Synthesis and Characterization of Novel Inorganic Nanoparticles for Diagnostic and Therapeutic Applications
A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy
by
Vindya S. Perera
December, 2014
Dissertation written by
Vindya S. Perera
B.S., University of Peradeniya, SriLanka, 2006
Ph.D., Kent State University, USA, 2014
Approved by
______Songping Huang, Professor, Ph.D., Department of Chemistry & Biochemistry, Doctoral Advisor
______Scott D. Bunge, Professor, Ph.D., Department of Chemistry & Biochemistry
______Mietek Jaroniec, Professor, Ph.D., Department of Chemistry & Biochemistry
______Qi-Huo Wei, Professor, Ph.D., Liquid Crystal Institute
______Ernest Freeman, Professor, Ph.D., Department of Biological Sciences
Accepted by
______Michael J. Tubergen, Professor, Ph.D., Chair, Department of Chemistry & Biochemistry
______James L. Blank, Professor, Ph.D., Dean, College of Arts and Sciences
TABLE OF CONTENTS
List of figures……………………………………………………………………….…..iii
List of schemes……………………………………………………………………….....ix
List of tables……………………………………………………………………………..x
Acknowledgments……………………………………………………………………...xi
Chapter 1: Introduction and background…………………………………………….…...1
1.1. Magnetic resonance imaging (MRI)………………………………………….…...1
1.1.1 Contrast agents for MRI……………………………………………………..…...6
1.2. X-ray computed tomography (CT)………………………………………….....….9
1.2.1 Contrast agents for X-ray computed tomography (CT)……………………....10
1.3. Contrast agents for dual MRI/CT modalities…………………………………...16
1.4. Targeting intracellular copper ions for selective removal………………….…..17
1.4.1 Intracellular copper removal as a potentially more effective treatment
for the Wilson’s disease……………………………………………………………...... 19
1.4.2. Copper depletion as a novel strategy for angiogenesis inhibition………....…23
Chapter 2: Materials and Methods ……………………………………………….……....28
2.1 Materials…………………………………………………………………….……..28
2.2 Experimental design and techniques…………………………………………...... 29
2.3 Experimental methodology………………………...... 29
2.4 Instrumentation…………………………………………………………………....30
2.4.1 Powder X-ray diffraction spectroscopy (PXRD)……………………...30
2.4.2 Transmission electron microscopy (TEM) and energy dispersive
X-ray (EDX)…………………………………………………………………...30
2.4.3 Thermogravimetric analysis (TGA)……………………………………31
iii
2.4.4 Fourier transform infrared spectroscopy (FTIR)…………………….31
2.4.5 Confocal microscopy…………………………………………………....31
2.4.6 T1 and T2 measurements…………………………………………….….32
2.4.7 Atomic absorption measurements……………………………………..32
2.5 Experimental calculations………………………………………………………...33
2.5.1 Relaxivity (r1 and r2 values)………………………………………….....33
Chapter 3: Contrast agents for magnetic resonance imaging………………………34
3.1. Gadolinium-based MRI contrast agents………………………………………...34
3.2. Biocompatible nanoparticles of KGd(H2O)2[Fe(CN)6]•H2O with extremely
high T1-weighted relaxivity owing to two water molecules directly bound to the
Gd(III) center...... 38
3.3. Manganese-based MRI contrast agents………………………………………....64
3.4. PVP-coated KMn[Fe(CN)6] nanoparticles as a potential contrast agent
for MRI...... 66
Chapter 4: Contrast agents for X-ray computed tomography……………………...81
4.1 Nanoparticles of the novel coordination polymer KBi(H2O)2[Fe(CN)6].H2O
as a potential contrast agent for computed tomography…………………………...83
4.2. Nanoparticles of KBiXGd(1-X)[Fe(CN)6] as a potential bimodal contrast
agent for MRI and CT………………………………………………………………...95
Chapter 5: Nanoparticles for therapeutic applications……………………………….105
5.1. Cell permeable Au@ZnMoS4 core-shell nanoparticles: Towards a novel cellular
