated or deprotonated. The balance between the forces in the hydrophobic and hydrophilic regions dictates the pH and concentration at which these transitions occur.

It was recently observed that specific PA sequences were able to undergo spherical micelle to cylindrical fiber transitions under physiological conditions using enhanced electrostatic screening with Ca2+ as the trigger (Figure 3.2). Goldberger et. al. studied a class of molecules containing an IKVAV44 epitope for its application in spinal cord regeneration. They showed the sequence palmitoyl-VVAAEEEEGIKVAV-NH2 assembled into spherical micelles 5 nm in diameter in 150 mM NaCl and 3 mM KCl, and cylindrical nanofibers, 10 nm in diameter and 1-10 µm in length when 5 mM CaCl2 was added to 1 wt% solutions. The self-assembly morphology and size were characterized using small-angle x-ray scattering (SAXS), cryo-TEM and conventional TEM (Figure

3.3). When the molecules were organized into spherical micelles, the CD spectra displayed a random-coil secondary structure. In the cylindrical nanofiber state, the presence of β-sheets was evidenced by the minimum at 218 nm and a maximum at 196 nm in the CD spectra. Thus, for this sequence a random coil secondary structure was indicative of a spherical micelle while a β-sheet secondary structure indicated nanofiber morphology. A minimum of four charged amino acid residues were found to be essential for the peptide amphiphile material to undergo a spherical micelle to cylindrical nanofiber transition at pH of 7.4.

We hypothesized that it may be possible to design a sequence with a spherical micelle to cylindrical nanofiber transition, which can be triggered by the protonation state of the glutamic acid residues under constant physiological salt concentrations. A lower pH would reduce the amount of charge on the glutamic acid region, causing a reduced headgroup size in an Israelachvili model51, enabling self-assembly into nanofibers.

Consequently, by balancing the attractive and repulsive forces of the hydrophobic and charged regions by amino acids in the hydrogen bond forming region; fine control of the self-assembly may be possible.

3.1 Molecular Tunablity of PAs

The hydrogen bond-forming region is typically composed of amino acids known to form hydrogen bonds in the form of β-sheets that bridges the carbonyl and amine groups of neighboring molecules in the peptide backbone. Berg et. al. examined the

Zinc-finger peptide52 through a series of mutations at a solvent exposed area capable of forming a β-sheet secondary structure. They were able to rank each amino acid’s propensity for β-sheet formation by calculating the ΔΔG from a titration study using metal ions, Cobalt (II) and Zinc (II). The generated data (ΔΔG) corresponded well with the statistical propensity for each amino acid residue from crystallographic data of amino acids that form β-sheets in 64 different proteins. The ΔΔG and the probabilities of the amino acids to form β-sheets have been tabulated in Table 3.1 and have been used as a guide to develop the dynamic systems here. This table allowed us to generate a series of

PA molecules (Table 3.2) featuring amino acids with high β-sheet propensities to help us reach our goal of constructing a dynamic system that can transform into cylinders at 10

µM and at pH between 6.4-7.0.

3.2 Library Screening

A major focus of this thesis is the development of materials capable of functioning in vivo, thus mimicking physiological conditions is crucial. The difficulty here is providing the most physiologically relevant and applicable analysis of PAs. CD,

SAXS, and cryo-TEM are capable of correlating the morphology of molecules for self- assembly but it would be impractical to use these techniques in blood serum due to noise from other proteins in the blood. Human blood serum53 contains 2.2-2.6 mM Ca2+, 150 mM Na+, other trace metals, and various proteins. For accurate analysis, characterization of PAs must be done in similar conditions. Thus, we chose an aqueous system consisting of 2.2 mM CaCl2 and 150 mM NaCl for the screening of peptides. For colloidal systems, it is well known that the concentration of electrolytes for colloids to overcome their repulsive charge surface forces and precipitate (critical coagulation concentration), depends on the concentration and charge of the electrolytic counter-ions.

According to the Schulze-Hardy54 rule, the critical coagulation concentration is inversely proportional to the sixth power of the charge of the salt counter-ion. Thus, a solution with 2.3 mM Ca2+ has the same effective electrostatic screening strength as a solution with 150 mM Na+. Finally, the limit of detection for Gd3+ based contrast agents is 10 µM

21 for MRI imaging applications55, we decided that this would be an appropriate concentration to begin molecule development. This concentration of molecules is below the detectable limit for characterizing morphology using cryo-TEM and SAXS.

However, utilizing CD it is possible to detect the secondary structure at 10 µM concentrations and was employed as the primary technique to elucidate the morphology of molecules in solution.

3.3 Influence of the Hydrogen Bond Forming Region

The hydrogen bond-forming region56 has been shown to play a significant role in the formation of self-assembled structures. To study the effect of incorporating this region, the amphiphilic molecule palmitoyl-EEEE-NH2 lacking the hydrogen bond- forming region was synthesized. The pH-dependent CD spectra from pH 4 to pH 10 are superimposable, and nearly zero between 210-220 nm with a decrease in ellipticity below

205 nm, indicative of random coil behavior57 (Figure 3.4.A). Therefore, the secondary structure does not change with respect to pH. Pyrene58 titration experiments were also performed to determine the CAC at a pH of 7.4. (Figure 3.4.B) The absence of concentration dependence on the I1/I3 ratio indicates this molecule does not self-assemble at concentrations ranging from 245 μM to 95 nM. This provides insight to the importance of the hydrogen bond-forming region and molecular self-assembly of these

PAs; nanofiber assembly of this class of molecules is largely due to the hydrogen bond forming region.

