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Synthesis of Gadolinium Phosphonate (Gd-Hedp) as a New MRI Contrast Agent and Measurement of Its Relaxivity Resonance Properties

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Title:
Synthesis of Gadolinium Phosphonate (Gd-Hedp) as a New MRI Contrast Agent and Measurement of Its Relaxivity Resonance Properties
Creator:
Al-Marzooq, Fatimah Yousef
Place of Publication:
[Gainesville, Fla.]
Florida
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University of Florida
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english
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1 online resource (42 p.)

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Chemistry
Committee Chair:
TALHAM,DANIEL R
Committee Co-Chair:
SMITH,BEN W
Committee Members:
TOTH,ANNA F
Graduation Date:
8/9/2014

Subjects

Subjects / Keywords:
Gadolinium ( jstor )
Imaging ( jstor )
Ions ( jstor )
Ligands ( jstor )
Magnetic resonance imaging ( jstor )
Nanoparticles ( jstor )
Phosphates ( jstor )
Protons ( jstor )
Signals ( jstor )
Solar X rays ( jstor )
Chemistry -- Dissertations, Academic -- UF
agent -- contrast -- gadolinium -- mri
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Chemistry thesis, M.S.

Notes

Abstract:
In this thesis, two sets of experiments will be discussed: synthesizing a T1 contrast agent as nanosize particles and in bulk. In the first set of experiments, a T1 MRI contrast agent was synthesized as nanoparticles. The goal was to prepare a new gadolinium based MRI contrast agent with a stable structure in aqueous solution and a low relaxivity (r2/r1) ratio compared to those commercially available. Gadolinium Phosphonate nanoparticles were prepared using a reverse microemulsion method with IGEPAL-520 as the surfactant. The nanoparticles varied in size from 38 nm to 1000 nm. A surface modification with PMIDA was performed to achieve particle stability in aqueous solution. FTIR (Fourier Transform Infrared Spectroscopy), C/H/N analysis (carbon, hydrogen, nitrogen analysis), XRD (X-Ray Diffraction Spectroscopy), EDS (Energy Dispersive X-Ray Spectroscopy) and TEM (Transmission Electron Microscopy) images were recorded to visualize the nanoparticles. Magnetic resonance relaxivity properties were measured to compare those of the nanoparticles to available gadolinium based contrast agents (GBCA). If GP nanoparticles prove stable at room temperature in aqueous solution and generate higher resolution MRI images, this would represent a significant improvement over existing GBCA. In the second part, the same particles were synthesized in bulk. The goal of this part was to make a comparison between the bulk and nanoparticles materials. The comparison was made using FTIR, TEM and EDS analysis for both parts. An interesting result was observed with the EDS assay; the Gd atoms in the nanoparticles bound to more ligands than the gadolinium in the bulk materials. Although several attempts were made to grow crystals from the GBCA, they were not successful. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (M.S.)--University of Florida, 2014.
Local:
Adviser: TALHAM,DANIEL R.
Local:
Co-adviser: SMITH,BEN W.
Statement of Responsibility:
by Fatimah Yousef Al-Marzooq.

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UFRGP
Rights Management:
Copyright Al-Marzooq, Fatimah Yousef. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
968785875 ( OCLC )
Classification:
LD1780 2014 ( lcc )

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SYNTHESIS OF GADOLINIUM PHOSPHONATE (GD HEDP) AS A NEW MRI CONTRAST AGENT AND MEASUREMENT OF ITS RELAXIVITY RESONANCE PROPERTIES By FATIMAH YOUSEF AL MARZOOQ A THE SIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FL ORIDA IN PARTIAL FULFILLMENT OF THE REQUI REMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2014

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© 2014 Fatimah Yousef AL Marzooq

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To my f amily members who supported me during my study

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4 ACKNOWLEDGMENTS I would l ike to acknowledge my advisor Dr. Dan Talham for giving me the opportunity to work in his research group as well as for his guidance, good advice, patience and follow up throughout the period I spent in his labs. I would also like to thank my committee members, Dr. Talham, Dr. Smith, and Dr. Toth for their valuable advi ce , for their time spent reading my thesis and attending my defense. Special thanks go to all the group members for their support and friendship. Last but not least, to my great family in Saudi Arabia for their encouragement during my years of study.

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5 TABLE OF CONTENTS P age ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 12 1.1 Imaging Techniques ................................ ................................ .......................... 12 1.2 Magnetic Resonance Imaging (MRI) ................................ ................................ . 12 1.3 Nanoparticles as MRI Contrast Agents ................................ ............................. 13 1.4 Types of MRI Contrast Agents ................................ ................................ .......... 14 1.5 Devising New Gadolinium Based Contrast Agents ................................ ........... 15 2 MATERIALS AND EXPERIMENTAL METHODS ................................ ................... 19 2.1 Materials and Methods ................................ ................................ ...................... 19 2.2 Part 1 ................................ ................................ ................................ ................ 20 2.2.1 Method ................................ ................................ ................................ .... 20 2.2.2 Characterization ................................ ................................ ...................... 20 2.2.2.1 Fourier transform infrared spectroscopy (FTIR) [26]: ..................... 20 2.2.2.2 High resolution transmission electron microscopy (HR TEM) [25]: ................................ ................................ ................................ ......... 21 2.2.2.3 Energy dispersive X ray spectroscopy (EDS) [23, 24]: .................. 21 2.2.2.4 Carbon, Hydrogen And Nitrogen analysis [27]: .............................. 25 2.2.2.5 X Ray powder diffraction (XRD) [22]: ................................ ............. 25 2.3 Surface Modification with N (Phosphonomethyl) Iminodiacetic Acid (PMIDA): ................................ ................................ ................................ .............. 25 2.4 Part 2 ................................ ................................ ................................ ................ 27 2.4.1 Method ................................ ................................ ................................ .... 27 2.4.2 Characterization ................................ ................................ ...................... 27 2.4.2.1 Fourier transform infrared spectroscopy (FTIR): ............................ 27 2.4.2.2 High resolution transmission electron microscopy (HRTEM): ........ 29 2.4.2.3 Energy dispersive X ray spectroscopy (EDS): ............................... 29 3 RELAXATION DATA ................................ ................................ ............................... 32 3.1 T1 and T2 Relaxation Times ................................ ................................ ............. 32 3.2 T1 and T2 Measurement ................................ ................................ ................... 33

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6 4 DISCUSSION AND CONCLUSIONS ................................ ................................ ...... 37 4.1 Discussion ................................ ................................ ................................ ........ 37 4.2 Conclusions / Future Directions ................................ ................................ ........ 38 LIST OF REFERENCES ................................ ................................ ............................... 40 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 42

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7 LIST OF TABLES Table P age 2 1 EDS data of Gadolinium to Phosphorus ratio. ................................ .................... 24 2 2 Carbon, Hydrogen and Nitrogen analysis of two samples of Gd HEDP. ............ 25 2 3 EDS data using the method described in part 2. ................................ ................ 30 3 1 Comparison of r2/r1 values for different contrast agents. ................................ ... 35

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8 LIST OF FIGURES Figure P age 1 1 T1, T2 weighted image of transverse sections of human brain and proton density weighted images of human brain. ................................ .......................... 16 1 2 HEDP structure (1 hydroxyethane 1, 1 diphosphonic acid) ................................ 17 2 1 The structure of PMIDA. ................................ ................................ ..................... 19 2 2 In frared absorption spectrum of etidronic acid (HEDP) ................................ ..... 22 2 3 Infrared spectrum of three different samples of Gd HEDP. ................................ 23 2 4 TEM images of Gd HEDP. ................................ ................................ ................. 24 2 5 HR TEM images of Gd HEDP with the altered ratio of cyclohexane to IGEPAL CO 520. ................................ ................................ ................................ 24 2 6 X ray powde r diffraction of Gd HEDP. ................................ ................................ 26 2 7 FTIR spectroscopy of Gd HEDP in bulk ................................ ............................. 28 2 8 FTIR for Gd HEDP synthesized in part 1 (FY005) an d Gd HEDP synthesized in part 2 (FY012). ................................ ................................ ................................ 28 2 9 TEM images of Gd HEDP synthesized with method in part 2. ........................... 29 3 1 Mz recovery in T1 relaxation (Left) and Mxy decay in T2 relaxation (Right). ...... 33 3 2 T1 measurement of Gd HEDP of FY005 sample ................................ ................ 34 3 3 T2 measu rement of Gd HEDP of FY005 sample. ................................ ............... 34 3 4 T1 measurement of Gd HEDP of FY019. ................................ ........................... 36 3 5 T2 measurement of Gd HEDP of FY019. ................................ ........................... 36

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9 LIST OF ABBREVIATIONS AKI BBB CA Acute Kidney Injury Blood Brain Barrier Contrast A gent C/H/N Carbon, Hydrogen, Nitrogen CKD CT DTAP EDS FTIR GBCA GFR GP HRTEM MRI NSF PMIDA PPM SPIO XRD Chronic Kidney Disease Computed Tomography Diethylenetriamine P e nta a cetic A cid Energy Dispersive X Ray S pectroscopy Fourier Transform Infrared S pectroscopy Gadolinium Based Contrast A gent Glomerular Filtration Rate Gadolinium P articles High Resolution Transmission Electron M icroscop y Magnetic Resonance I maging Nephrogenic Systemic F ibrosis N (Phosphonomethyl) Iminodiacetic A cid Parts Per M illion Superparamagnetic Iron O xide X Ray Diffraction S pectroscopy

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10 Abstract of Thesis Presented to the Graduate School of the University o f Florida in Partial Fulfillment of the Requi rements for the Degree of Master of Science SYNTHESIS OF GADOLINIUM PHOSPHONATE (GD HEDP) AS A NEW MRI CONTRAST AGENT AND MEASUREMENT OF ITS RELAXIVITY RESONANCE PROPERTIES By Fatimah Yousef Al Marzooq August 2014 Chair: Dan Talham Major: Chemistry In this thesis, two sets of experiments will be discussed: synthesizing a T1 contrast agent as nanosize particles and in bulk. In the first set of experiments, a T1 MRI contrast agent was synthesiz ed as nanoparticles. The goal was to prepare a new gadolinium based MRI contrast agent with a stable structure in aqueous solution and a low relaxivity (r2/r1) ratio compared to those commercially available. Gadolinium Phosphonate nanoparticles were prepar ed using a reverse microemulsion method with IGEPAL 520 as the surfactant. The nanoparticles varied in size from 38 nm to 1000 nm. A surface modification with PMIDA was performed to achieve particle stability in aqueous solution. FTIR (Fourier Transform In frared Spectroscopy), C/H/N analysis (carbon, hydrogen, nitrogen analysis), XRD (X Ray Diffraction Spectroscopy), EDS (Energy Dispersive X Ray Spectroscopy) and TEM (Transmission Electron Microscopy) images were recorded to visualize the nanoparticles. Mag netic resonance relaxivity properties were measured to compare those of the nanoparticles to available gadolinium based contrast agents (GBCA). If GP nanoparticles prove stable at room temperature in aqueous solution and generate

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11 higher resolution MRI imag es, this would represent a significant improvement over existing GBCA. In the second part, the same particles were synthesized in bulk. The goal of this part was to make a comparison between the bulk and nanoparticles materials. The comparison was made us ing FTIR, TEM and EDS analysis for both parts. An interesting result was observed with the EDS assay; the Gd atoms in the nanoparticles bound to more ligands than the gadolinium in the bulk materials. Although several attempts were made to grow crystals fr om the GBCA, they were not successful.