3.4 Screening of Electrostatic Repulsive Forces

The sequence palmitoyl-VVAAEEEE-NH2, without the biologically active epitope served as a starting point for this research based upon its similarity to the system discovered by Goldberger et al. To illustrate the sensitivity of the electrostatic screening strength on the pH-dependent self-assembly properties, samples were prepared containing

10 μM of PA and 150 mM NaCl, at two concentrations of CaCl2; 2.2 mM CaCl2 and 1.1 mM CaCl2. The comparison of the CD spectra is shown in Figure 3.5, indicating that palmitoyl-VVAAEEEE-NH2 undergoes a transition from random coil to β-sheet between pH 6 and 8 (by arrows in Figure 3.5) in the presence of 1.1 mM CaCl2. At pH 8 and 10, the CD spectra showed a superimposable random coil morphology. When the CaCl2 concentration was increased to 2.2 mM, the CD spectra at pH 8 and 10 showed a minimum at 218 nm and a maximum at 205 nm but the intensity was distinctly different from samples at pH 4 and 6. At this CaCl2 concentration, a purely random coil morphology is not observed in the CD, demonstrating some β-sheet secondary structure at all pH values. For the PA in the presence of 2.2 mM CaCl2, K2D2 simulations calculated a decrease in β-sheet character from 29% to 21% from pH 6 to 8. From these simulations, the percentage of random coil morphology increases with pH but does not reach 100%. PAs are more prone to self-assemble to the nanofibers at higher concentrations of calcium due to the screening of the charged glutamic acids by the divalent calcium ions.

3.5 Effect of Electrostatic Repulsion

The ability of the hydrogen bond-forming region to overcome electrostatic repulsion at higher pH exemplifies the role of the charged residues in self-assembly. The comparison of palmitoyl-VVAAEEEE-NH2 with palmitoyl-VVAAEEEEE-NH2 allows the investigation of elongating the hydrophilic region. The only difference between the two molecules is the number of charged residues, or the electrostatic repulsion between neighboring molecules. Figure 3.6 shows a comparison of the CD spectra for these molecules at pH 6, 8, and 10 of both sequences. The addition of one charged residue to the hydrophilic region decreases the intensity of the minimum at 218 nm and the maximum at 205 nm. This decrease corresponds to fewer molecules containing the β- sheet secondary structure at each pH point. Thus, the additional electrostatic repulsion by the additional glutamic acids, drives the transition to more acidic pH points.

3.6 Role of Amino Sequence of H-Bond Region

Another key factor in tuning the pH dependent self-assembly behavior is the amino acid sequence59 of the hydrogen bond forming region. To determine the influence of amino acid order on the transition pH, the location of the β-sheet forming valine was varied, and palmitoyl-AAVVEEEE-NH2 was synthesized. In contrast to palmitoyl-

VVAAEEEE-NH2, when valine is positioned next to the hydrophilic residues, the CD spectra (Figure 3.7) are indicative of a predominately β-sheet secondary structure, and

24 there is no change in the CD spectra with respect to pH. The results shown here, indicate moving the valine closer to the charged residues increases the attractive strength of the lipophilic tail and hydrogen bond region of the molecule. This allows self-assembly to occur at a higher pH and overcome the electrostatic repulsion seen for charged glutamic acid residues at pH of 8 and 10.

The experiments thus far have shown that incorporation of the hydrogen bond forming region is necessary for self-assembly in this class of molecules. Yet, they did not take into account the relative strength of this region and its implications in designing organized structures. A series of molecules with the sequence, palmitoyl-AAXXEEEE-

NH2, where X is valine, isoleucine, tyrosine, and threonine were prepared to investigate the effect of varying the relative strength of the H-Bonding region. Berg et al. determined among the naturally occurring amino acids, valine and isoleucine have similar affinities to form β-sheet like structure. Tyrosine has a weaker β-sheet propensity than the highly lipophilic residues valine and isoleucine but is stronger than threonine. The high propensity of these amino acids for β-sheet like structures arises from the aliphatic nature of the side chain and the effective size of the side chain60. The large aliphatic side chains introduce a steric blocking effect that limit the conformations the peptide backbone can adopt due to steric hindrance from side chain. In isoleucine, valine, and threonine the β-carbon is branched61, with more of the bulky side chain near the peptide backbone, decreasing the allowable confomations of the mainchain. The branched amino acids tend to adopt β-sheet secondary structure due to the favorable dihedral

25 angles (,) adopted by the peptide (Figure 3.8). However, for branched amino acids the dihedral angles are unfavorable due to the steric clash between the side chain and backbone. The aromatic amino acids are not β-branched but also show high tendencies for β-sheet like secondary structure. Despite being branched, the large aromatic rings are bulky and undergo a large steric hindrance with the peptide backbone, therefore limiting accepted conformations and increasing preference for β-sheet secondary structure.