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12 CHAPTER 1 INTRODUCTION 1 .1 Imaging T echniques A critical step in the diagnosis of many diseases involves internal imaging of the body. Several non invasive imaging modalities are in use today, including plain film X ray, computed tomography (CT), and Magnetic Resonance Imaging (MRI) [17]. Each form of imaging has advantages and drawbacks. On the one hand, X rays and CT scans can be performed in a matter of minutes; however, the resulting images are often time consum ing for radiologists to interpret, with the exceptions of gross bone fractures and acute cerebral hemorrhages. On the other hand, an MRI scan may take 30 60 minutes to complete but generally provides higher quality images than the other two techniques, esp ecially of most soft tissues. This property makes MRI the preferred choice for imaging the brain [1], heart, skeletal muscles, abdominal cavity as well as a majority of tumors [10]. Additional advantages of using MRI for body imaging are that the process i s painless and entails no exposure to X ray radiation. Excessive exposure to ionizing radiation including X rays can induce mutations in DNA leading to cancer and birth defects [5]. 1 .2 Magnetic Resonance I maging (MRI) MRI uses a powerful magnetic field an d various frequencies of radio waves to scan the body and produce images. The magnetic field aligns the spin axes of water protons (and other atomic nuclei with an odd number of protons and neutrons) [2]. Once aligned, the protons absorb radio waves, which or change direction. After the excitation step, relaxation occurs several milliseconds later as the protons return to their baseline spin state. This process generates a signal

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13 detected by a computer [8]. Using a program based on Fourier transformations, the computer converts the signal into an image corresponding to that part of the body. The final image is three dimensional [19], which is very desirable in radiology. This is the ideal scenario; in practice, ran dom signals arise from hydrogens in surrounding molecules, e.g. proteins and lipids. To minimize this interference, contrast agents are used to enhance the signal to noise ratio and make the resulting images clearer. Without a contrast agent, the water pro background magnetic fields. When a soluble contrast agent is added, however, the MRI metal atoms. The CA signal dwarfs a proton sign al by a factor of several thousand. So, when the contrast agent is added the sensitivity and the ability to detect lesions increases [11,14]. This will help and improve imaging in the fields of biology and medicine. 1 .3 Nanoparticles as MRI Contrast A gent s Advances in nanotechnology have led to improvements and new directions in the development of contrast agents. Nanoparticle contrast agents, especially those less than 100 nm in diameter, have several advantages. Their two most important properties are, f irst the large surface area increases the reactivity of the material, and second, the core metal atom has a greater ability to bind to other functional groups [8]. The superiority of nanotechnology is obvious compared to traditional contrast agents, the ferromagnetic iron oxides. Ferromagnetic iron oxides distort the magnetic field due to their huge magnetic susceptibility from neighboring normal tissues. This background distortion is known as the blooming effect [8]. This effect increases the signals in the background which decreases the resolution of the images, in turn leading

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14 to inaccurate or inconclusive disease diagnoses. On the other hand, at nanoparticle sizes, the ferromagnetic iron oxides will lose the large susceptibility and become superparamag netic. That will shorten T2 and increase the contrast in the resulting images. 1 .4 Types of MRI Contrast A gents MRI contrast comes from the interaction between the contrast agent and the neighboring water molecules in the body. MRI contrast agents are gea red toward enhancing one of two imaging parameters, known as T1 and T2. T1 and T2 weighting use two entirely different types of contrast agents (CA). T1 contrast agents usually consist of paramagnetic complexes. They can be called Longitudinal or positive contrast agents. The other type of contrast agent is a T2 contrast agent. They can be called transverse or negative contrast agents. Most of T2 contrast agents are based on transition metal agents contain Fe (III) or Mn (II). One well known example of a T2 contrast agent is Superparamagnetic iron oxide (SPIO). This type of contrast agent is based on spin spin relaxation time, or the magnetic interaction of protons with neighboring protons [15]. Although T2 contrast agents have been used for a long time in MRI imaging, they have several disadvantages which limit their usefulness in clinical applications. First, the fast absorption of SPIO agents by the liver leads to a correspondingly rapid excretion from the body. To avoid this limitation, particles less th an 50 nm in diameter are necessary to prolong hepatic excretion time. Another disadvantage is that SPIO agents tend to appear darker than the background tissue; these areas can easily be confused with opacities in the surrounding tissue [8]. Today, SPIO co ntrast agents are still

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15 available for human use; however, their sole indication is to confirm the presence of cancer metastases to the liver [19]. The second type is the Gadolinium based contrast agents. They can be thought of as T1 or Longitudinal contras lattice it returns to its baseline state. Paramagnetic gadolinium complexes are used because this element has seven unpaire d electrons in its 4F orbital, which has a total capacity of 14 electrons. Each unpaired electron has a local magnetic field almost 660 times more potent than a proton. In addition, Gadolinium can induce relaxivity in 1 million water molecules per second, making it an ideal contrast agent [11]. A good example of a gadolinium contrast agent is Gd DTAP [7]. It has been widely used in detecting breakage of the blood brain barrier (BBB) [8]. More recently, a Gd based contrast agent has been used in liver imagin g in addition to SPIO [12]. 1 .5 Devising New Gadolinium Based Contrast A gents In recent years and for the above mentioned reasons, there has been a great deal of interest in developing new Gadolinium contrast agents in order to provide higher resolution and better quality T1 images. In practical terms, this will make MRI scans even more sensitive at detecting lesions like tumors at smaller sizes than currently possible [21]. The in vivo resolution limit of MRI is approximately 0.5 1 millimeters [3]. Altho ugh this sounds tiny, a tumor that reaches 2 mm in diameter (and is probably still asymptomatic at that size) may already contain 1 x 10 7 or 10 million cancer cells [20]. Clearly, much room for improvement exists in the realm of MRI imaging. Although T2 im aging is preferred for highlighting a tumor or a fluid collection, T1 imaging plays a complementary role in most diagnostic situations.

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16 Figure 1 1. T1, T2 weighted image of transverse sections of human brain and proton density weighted i mages of human b rain. From a safety perspective, a solution containing free Gadolinium (III) is too toxic for use in humans; hence, it is necessary to chelate Gd with other compounds to minimize the risk of heavy metal poisoning, especially hepatotoxicity (liver damage) a nd nephrotoxicity (kidney damage). The big concern for researchers when synthesizing a new gadolinium contrast agent is to choose a stable chelate that will hold the Gd 3+ ion and not release it or allow it to exchange inside the body. There are three type s of stability classes: non ionic linear agent, ionic linear agents, and macrocyclic agents. In the Anzalone paper [16], data suggests that the macrocyclic agents are the most stable, as they contain a core of four atoms that bind covalently to the central Gd. After that, ionic linear ligands come next in order from a safety perspective. In this res earch we used HEDP (F igure 1 2 ) which is an ionic linear ligand. The reason for our choice was because we wanted to get solid nanoparticles which is not the case with macrocyclic; they are soluble molecules. As will be discussed later, this ligand has an interesting property that could make it more stable than other ionic linear ligands.

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17 Figure 1 2. HEDP structure (1 hydroxyethane 1, 1 diphosphonic acid) One ris k factor associated with GBCA deserves special mention, and that is nephrogenic systemic fibrosis (NSF). NSF occurs almost exclusively in patients with acute kidney injury (AKI) or chronic kidney disease (CKD). In severe renal impairment, a low GFR (glomer ular filtration rate), corresponding to a creatinine clearance less than reasonable amount of time [6]. Although the cause(s) of NSF remains obscure, it seems after several day s in the body, Gd 3+ ions dissociate from their ligand shell and deposit systemically, triggering a serious, sometimes fatal dermatological reaction. With the above in mind, the advent of a GBCA in the form of a chelated gadolinium nanoparticle should mean a lower dose of contrast agent needed for human imaging studies. This, in turn, should lower the risk of nephrotoxicity, NSF, or other complications arising from Gd Considering that renal elim most contrast agents, the consensus is that GBCA are safer than the iodinated contrast agents still used in X ray radiography and CT scans [17].

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18 The strategy for developing a new GBCA was based on a 1997 pa per entitled DNA Surface Modified Gadolinium Phosphate Nanoparticles as MRI Contrast Agents [15]. IGEPAL CO520 would be used as a surfactant to control the sizes of the nanoparticles. The next step was to modify the surface with PMIDA to make the particles soluble in aqueous solution. Finally, the resulting nanoparticles would be subjected to a magnetic field and radio frequencies to measure their r2/r1 ratio as an indicator of relaxivity. An r2/r1 ratio less than 2 is considered desirable for a T1 contrast agent [9].

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19 CHAPTER 2 MATERIALS AND EXPERIMENTAL METHODS 2 .1 Materials a nd Methods The following three compounds were purchased from Sigma Aldrich and used as received. 1) Gadolinium nitrate (Gd (NO 3 ) 3 ) was dissolved in water to allow the Gd 3+ ions to di ssociate. 2) Etidronic acid monohydrate (1 hydroxyethane 1, 1 diphosphonic acid): This compound was used to chelate the Gd 3+ ions. It has two phosphate groups on each side (bisphosphonate) as shown in Figure 1 1 . 3) IGEPAL CO 520 is a non ionic surfactant used to control the particle size. Additional reagents: Cyclohexane: Cyclohexane was used as a non polar solvent. Potassium bromide (KBr): 200 mg of this compound was used for FTIR background. N (Phosphonomethyl) iminodiacetic acid (PMIDA): This compound used for the surface modification. It has two carboxylic acid groups on one side and a phosphate group on the other side. The phosphate group binds to and stabilizes the Gd 3+ on the particle's surface ( Figure 2 1 ). Figure 2 1. The structure of PMIDA. Ga dolinium HEDP contrast agent was synthesized and characterized in this research. The two parts of this chapter will discuss the synthesis of Gd HEDP. Part 1

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20 will explain how the Gd HEDP was synthesized as nanoparticles. Some techniques that have been used to characterize the synthesize material such as FTIR, TEM, EDS, C,H,N analysis and XRD will also be shown. Next, surface modification with PMIDA will be discussed. Part 2 will discuss synthesis of the Gd HEDP in bulk. Next, a review of some techniques use d to characterize the new material will be highlighted. After that, a detailed comparison between the two different parts will be summarized. 2 .2 Part 1 2 .2.1 Method Gd HEDP was prepared using the same method as described in the article DNA Surface Modifie d Gadolinium Phosphate Nanoparticles as MRI Contrast Agents (10). The first beaker (A) was prepared by adding a solution of 500 mg Gadolinium nitrate (Gd (NO 3 ) 3 ) in 5 mL of H 2 O to 100 mL of cyclohexane combined with 20 mL of IGEPAL CO 520. The second beake r (B) was prepared with the addition of a solution of 568 mg of HEDP to 5 mL of H 2 O. That was added to 100 mL of cyclohexane mixed with 20 mL of IGEPAL CO 520. Beakers A and B were stirred for 30 minutes. Afterwards, the contents of beaker B were added dro p by drop to beaker A. After three hours of stirring, the microemulsion was broken with 250 mL of Acetone. The washing steps consisted of three centrifugations with acetone then twice with water. After synthesizing the material several techniques were use d to characterize the product. 2 .2.2 Characterizat ion 2 .2.2.1 Fourier transform infrared s pectroscopy (FTIR) [26]: The infrared spectrum was taken on KBr background. 1 mg of Gd HEDP was added to 200 mg of KBr. The mixture was ground for 3 minutes and put it into the die.