Figure 3.9. A, B, and C, show the CD spectra for molecules containing the sequence palmitoyl-AAIIEEEE-NH2, palmitoyl-AAVVEEEE-NH2, and palmitoyl-

AAYYEEEE-NH2 respectively. CD spectra for all three molecules are highly similar, showing a negative peak at 218 nm and a positive ellipticity having a maximum below

205 nm, with the spectra being independent of pH from 4 to 10. The lack of pH independent behavior of the CD spectra indicate that these molecules contain similar β- sheet like secondary structure and the strength of the H-bond forming region is too strong for the molecules to be in an unassembled state at this ionic strength. Figure 3.4. D shows the CD spectra for palmitoyl-AATTEEEE-NH2, a random coil to β-sheet transition is observed between pH 5 and 6. Substituting the amino acids in the hydrogen bond forming region to residues with weaker propensities for β-sheet structures, such as from valine to threonine, enables a pH dependent morphological transition. TEM images taken of palmitoyl-AATTEEEE-NH2 can be seen in Figure 3.10 at pH 4, nanofibers 200-300 nm in length are formed at a concentration ~1mM.

3.7 Effect of Hydrophobic Tail Length

To determine the effect of decreasing the chain length62 of the hydrophobic tail and analyze its role in self-assembly, the PAs palmitoyl-AAYYEEEE-NH2, myristoyl-

AAYYEEEE-NH2, and lauryl-AAYYEEEE-NH2 were synthesized. In Figure 3.11. A, the

CD spectra for palmitoyl-AAYYEEEE-NH2 show a predominately β-sheet like behavior indicative of little morphology change with respect to pH. In this PA, the hydrophobic component is strong enough to overcome the electrostatic repulsion that occurs at higher pH values and nanofibers are formed under all pH conditions. The molecule myristoyl-

AAYYEEEE-NH2 is only two carbons shorter than palmitoyl-AAYYEEEE-NH2 but undergoes a transition between pH 5 and 6 as shown in Figure 3.11. B. In lauryl-

AAYYEEEE-NH2, the transition is shifted down to pH 4 and 5 (shown in Figure 3.11.

C). Therefore, decreasing the attractive strength of the hydrophobic component causes the self-assembly transition to be shifted to more acidic pH values.

3.8 Decreasing the Strength of β-sheet propensity of the H-Bond Region

In the search for the target molecule, the role of the strength of the hydrogen bond forming region has been probed extensively and determined to play an intricate role in self-assembling properties for this class of molecules. Understanding that palmitoyl-

VVAAEEEE-NH2’s hydrogen bond forming region was too strong, we decided to decrease the strength of the H-Bond forming region by removing a valine from this

27 region and we synthesized the molecule palmitoyl-VAAAEEEE. The removed valine was replaced by alanine, an amino acid considered to be neutral in β-sheet propensity, therefore decreasing the attractive strength of this region. A comparison of the CD spectra for the molecules palmitoyl-VVAAEEEE-NH2 with palmitoyl-VAAAEEEE-NH2 can be seen in Figure 3.12. A and B. In the latter molecule, a random coil to β-sheet transition is now observed and determined to occur between pH 6 and 8. The CD spectra indicates at pH 8 and 10, molecules are random coil in nature, and at the more acidic pH values (pH 4 and 6) they self-assemble into structures containing β-sheets. To determine the pH transition point more precisely, CD spectra (Figure 3.12.C) were recorded at numerous pH points between pH 5.8 and 7.3. As the pH is raised to 7.3, the minimum at

218 nm decreases and the maximum at 205 nm also decreases, indicating less β-sheet character per residue. At pH 6.26, a minimum at 218 nm and maximum at 205 nm are no longer seen and the spectrum is indicative of random coil. This was the first molecule synthesized that displayed the self-assembly transition close to our target pH of 6.8.

In conclusion, the self-assembly behavior of these PA molecules is a delicate balance between the supramolecular forces of each region. Increasing the attractive hydrophobic component or the hydrogen-bond forming region causes self-assembly to occur at higher pH values. Introducing more charge into the head group increases the electrostatic repulsive forces, shifting the pH to acidic values to compensate for the increase in electrostatic repulsion. Therefore, it is possible to design molecules able to

28 undergo this transition at similar pH values but with molecules containing various chain lengths and distinctly different peptide sequences.

Hydrophobic Hydrogen Bond Hydrophilic

Region Forming Region Region

Amino Acid ΔΔG (kcal mol-1) Probability Isoleucine -0.56 1.57 Phenyalanine -0.55 1.23 Valine -0.53 1.64 Tyrosine -0.50 1.31 Threonine -0.48 1.33 Alanine -0.35 0.79 Glutamic Acid -0.41 0.51 Table 3.1: A list of Gibbs free energy and probability values for relevant amino acids to form a β-sheet in the Zinc-finger protein take from (Data taken from reference 51).