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21 Afterwards, the IR spectrum was measured. The selected range was between 400 4000 cm 1, and the number of scans was 16. With this method the infrared spectrum of HEDP (Etidronic acid) w as recorded as shown in Figure 2 2 . From this spect rum, we can tell that the signals from 1230 1260 cm 1 belong to phosphonate group. The second step consisted of recording the infrared spectrum of the synt hesis Gd HEDP, shown in Figure 2 3 . A consistent result was observed with three different samples. Th ey were prepared according to the same method described previously. The phosphonate stretch signal remains very sharp and intense. 2 .2.2.2 High resolution transmission electron m icroscopy (HR TEM) [25]: HRTEM images were taken on the prepared samples. The purpose of capturing the images was to take a close look at the shapes and sizes of the particles. TEM grids were purchased from the Ted Pella Company. The sample was prepared by dispersing 5 mg of Gd HEDP in 2 mL of acetone. Next, 20µL of the solution wa s added drop wise to the grid. Afterwards, the microscopy was performed on JEOL 2010F HRTEM at 200 kV. The s hapes of the samples in Figure 2 4 were well defined rectangles. The sizes varied a great deal between 160 nm 1000 nm. The goal was to achieve smaller crystal sizes with a more defined shape, wh ich is clearly shown in Figure 2 5 . In this image, I used the same preparation method as previously discussed but decreased the volume of cyclohexane to 50 mL. This change produced crystals as small as 38 nm i n diameter, as shown in Figure 2 5 . 2.2.2.3 Energy dispersive X ray s pectroscopy (EDS) [23, 24]: Analysis was performed on an EDS X ray microanalysis system coupled to an HRTEM microscope. Determining the ratio between gadolinium and phosphate was our

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22 goal in this step. Table 2 1 shows the ratio of gadolinium to phosphate for the two different samples. Figure 2 2. I nfrared absorption spectrum of e tidronic acid (HEDP )

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23 Figure 2 3. Infrared spectrum of three different samples of Gd HEDP .

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24 Figure 2 4. TEM images of Gd HEDP . Figure 2 5. HR TEM images of Gd HEDP with the altered ratio of cyclohexane to IGEPAL CO 520. Table 2 1. EDS data of Gadolinium to Phosphorus ratio. Sample ID Gadolinium Phosphorus Sample 1 21.00 79.00 Sample 2 21.60 78.40

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25 Sample 1 and sample 2 show the same ratio between the two elements; these numbers are consistent with a Gd to P ratio of 1:4. Essentially, we are chelating the gadolinium with two bidentate molecules of HEDP, which will stabilize the complex inside the b ody and minimize the risk of free Gd 3+ ions being released. 2 .2.2.4 Carbon, Hydroge n A nd Nitrogen a nalysis [27]: This step characterized the sample content to ensure that we were able to accurately synthesize the material on a consistent basis. In Table 2 2 , which shows the percentage of carbon, hydrogen and nitrogen for two different samples, a consistent result from the two samples can be seen. Table 2 2. Carbon, Hydrogen and Nitrogen analysis of two samples of Gd HEDP. Sample ID Carbon Percentage Hydrog en percentage Nitrogen Percentage Sample 1 7.081% 2.252% 0.000% Sample 2 7.383% 2.377% 0.081% 2 .2.2.5 X R ay p owder d iffraction (XRD) [22]: This technique was used to determine if the sample is crystallized. We obtained X ray data for both sa mples that is shown in Figures2 4 and 2 5 . Both of the two samples show exactly the sa me result, as shown in (Figure 2 6 ). Powder data were obtained using X'Pert Powder instrument that was purchased from the "PANalytical" company. We weighed out 100 mg of the nanopa rticles then applied a very thin layer of them to double sided tape backed by a glass slide. D efine d crystalline structures usually produce very sharp peaks. This is not the case with my material; in angle 22 we can see a very broad peak ( Figure 2 6 ). 2 .3 Surface M odification w ith N (Phosphonomet hyl) Iminodiacetic A cid (PMIDA) : To make this contrast agent comparable to the other contrast agent, we needed to see how these particles disperse in water. In their native state, the particles are not

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26 soluble in wa ter so the idea was to modify their surfaces to make them more soluble in aqueous solution. For this step we added 20 mg of Gd HEDP Nanoparticle to 1 mL of water. Then 30 mg of Gd (NO 3 ) 3 was added to the solution. After that, we placed the solution in the ultrasonicator for 30 minutes. This step allowed us to coat the particle surface with Gd 3+ ions. In another vial 60 mg of PMIDA was dissolved in 3 mL of water. Then, the pH of the solution was adjusted to around 7. To raise the pH, we first measured the p H of the PMIDA solution; it was extremely acidic. As such, we added 100 µL of NaOH and measured the resulting pH. We repeated this step until the solution reached pH 7. Next, a Gd HEDP vial was added to the PMIDA vial and placed in the ultrasonicator for 6 0 minutes. The new particles were collected by centrifuge and washed three times with nanopure water. After completing these steps, we visually inspected the dispersal properties of the particles in water and found that they disperse very well. Figure 2 6. X ray powder d iffraction of Gd HEDP.

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27 Next we wanted to see how these nanoparticles compare to bulk particles. So, I performed another series of experiments which will be discussed in Part 2. 2 .4 Part 2 2 .4.1 Method The method used was similar to the me thod described by Nash, K. (13). This experiment basically involves mixing the lanthanide with the HEDP in a 1:2 molar ratio. For this method two vials were mixed. The first one, which we will call vial A, contained 0.295 g of Gd (NO 3 ) 3 dissolved in 5 mL o f H 2 O. The second vial (vial B) contained 0.448 g of HEDP dissolved in 5 mL of H 2 O. Then we added vial A to B. After that I tried several ways to collect the particles. Two ways were ultimately successful. The first one was to allow the mixture to stand a t room temperature for three days until the particles form then collect them by four rounds of centrifugation with water. The result of this method will be shown in sample FY012. The second way was to stir the solution for 60 minutes, let it stand for anot her hour, and then centrifuge it with water 4 times. The result of this approach will be shown with the sample FY013. We characterized the results of the two methods FY012 and FY013 by several techniques in order to compare it to the nanomaterials. 2 .4.2 Characterization 2 .4.2.1 Fourier transform infrared s pectroscopy (FTIR): The same setup of Part 1 was used. 200 mg KBr was ground and combined with 1 mg of the synthesized material. The result is shown in Figure 2 7 , which shows the FTIR spectroscopy of the bulk materials of Gd HEDP. We still can see the P=O peak which confirms the presence of the phosphate group. Al so, in Figure 2 7 we can directly compare the two ways of collecting the materials FY012 and FY013; in both methods,

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28 identical material was c ollected. Finally, IR spectroscopy confirms the presence o f a phosphonate peak at 1230 cm 1 . Figure 2 7. FTIR spectroscopy of Gd HEDP in bulk Figure 2 8. F TIR for Gd HEDP synthesized in p art 1 (FY0 05) and Gd HEDP synthesized in p art 2 (FY012).

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29 For t he sake of comparison, Figure 2 8 shows two samples of FY005 (prepared in Part 1) with FY012 (prepared in Part 2). We can see many identical peaks that represent identical functional groups in both materials. The only difference between the two samples is a s mall shift in the phosphonate peak. 2 .4.2.2 High resolution transmission electron m icroscopy (HRTEM): The second step in characterizing these materials was obtaining high resolution TEM images. The same setup that preformed in Part 1 was used. By doing thi s step, we wanted to compare the shape and size of crystals produced by the two different methods. In Figure 2 9 , Gd HEDP synthesized by the method described in Part 2 is shown. We can see that it is much larger than the nanomaterial, which was expected. T he other noticeable feature is the rectangular shape of the particles. Figure 2 9. TEM images of Gd HEDP s ynthesized with method in p art 2. 2 .4.2.3 Energy dispersive X r ay s pectroscopy (EDS): The analysis was done coupled to the TEM instrument. We want ed to know the ratio between the Gd and P at oms in these particles. Table 2 3 shows the atomic percentage of both Gd and P.

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30 Table 2 3. EDS data using the method described in p art 2. Sample ID Gadolinium percentage Phosphorus percentage Sample 1 38.75 61.2 5 Sample 2 31.35 68.65 From Table 2 3 we can see that we were able to synthesize Gd HEDP with a different Gd to P ratio than the compound in Part 1. In this part, the Gd HEDP has a Gd to P ratio of 1:2, which means a lower number of P ligands around the core lanthanide atom. After using these various methods to characterize the two materials, we conclude that the nanomaterial and bulk material differ in terms of ligand composition, i.e. the Gd to ligand ratio. In FTIR for both methods the functional gro ups are present and had very sharp, intense peaks with only a sm all shift, as shown in Figure 2 8 . In the HRTEM images it is notable that the nanoparticles are much smaller that the bulk material. In both methods the particle shape was a rectangular prism . In the EDS technique, an important difference between the two methods was found. In the nanoparticles, more HEDP ligands are attached to the Gd whereas in the bulk material only one ligand is attached. More phosphate ligands are attached to the nanomate rial than to the bulk material; the implications of this finding will be discussed in Chapter 4. 2 .5 Crystal Growth One of the main goals of this research was to grow a crystal of Gd HEDP in order to obtain a more detailed structure and a better idea of arrangement. The following experimental protocol was taken from a 1997 paper [13]. I tried to grow different lanthanide HEDP crystals using Gd, Eu, and Dy HEDP. In the

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31 following experiment 0.448 g of HEDP dissolved in 5 mL of H2O was a dded to 0.295 g, 0.446 g and 0.377 g of Gd(NO 3 ) 3 , Eu(NO 3 ) 3 and DyCl 3 . 6 H 2 O dissolved in 5 mL of H 2 O, respectively. The solutions were left on the counter for almost seven months. During this time, white Gd HEDP and Eu HEDP particles as well as yellow Dy HE DP particles appeared, but ultimately no crystals formed. By the end of the experiment, the particles resembled a powder more than a crystal.

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32 CHAPTER 3 RELAXATION DATA 3.1 T1 and T2 Relaxation Times 1 H NMR signals arise from the coupling proton spin exci tation and relaxation in a strong magnetic field swept by radio frequencies. Proton spin relaxation can be enhanced in one of two ways depending upon whether a T1 or T2 contrast agent is used. T2 (the transverse or spin spin relaxation time) is defined as the time it takes for the synchronous magnetic spins of the 1 H nuclei to decay from their peak excitation state in the xy plane (Mxy decay) [8]. A plot of T2 appears as a downward sloping curve that falls to its original value after several seconds. Effe ctive T2 contrast agents are compounds with the ability to shorten T2 relaxation time (see image 12). This makes the contrast appear as a dark area on the resulting image; for this reason T2 agents are also called negative contrast agents. This property is considered a drawback, however, because it often makes image interpretation difficult. Conversely, T1 (the longitudinal or spin lattice relaxation time) is the time it takes the 1 H nuclei to realign or their magnetic spins along the longitudinal or z plane (Mz recovery) [8]. The T1 signal plot appears as a curve with a positive slope that plateaus after several seconds. T1 contrast agents shorten T1 relaxation time; this increases the signal and produces a correspondingly brighter region on the resulti ng image. For this reason, T1 agents are called positive contrast agents and are often preferred over T2 agents.

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33 Figure 3 1. Mz recovery in T1 relaxation (Left) and Mxy decay in T2 relaxation (Right). In an effective T1 contrast agent, the rati o between the transverse and longitudinal relaxivities (r2/r1) must be kept low. Ideally, the value of this ratio should be lower than 2 [9]. To obtain r2 values, we plot the concentration in mM vs. the T2 value in S 1 unit and measure the slope, which cor responds to r2; similarly, the value of r1 is obtained by plotting the concentration vs. T1. 3.2 T1 and T2 M easurement T1 and T2 measurements were done to assess the quality of our new synthesized contrast agent. Five T1 and T2 measurements were taken for each sample. Different concentrations of gadolinium were prepared by dissolving 10 mg, 5 mg, 2.5 mg, 1.25 mg, and 0.625 mg of Gd HEDP respectively into 1 mL of H 2 O. Another sample was prepared for ICP to measure the concentration. For the ICP measurement , 1 2 mg of the nanoparticles was dissolved in 3 5 mL of nanopure water. First, a sample of nanopure water is run to measure any background signal and calibrate the machine accordingly. Next, a series of standards is run containing 1, 10, and 100 ppm of Gd , respectively. Finally the sample solution is measured, and the instrument calculates the Gd concentration automatically.