PA sequence PA sequence PA sequence

Palmitoyl-VVAAEEEE-NH2 Palmitoyl-VAAAEEEE-NH2 Palmitoyl-VAAAEEEEK(DO3A)

Palmitoyl-AAVVEEEE-NH2 Palmitoyl-FAAAEEEE-NH2 Palmitoyl-FAAAEEEEK(DO3A)

Palmitoyl-AAYYEEEE-NH2 Palmitoyl-IAAAEEEE-NH2 Palmitoyl-IAAAEEEEK(DO3A)

Palmitoyl-AATTEEEE-NH2 Palmitoyl-YAAAEEEE-NH2 Palmitoyl-YAAAEEEEK(DO3A)

Palmitoyl-AAIIEEEE-NH2 Palmitoyl-EEEE-NH2 Myristoyl-AAYYEEEE-NH2

Palmitoyl-YYYYEEEE-NH2 Palmitoyl-TTTEEEE-NH2 Lauryl-AAYYEEEE-NH2

Palmitoyl-VVAAEEEEE-NH2 Table 3.2: Complete list of the molecules synthesized and analyzed.

pH 6.6

pH 7.4

Figure 3.2 Schematic of a pH responsive self-assembly PA in the unassembled and the self-assembled nanofiber states.

C D

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 determine the size and shape of molecules in solution. B. The CD spectra shown to emphasize the response to Ca2+. C. cryo-TEM image of the PA in a salt solution showing spherical micelles. D. cryo-TEM image of the PA showing cylindrical fibers formed (Figure taken from reference 32)

palmitoyl-EEEE-NH2 2

pH 4

1 pH 6 - 1 pH 8

pH 10

dmol

2 0 205 225 245

-1 [θ], deg cm deg [θ], -2 Wavelength (nm)

1.07 palmitoyl-EEEE-NH2

1.06

1.05

391

/I 1.04 375 I 1.03

1.02 4 5 6 7 8 9 log(palmitoyl-EEEE), log (pM)

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 presence of 150 mM NaCl and 2.2 mM CaCl2. B. The CAC determination of palmitoyl-EEEE-NH2 in physiological salt conditions.

5 palmitoyl-VVAAEEEE-NH2 2.2 mM CaCl2 pH 4

pH 6 1 2.5 - pH 8

pH 10

dmol

2 0 205 225 245

-2.5 Change in

[θ], deg cm deg [θ], % β-sheet

Wavelength (nm) -5 A

palmitoyl-VVAAEEEE-NH2 5 1.1. mM CaCl2 pH 4

Transition from random pH 6 1 - coil to β-sheet 2.5 pH 8

pH 10

dmol

2 0 205 225 245

-2.5 [θ], deg cm deg [θ],

-5 Wavelength (nm)

Figure 3.5: Circular Dichroism spectra of palmitoyl-VVAAEEEE-NH2 utilized for 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.

Effect of Electrostatic Repulsion 6 5-Glu pH 6

5-Glu pH 8 1 - 4 5-Glu pH 10

4-Glu pH 6 dmol

4-Glu pH 8

2 2 4-Glu pH 10 0 205 225 245

-2 [θ], deg cm deg [θ],

-4 Wavelength (nm)

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 are for the PA with 4 glutamic acids.

40 palmitoyl-AAVVEEEE-NH2

1 - pH 4

20 pH 6

dmol

2 pH 8 pH 10 0

205 225 245 [θ], deg cm deg [θ],

-20 Wavelength (nm)

Figure 3.7: CD spectrum for 10 μΜ palmitoyl-AAVVEEEE-NH2 in 2.2 mM CaCl2 and 150 mM NaCl.

 ω



Figure 3.8: The peptide backbone used to show the 3 types (ω,,) of dihedral angles seen in a peptide.

palmitoyl-AAIIEEEE-NH

12 2

1 - 8 pH 4

pH 6

dmol

2 pH 8 4 pH 10

0 [θ], deg cm deg [θ], 205 225 245 -4 Wavelength (nm)

40 palmitoyl-AAVVEEEE-NH2

1 - pH 4

20 pH 6

dmol

2 pH 8 pH 10 0

205 225 245 [θ], deg cm deg [θ],

-20 Wavelength (nm)

B Continued

Figure 3.9: Circular Dichroism spectra for examining the effect of peptide sequence in the H-Bonding region on self-assembly. Palmitoyl-AAXXEEEE-NH2 was used as a base molecule, where X is isoleucine, valine, tyrosine, and threonine. A. CD spectrum for 10 (D) μΜ palmitoyl-AAIIEEEE-NH2 . B. CD spectrum for 10 μΜ palmitoyl-AAVVEEEE. C. CD spectrum for 10 μΜ palmitoyl-AAYYEEEE. D. CD spectrum for 10 μΜ palmitoyl-AATTEEEE. Note: All CD spectra were recorded in the 2.2 mM CaCl2 and 150 mM NaCl.

Figure 3.9: Continued

d palmitoyl-AAYYEEEE-NH2 50

pH 4

1 - 30 pH 6

pH 8

dmol

2 10 pH 10

-10 205 225 245 [θ], deg cm deg [θ], -30 Wavelength (nm)

palmitoyl-AATTEEEE-NH2 4 pH 4

Transition between 1 - pH 5-6 pH 5 pH 6

dmol 2

pH 7 2 pH 8 (A) 0

205 225 245 [θ], deg cm deg [θ], -2 Wavelength (nm)

Figure 3.10: TEM images of palmitoyl-AATTEEE-NH2 at pH 4.

palmitoyl-AAYYEEEE-NH2 50

pH 4

1 - 30 pH 6

pH 8

dmol

2 10 pH 10

-10 205 225 245 [θ], deg cm deg [θ], -30 Wavelength (nm) A

myristoyl-AAYYEEEE-NH2 40 pH 4

pH 5

- pH 7 20 pH 8

dmol pH 9

2 pH 10

202 222 242 [θ], deg cm deg [θ], -20 Wavelength (nm)

B Continued

Figure 3.11: Circular dichroism spectra of palmitoyl-AAYYEEEE-NH2, myristoyl- AAYYEEEEE-NH2, and lauryl-AAYYEEEE-NH2 used for examining the impact of chain length of the lipophilic tail on self-assembly. A. 10 μM palmitoyl-VVAAEEEE- NH2 in 2.2 mM CaCl2 and 150 mM NaCl. B. myristoyl-AAYYEEEE-NH2 2.2 mM CaCl2 and 150 mM NaCl. C. lauryl-AAYYEEEE-NH2 2.2 mM CaCl2 and 150 mM NaCl.

Figure 3.11: Continued

lauryl-AAYYEEEE-NH 5 2 pH 4

pH 5

1 - pH 6

0 pH 8

dmol

2 200 220 240 260 pH 10

-5 [θ], deg cm deg [θ], -10 Wavelength (nm)

5 palmitoyl-VVAAEEEE-NH2 pH 4

pH 6 1 2.5 - β-sheet at pH 10 pH 8

pH 10

dmol

2 0 205 225 245

-2.5 [θ], deg cm deg [θ],

Wavelength (nm) -5

palmitoyl-VAAAEEEE-NH2 20

Transition pH 4

1 - between pH 6-8 pH 6 10 pH 8

dmol pH 10

2 0 205 225 245

-10 [θ], deg cm deg [θ], -20 Wavelength (nm)

B Continued Figure 3.12: CD spectra investigating the role of the relative strength of the H-Bond forming region and its implication in self-assembly in peptide amphiphiles. A. 10 μΜ palmitoyl-VVAAEEEE-NH2 in 2.2 CaCl2 and 150 mM NaCl. B. 10 μΜ palmitoyl-VAAAEEEE-NH2 in 2.2 CaCl2 and 150 mM NaCl. C. 10 μΜ palmitoyl- VAAAEEEE-NH2 in 150 mM NaCl and 2.2 mM CaCl2 used to precisely define the pH required for self-assembly. 42

Figure 3.12:Continued

40 palmitoyl-VAAAEEEE-NH2 5.85 5.95

30 6.07 1 - Transtion between pH 6.16 6.16

20 and 6.26 6.26

dmol 6.35

2 6.44 10 6.54 6.62 6.73 0 6.83 6.91

[θ], deg cm deg [θ], 205 225 245 -10 7.04 7.15 Wavelength (nm) 7.3 -20 C

Chapter 4

pH dependent Peptide Amphiphile Contrast Agents

The creation of smart, self-assembling materials capable of morphological transitions in response to specific physiological environments can allow for the enhanced accumulation of imaging or drug delivery agents, based on differences in diffusion kinetics. Here, we have developed a series of self-assembling peptide amphiphile molecules which transform from isolated molecules and assemble into nanofibers when the pH is slightly reduced from 7.4 to 6.6, in isotonic salt solutions that simulate 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 balance of attractive hydrophobic and hydrogen bonding forces with repulsive electrostatic forces, the single molecule to nanofiber transition pH can be systematically shifted. This transition still occurs upon incorporating MRI imaging moieties onto these molecules, making them prospective candidates as cancer imaging agents

4.1 Discovery of a Possible pH Dependent PA Contrast Agent

We have developed a series of self-assembling PA molecules that undergo a molecule-to-nanofiber morphological transition at a pH between 6.0-6.6, indicated by the presence of β-sheet secondary structure from the CD spectra (shown in Figure 4.1). The

PAs in this study contain a palmitic acid tail, an XAAA β-sheet-forming region, where X is an amino acid with a nonpolar side chain, and four glutamic acid residues. We found under these conditions, a ratio of one strongly hydrophobic amino acid tyrosine (Y), valine (V), phenylalanine (F), or isoleucine (I), three alanine residues and four glutamic acids were essential for this transition in the desired pH range.