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34 Figure 3 2. T1 measurement of Gd HEDP of FY005 sample Figure 3 3. T2 measurement of Gd HEDP of FY005 sample. T1 and T2 measureme nts were done on the product synthesized according to the two methods described in Part 1 of the Experimental section. The first sample, represented by FY005, was synthesized by the original method. The other sample,

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35 represented by FY019, was synthesized u sing the new ratio between cyclohexane and IGEPAL 520 and corresponds to sample FY018. In Figures 12 and 13, a plot of T1 and T2 vs. concentration is shown. All five data points appear distinctly on the plot. From the slope of the linear equation, r1 and r2 can be calculated. In Figures 12 and 13, the ratio of r2/r1 was around 2.495, which is higher than the desired result. The other measurements are shown in Figures 14 and 15, which show the plot for the FY019 sample. Here the value of r2/r1 is around 1. 800. By changing the ratio between cyclohexane and IGEPAL 520, we were able to achieve an r2/r1 ratio within the acceptable range (lower than 2). To compare this result with the available result for Gd DTPA, see Table 3 1 . In this t able our relaxation data is compared to Gd DTPA which is a complex of gadolinium with a chelating agent, diethylenetriamine penta acetic acid (DTPA). Also, we compare it with Gd PO 4 DNA which is a complex of gadolinium phosphate with DNA surface modification. Table 3 1. Compariso n of r2/r1 values for different contrast agents. Contrast agent Structure a Magnetism r1 mM 1 s 1 r2 mM 1 s 1 r2/r1 Gd HEDP(FY019) NP Paramagnetic 9.9 17.8 1.8 Gd HEDP(FY005) NP Paramagnetic 2.5 6.2 2.4 Gd PO 4 DNA NP Paramagnetic 0.2 12.8 60.9 Gd DTP A C Paramagnetic 4.6 5.4 1.1 a NP: nanoparticles C: chelate As Table 3 1 shows, it is clear we were able to get an acceptable r2/r1 ratio with our new contrast agent compared to the other agents.

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36 Figure 3 4. T1 measurement of Gd HEDP of FY019. Figu re 3 5. T2 measurement of Gd HEDP of FY019.

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37 CHAPTER 4 DISCUSSION AND CONCLUSIONS 4.1 Discussion The main objective of this project was to develop a novel T1 gadolinium based contrast agent. Since free Gd 3+ ions are too toxic to be used in vivo, it was n ecessary to select a strong chelating agent to prevent the release or exchange of ions in solution. As noted previously, as a class, macrocyclic agents reliably chelate lanthanide ions by forming covalent bonds with them. However, since linear chain ionic agents form comparatively stable complexes with Gd 3+ ions in solution, HEDP was chosen as the gadolinium chelating agent. Gd HEDP nanoparticles were synthesized using a reverse microemulsion method. Many techniques were employed to characterize the result ing product including FTIR, C,H,N analysis, XRD, HR TEM images, and EDS. Nanoparticles ranged in size from 160 to 1000 nm in diameter. To achieve a smaller particle size, the synthesis was repeated using the same protocol but decreasing the volume of cyclo hexane by 50%. After making this change, we obtained complexes as small as 38 nm in diameter. TEM images revealed a rectangular prism shape for the nanoparticles. A second set of experiments was done using the bulk synthesis product for comparison, as desc ribed in Chapter 2. FTIR spectroscopy of both nanoparticles and bulk material showed the same peaks with a small shift in the (P=O) peak seen in the bulk product. TEM images of the bulk material also revealed rectangular prisms. The size of the bulk produc t was much larger than the nanoparticles, as we anticipated. The EDS analysis revealed a surprising result. In the nanoparticles, the Gd to P ratio was 1:4 whereas the ratio for the bulk material was 1:2. This means twice as many

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38 phosphonate ligands are p resent in each nanoparticle compared to their bulk material counterparts. A chelation shell consisting of two ligands would bind Gd 3+ ions more effectively and provide greater stability in solution compared to an agent with only one ligand. Although bulk s ynthesis of Gd HEDP requires less time than nanoparticle preparation, use of the bulk contrast agent would be confined to in vitro imaging. Gd HEDP nanoparticles, on the other hand, are far more likely to meet the safety criteria for future in vivo experim ents using animal models. XRD analysis showed that the nanomaterial lacks a well defined crystal structure. Several attempts were made to grow crystals using various lanthanide HEDP combinations (specifically Gadolinium, Europium, and Dysprosium); howev er, none of these attempts proved successful. To evaluate the effectiveness of the contrast agent, T1 and T2 measurements were performed on five different product concentrations. Afterwards, the r2/r1 ratios were calculated. We were able to achieve a rati o of 1.8, which is lower than the threshold value of 2 considered to be the acceptable upper limit of relaxivity for a T1 contrast agent. 4.2 Conclusions / Future Directions In summary, we have developed a novel GBCA with optimal dispersal properties in a queous solution. EDS results for the nanoparticles confirm that each Gadolinium ion is chelated by two HEDP complexes rather than one, which minimizes the risk of free Gd 3+ ions being released inside the body. Relaxation measurements were especially promis ing, as we achieved an r2/r1 value less than 2. On the other hand, we were unable to crystallize the Gd HEDP complex; consequently, we have not yet worked out its exact structure at the atomic level.

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39 Crystallization experiments based on alternate lanthani de series elements may yet prove successful. Also, measure the r2/r1 for the bulk material will be interesting to know and compare to our NP result. Beyond in vitro experiments, using this contrast agent to obtain real time images in animal models repre sen ts a truly exciting prospect.