Palmitoyl-IAAAEEEE-NH2 (Figure 4.2. A) was the first successful molecule to self-assemble in our desired pH range. In 1 wt% solutions, conventional transmission electron microscopy (Figure 4.2. B) showed this molecule assembles into nanofibers with an average diameter of 12 nm (ranging from 8-14 nm) under acidic conditions in samples prepared from water at pH 6.0. The nanofiber diameter corresponds roughly to twice the molecular length based on MM+ molecular simulations, approximately corresponding to the expected diameter of hydrophobically collapsed β-sheets cylindrical fibers . All the molecules studied were observed to form nanofibers under conventional TEM. Cryo-

TEM images indicated that 1 mM of palmitoyl-IAAAEEEE-NH2 indeed formed nanofibers in a solution of 150 mM NaCl, and 2.2 mM CaCl2, at pH 6.0 (Figure 4.2. C).

4.2 Dynamic Reversibility of Self-Assembly

CD spectra of palmitoyl-IAAAEEEE-NH were collected in salt solutions of 150 mM NaCl and 2.2 mM CaCl2 at various pH ranges. The secondary structure exhibited a superimposable random coil morphology at pHs above 6.82, determined from the collected fine scan CD spectra (Figure 4.1. A). At more acidic pHs, the secondary structure starts to have more β-sheet character. The transition between random coil structure and β-sheet structure is rapid and reversible. At pH 7.4, HCl was added until the pH was 6.2, and the resulting CD spectra (Figure 4.3. A) were collected within three minutes. An appropriate amount of NaOH was then added to reverse the pH back to 7.4, resulting in random coil morphology. This process was repeated three times and the CD spectra were observed to be superimposable with respect to pH. This indicates the transition is in thermodynamic equilibrium, and occurs in three minutes or less. This rapid, reversible transition was also observed for palmitoyl-VAAAEEEE-NH2 (Figure

4.3. B).

4.3 Correlation of Peptide Sequence to Self-Assembly

By varying the thermodynamic β-sheet propensity of the amino acids in the β- sheet forming segment, the transition pH can be shifted. For the molecules listed in Table

4.1, the isoleucine was substituted with other hydrophobic amino acids F, V, and Y. CD spectra of the PAs also showed a β-sheet to random coil transition at pH’s between 6.0-

6.6 (Figure 4.1). Previous studies have shown the propensity for β-sheet formation of the following amino acids follows the trend; I > F > V > Y52. The greater the propensity for

β-sheet formation, the higher the transition pH needed for self-assembly.

4.4 Determination of the Effect of Protonation of Glutamic Acid and its pKa

To establish if the pH transition of self-assembly occurred as a direct result of protonation, the pKa of these molecules were measured through titrations. Titrations were done at 10 μΜ to accurately correlate the pKa with the recorded CD spectra. The pKa for glutamic acid is 4.07; however the synthesized molecules displayed a slightly higher pKa than accepted values, but were not significantly different from one another, with all the pKa values around 4.9-5.0 (Figure 4.4). From CD, the pH transition of palmitoyl-FAAAEEEE and palmitoyl-VAAAEEEE occurred at 6.6 and 6.2, respectively.

We reasoned the shift in pH for self-assembly was not due to perturbation of the glutamic acid side chains. We also speculated if the self-assembly was solely due to protonation of the glutamic acids then a corresponding shift in the pKa would be seen for PAs with a higher pH transition. Consequently, the pH responsive self-assembly is not necessarily dictated by the protonation of the charged amino acids but suggestive of a more complex mechanism.

4.5 Critical Aggregation Concentration of PAs

Based on the CD spectra, it is certain that at a particular pH a morphological transition occurs, but at this time it is uncertain whether it is a spherical micelle to nanofiber transition or even an isolated molecule to nanofiber transition. In an attempt to elucidate the Critical Aggregation Concentration (CAC), we used pyrene fluorescence. In

Figure, 4.5.A and B, the pyrene 1:3 fluorescence of palmitoyl-IAAAEEEE-NH2, and palmitoyl-VAAAEEEE-NH2 was collected and plotted against concentration between 95 nM and 245 μM. By monitoring the I1/I3 change, it is possible to determine when a phase

st rd transitions occurs. The I1/I3 refers to the intensity of the 1 and 3 fluorescence emissions of pyrene when it is excited at 335 nm. A change in the intensity of I1/I3 occurs when the hydrophobicity of the pyrene environment is changed. For these molecules, I1/I3 transition is constant until it reaches the CAC, the concentration at which the PA molecules start assembling into self-assembled aggregates. Above the CAC, the I1/I3 begins to decrease linearly until it reaches a point where all the pyrene is encapsulated by nanofibers. For Palmitoyl-IAAAEEEE-NH2, and Palmitoyl-VAAAEEEE-NH2, the

CAC was determined at pH 8 to be 8 µM and 15.8 µM, respectively. This suggests that the random coil structure of both of these molecules observed in the CD at 10 µM corresponded to isolated, unassembled molecules. However, it is rather unlikely that the

CMC is below 95 nM, as the CMC of single-charged palmitoyl surfactants typically is in the range of 0.5-1.5 mM51. Further characterization is needed to confirm that the random coil structure seen by the CD is in fact a single molecule.

4.6 Incorporation of an MRI Imaging Moiety

We have successfully created a system that transitions from single molecule to nanofibers assembling at pH’s and ionic strengths simulating the extracellular vasculature of tumor tissue. We incorporated an MRI imaging moiety on the outer surface of the nanofibers to determine its effect on self-assembly. An additional lysine, conjugated to a

DO3A moiety was linked to the C-terminus of palmitoyl-IAAAEEEE-NH2 and palmitoyl-VAAAEEEE-NH2. The molecule-to-nanofiber transition was still observed, however, the transition pHs of the PA-DO3A conjugates were more acidic, and shifted to

5.7 and 5.3, respectively (Figure 4.5). Since this imaging moiety does not add excess charge, this shift towards acid pH is likely due to the greater steric hindrance63 of the PA-

DO3A molecules in the self-assembled state. Further adjustment of the strength of the attractive and repulsive forces is necessary to fine tune the self-assembly transition to occur near the slightly acidic pH of tumor tissue; to create functional imaging agents.

In summary, we have shown that through judicious design it is possible to use self-assembly to develop dynamic materials to spontaneously change shape and size in response to slight pH changes. This morphological change is rapid, reversible, and occurs under thermodynamic equilibrium, which is ideal for in-vivo imaging and drug delivery applications. With the creation of systems such as these it is now possible to create a system whose biodistribution can be reversibly modulated with respect to specific physiological stimuli.

Peptide sequence pH transition point pKa Palmitoyl-IAAAEEEE-NH2 6.6 4.7 Palmitoyl-FAAAEEEE-NH2 6.6 4.9 Palmitoyl-VAAAEEEE-NH2 6.2 4.9 Palmitoyl-YAAAEEEE-NH2 6.0 5.0 Palmitoyl-IAAAEEEEK-DOTA(Gd) 5.7 - Palmitoyl-VAAAEEEEK-DOTA(Gd) 5.3 - Table 4.1: List of synthesized molecules with pH for self-assembly and pKa values.

palmitoyl-VAAAEEEE-NH2 40 5.85 5.95

6.07

1 30 - 6.16 Transition between pH 6.16 6.26

20 and 6.26 6.35

dmol

2 6.44 10 6.54 6.62 6.73 0 6.83 6.91

[θ], deg cm deg [θ], 205 225 245 -10 7.04 7.15 Wavelength (nm) 7.3 -20

7.5 palmitoyl-FAAAEEEE-NH2 6.03

6.19

1 - 5 Transition between pH 6.32 6.63 and 6.73 6.43

6.54

dmol

2 2.5 6.63 6.73 0 6.83 205 225 245 6.93 7.01 [θ], deg cm deg [θ], -2.5 7.17 Wavelength (nm) 7.35 -5 8.61

B Continued Figure 4.1: Fine scan CD spectra used to precisely determine the pH responsive self- assembly. A. Palmitoyl-VAAAEEEE-NH2 in 150 mM NaCl and 2.2 mM CaCl2. B. Palmitoyl-VAAAEEEE-NH2 in 150 mM NaCl and 2.2 mM CaCl2. C. Palmitoyl- VAAAEEEE-NH2 in 150 mM NaCl and 2.2 mM CaCl2. D. Palmitoyl-VAAAEEEE-NH2 in 150 mM NaCl and 2.2 mM CaCl2.

Figure 4.1: Continued 20 palmitoyl-IAAAEEEE-NH2 6.01 6.16

6.26

1 6.39 - Transition between pH 6.49 and 6.6 6.49

6.6 dmol

6.71 2 6.82 5 6.91 7.01 7.18 7.32 7.41 [θ], deg cm deg [θ], 205 225 245 7.52 7.67 7.88 10 -10 Wavelength (nm)

palmitoyl-YAAAEEEE-NH2 5.51 20 5.66 Transition between 5.78

pH 5.96 and 6.02 5.88 1 - 5.96 6.02

dmol 10 6.12 2 6.2 6.29 6.38 6.47 0 6.55 [θ], deg cm deg [θ], 205 225 245 6.66 6.77 6.89 Wavelength (nm) -10 7.02

100 nm

Figure 4.2: A. The structure of the molecule of interest, palmitoyl-IAAAEEEE, the molecule undergoes a pH responsive self-assembly near pH 6.6. B. TEM image of self- assembled palmitoyl-IAAAEEEE-NH2 in 150 mM NaCl and CaCl2.

10 palmitoyl-VAAAEEEE-NH2 pH 6.0 1st pH 7.5 1 st (Reversibility) pH 6.0 2nd pH 7.5 2nd

6 1 - pH 6.0 3rd Unassembled pH 7.5 3rd

dmol 2

-2 205 225 245

[θ], deg cm deg [θ], -6 Self-assembled

-10 Wavelength (nm)

palmitoyl-IAAAEEEE-NH2 15 (Reversibility)

pH 6.1 1st

1 - 10 pH 7.8 1st pH 6.1 2nd

pH 7.8 2nd dmol

Unassembled 2 5 pH 6.1 3rd

0 205 225 245 [θ], deg cm deg [θ], -5 Self-assembled -10 Wavelength (nm)