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40 LIST OF REFERENCES [1] MRI: T1 and T2 relaxation http://onderwijs1.amc.nl/medfysica/doc/MRI%20T1%20and%20T2%20Comp.htm [2] Aoife O'Brien, MRI, Physics Review , 2009, 18, 16 17. [3] Bradley P. Thomas et al. High Resolution 7T MRI of the Human Hippocampus In Vivo , Journal of Magnetic Resonance Imaging . Nov 2008; 28(5): 1266 1272. doi: 10.1002/jmri.21576 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2669832/ [4] Deb. A, Way to PG, http://prep pg.blogspot.com/2012/04/undestanding mri in simplest way part.html [5] Environmental Health and Safety, Open Source Radiation Safety Training, Module 3: Biological Effe cts, 2014, Princeton University h ttp://web.princeton.edu/sites/ehs/osradtraining/biologicaleffects/page.htm [6] Jeffery D. Schlaudecker and C. R. Bernheisel, Gadolinium Associated Nephrogenic Systemic Fibrosis , American Family Physician , 2009, 80, 711 714 [7] Jaap Valk l, Ragnhild G. M ., De Slegte 1, Frank C. Crezee l, Govert J. Hazenberg 2, and Stephan I. Thjaha, Editorial Gadolinium Chelate MR Contrast Agents, Clinical Radiology, 1994, 49, 439 442. [8] Hyon Bin Na, In Chan Song, and Taeghwan Hyeon, Inorganic Nanoparticles for MRI Co ntrast Agents , Advanced Materials , 2009, 21, 2133 2148. [9] Hifumi, H; Yamaoka, S; Tanimoto, A; Citterio, D; Susuki, K, Gadolinium Based hybrid nanoparticles as a positive MR contrast agent , J. AM.CHEM.SOC , 2006, 128, 15090 15091. [10] H. Shokrollahi, Contrast agents for MRI , Materials Science and Engineering C, 2013, 33, 4485 4497. [11] Gadolinium contrast agents and relaxivity (2011) ICPM Education Video https://www.youtube.com/watch?v=Osx8 Ced9Eyw [12] Gustav J. Strijkers, willem J.M.Mulder, Geralda A.F. van Tilborg, Klaas Nicolay, MRI Contrast Agents: Current Status and Future Perspectives, Anti Cancer Agents in Medicinal Chemistry , 2007, 7, 291 305. [13] Kenneth L. Nash, F Element compl exation by diphosphonate ligands, Journal of Alloys and Compounds , 1997, 249, 33 40. [14] Lucie Paulet, Contrast Agents in MRI (2013) PDF http://epileptologie bonn.de/cms/upload/homepage/lehnertz/LPaulet_CA_MRI.pdf

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41 [15] Matthieu F. Dumont, Celine Baligand, Yichen Li, Elisabeth S. Knowles, Mark W. Meisel, Glenn A. Walter, and Daniel R. Talham, DNA Surface Modified Gadolinium Pho sphate Nanoparticles as MRI Contrast Agents, Bioconjugate chemistry , 2012, 23, 951 957. [16] Nicolette Anzalone, Are All Gadolinium based Contrast Agents Similar? The Importance of High Stability, High Relaxivity and High Concentration, European Neurologi cal Review , 2009, 4(2), 98 102. [17] Orthoinfo, http://orthoinfo.aaos.org/topic.cfm?topic=a00188 . [18] ReviseMRI.com, http://www.revisemri.com/questions/basicphysics/t1contrast [19] Saravanan Namasivayam, Diego R Martin and Sanjay Saini, Imaging of liver metastases: MRI, cancer imaging, 2007, 7, 2 9. [20] Sten Friburg and Stefan Mattson, On the Growth Rates of Human Malignant Tumors: Implications for Medical Decision Making, Journal of Surgical Oncology , 1997 ; 65:284 297 [21] Thomas E. McCann,Nob uyuki Kosakaa, Baris Turkbeya, Makoto Mitsunagaa, Peter L. Choykea and Hisataka Kobayashi, Molecular Imaging of Tumor Invasion and Metastases: the Role of MRI, NMR in Biomedicine , 2010, 7, 561 568. [22] Barbara L Dutrow, Louisiana State University, Chris tine M. Clark, Eastern Michigan University, 2013, Geochemical Instrumentation and Analysis, X Ray Powder Diffraction http://serc.carleton.edu/research_education/ge ochemsheets/techniques/XRD.html [23] Introduction to Energy Dispersive X ray Spectrometry (EDS), University of California Riverside http://micron.ucr.edu/public/manuals/EDS intro.pdf [ 24] Sayeeda Ghaffari, Nanofabrication PPT (2011) University of Victoria http://www.stehm.uvic.ca/using/training/workshops/CAMTEC/2011/xray%20presentatio n.p pt [25] Miroslav Karlík, Introduction to high resolution transmission electron microscopy, Czech Technical University in Prague http://www.tu chemnitz.de/physik/AFKO/eml/praktika/HREM_AN.pdf [26] Thermo Nicolet Corporation, Introduction to FTIR (2001) California Institute of Technology http://mmrc.caltech.edu/FTIR/FTIRintro.pdf [27] V. P. Fadeeva, V. D. Tikhova, and O. N. Nikulicheva, Elemental Analysis of Organic Compounds with the Use of Automated CHNS Analyzers , Journal of Analytical Chemistry , 2008, Vol. 63, No. 11, pp. 1094 11 06

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42 BIOGRAPHICAL SKETCH Fatimah Yousef Al Marzooq earned her Bachelor of Science degree in C hemistry from King Faisal University in Saudi Arabia in 2010. In 2012 she joined the graduate program at the University of Florida under the King Abdullah scholarsh ip program to study her master degree in Analytical Chemistry . synthesis a new T1 MRI contrast agent. Mrs. Fatimah Al Marzooq thesis was supervised by Dr. Dan Talham.