Figure 4.3: CD spectra used to illustrate the reversibility of self-assembly by the alternating the additions of NaOH and HCl. A. 10 uM palmitoyl-VAAAEEEE-NH2 in 150 mM NaCl and 2.2 mM CaCl2. B. 10 uM palmitoyl-IAAAEEEE-NH2 in 150 mM NaCl and 2.2 mM CaCl2.

palmitoyl-VAAAEEEE-NH2 titration 10

8 pH 6

4 0 100 200 300 400 Volume of NaOH (uL)

st 0.1 1 der. of palmitoyl-VAAAEEEE Peak at 176 μL

half is 88 μL

1) 0.075 corresponding to a - pH of 4.97 0.05

dpH/dV (μL dpH/dV 0.025

0 0 100 200 300 400 Volume of NaOH (μL)

B Continued

Figure 4.4: Titration and 1st derivative plots of peptide amphiphiles used to determine the pKa at 10 μM PA, 150 mM NaCl, and 2.2 CaCl2. A. Titration of palmitoyl- ST VAAAEEEE-NH2 . B. 1 derivative of palmitoyl-VAAAEEEE-NH2titration data. C. ST Titration of palmitoyl-FAAAEEEE-NH2. D. 1 derivative of palmitoyl-FAAAEEEE- NH2 titration data.

Figure 4.4: Continued

palmitoyl-FAAAEEEE-NH2 10 titration

8 pH 6

4 0 200 400 600 800 1000 1200 Volume of NaOH (uL)

1st der. of palmitoyl-FAAAEEEE-NH2 0.02

0.015 Peak at 513 μL half is 256 μL, falling between 0.01 pH points 4.87 – 4.96

dpH/dV (1/uL) dpH/dV 0.005

0 0 250 500 750 1000 Volume of NaOH (uL)

1.1 palmitoyl-VAAAEEEE-NH2

1.05

391

/I 377 I 1 (CAC 15.8 μΜ)

0.95 4 5 6 7 8 9 log (palmitoyl-VAAAEEEE), log (pM)

palmitoyl-IAAAEEEE-NH2 1.1

1.05

391

/I 377 I 1 (CAC 8 μΜ)

0.95 4 6 8 10 log (palmitoyl-IAAAEEEE-NH2), log (pM)

Figure 4.5: Critical aggregation concentration determination using the pyrene 1:3 method. All fluorescence measurements were done at 150 mM NaCl, CaCl2, 0.3 μM pyrene with 1% MeOH. A. Palmitoyl-VAAAEEEE-NH2 B. Palmitoyl-IAAAEEEE-NH2 .

40 palmitoyl-IAAAEEEE-NH 6.01 2 6.16 6.39 6.49 Transition between pH 6.6 and 6.6 20 6.71 6.71 6.82 6.91 7.01 7.1 7.18 0 7.32 7.41

Ellipticity (mdeg) Ellipticity 205 225 245 7.52 7.67 7.88 -20 Wavelength (nm) 10

3+ palmitoyl-IAAAEEEEK-DOTA(Gd ) 5.19 20 5.35

5.49

5.62

10 Transition between pH 5.71 5.71 and 5.62 5.81 5.89 5.98 0 6.1 205 225 245 6.21 6.31

Ellipticity (mdeg) Ellipticity -10 6.44 6.64 Wavelength (nm) 6.73 -20 6.91

Figure 4.6: CD fines scans of A. palmitoyl-IAAAEEEE-NH2 , self assembly occurs at pH 6.6 in simulated physiological salts. B. palmitoyl-IAAAEEEEK-DOTA(Gd3+), assembly occurs at pH 5.7

Chapter 5

Conclusions and Future Directions

Each year the number of cases of cancer grows and the need for non-invasive detection techniques also increase. Designing nanomaterial imaging agents with controllable shape and size in response to physiological stimuli, such as pH can potentially exploit differential diffusion kinetics to amplify the local accumulation of these agents. We have developed a library of peptide-amphiphile molecules responsive to physiologically relevant pH changes, and developed a systematic understanding of the means to balance the supramolecular forces to control the self-assembly transitions.

There are numerous directions in which the work presented here can be taken.

First, it may be possible to develop an empirically derived model that predicts the CAC and transition pH in this class of molecules according to the relative strength of the attractive and repulsive forces of the peptide sequence. Here, we have synthesized a library of closely related molecules, and discerned that there are systematic trends in the self-assembly behavior associated with peptide sequence. Consequently, it may eventually be possible to design structures that feature this transition de novo. With this work, it may also be possible to incorporate additional components to this class of 59 molecules, such that transitions can be triggered by other biological stimuli (e.g. enzymatically, or in a hypoxic environment).

The task ahead lies in confirming this transition can occur in vivo. Designing different characterization approaches to show that this transition can occur in serum is the next step forward. Possible avenues include exploiting differences in the magnetic relaxivity, or fluorescence anisotropy to determine the pH-dependent self-assembly behavior of these molecules in a protein-rich environment. If a pH-dependent morphology can be observed, it may yield vastly different biodistribution properties between materials existing solely as isolated molecules, and those existing as nanofibers.

The development of next generation drug delivery and tumor imaging vehicles lies within the proof that a significant accumulation of material in the extracellular tumor environment can occur under relevant biological and observable conditions.

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