copper detoxifying drug for Wilson’s disease…………….……………………...... 106
5.2. Nanoparticles of ZnMoS4 as novel inhibitors of angiogenesis………………...125
Chapter 6: Conclusions…………………………………………………………………….148
References……………………………………………………………………………….……150
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List of figures
Figure 3.1: TEM image of as prepared nanoarticles of KGdPB nanoparticles…………………40
Figure 3.2: EDX spectrum on a PVP-coated nanoparticle……………………………………...41
Figure 3.3: The FT-IR spectra of sodium citrate, PVP, bulk compound and nanoparticles alone……………………………………………………………………………………………..42
Figure 3.4: Powder X-ray diffraction spectra for the as-prepared KGd [Fe(CN)6].3H2O………44
Figure 3.5: The unit-cell packing diagram of KGd(H2O)2[Fe(CN)6]•H2O (left) with the K+ ion and zeolitic water molecule omitted for clarity. The coordination environment of the Gd3+ ion (middle and right) showing two water molecules directly bound to the
metal center……………………………………………………………………………………...44
II Figure 3.6: The TGA curve of bulk KGd(H2O)2[Fe (CN)6] H2O sample……………………....45
3+ Figure 3.7: The graph of 1/T1 (i=1,2) versus Gd -concentration at the magnetic field strength of 1.4 T………………………………………………………………………………....47
3+ Figure 3.8: The graph of 1/T1 (i=1,2) versus Gd -concentration at the magnetic field strength of 7.0 T………………………………………………………………………………....48
3+ Figure 3.9: The plot of 1/Ti (i=1,2) versus Gd -concentration at the magnetic field strength of 1.4 T for PVP coated nanoparticles……………………………………………….....49
Figure 3.10: The plot of r1 vs the hydrodynamic particle size for KGdPB nanoparticles……..50
Figure 3.11: The plot of r2 vs the hydrodynamic particle size for KGdPB nanoparticles……...50
Figure 3.12: T1-weighted MR phantom images of KGdPB nanoparticles with various
Gd3+-concentrations using a 7.0-T scanner………………………………………………….…...53
Figure 3.13: T2-weighted MR phantom images of KGdPB nanoparticles with various
Gd3+-concentrations using a 7.0-T scanner……………………………………………………....55
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Figure 3.14: Fluorescence spectra of carboxyfluorescein dye and dye labeled nanoparticles....56
Figure 3.15: Confocal microscopic images of Hela cell line (A) Fluorescence image of cells incubated with dye conjugated nanoparticles for 3 hrs (B) Bright field image of cells incubated with dye conjugated nanoparticles for 3 hrs (C) flurescence image of untreated cells. (D) Bright field image of untreated cells………………………….………………………………….……..58
Figure 3.16: Viability of Hela cells after incubation with KGdPB nanoparticles for 24 hrs and 48 hrs (Trypan Blue exclusion assay)…………………………………………………….....59
Figure 3.17: CN- releasing test for different conditions…………………………………………61
Figure 3.18: Gd3+ releasing test for different conditions………………………………………..62
Figure 3.19: T1-weighted MRI phantoms of PC3 cells incubated in PBS buffer (left), 0.13 mM nanoparticles (central), and 0.25 mM nanoparticles (right) for 6 hours. The images were
collected using a Bruker 9.4-T scanner………………………………………………………....63
Figure 3.20: TEM images of PVP-coated KMn[Fe(CN)6 nanoparticles……………………...... 68
Figure 3.21: PXRD pattern of bulk MnPB……………………………………………………...69
Figure 3.22: Crystal structure of bulk MnPB, color code: red=K, dark yellow=Fe and Mn, yellow=C, blue=N…………………………………………………………………………….....70
Figure 3.23: Overlay of the IR spectra of bulk (red) and nanoparticles (blue) of MnPB and PVP (green) alone…………………………………………………………………………...71
−1 Figure 3.24: Longitudinal relaxation rates (1/T1, s ) of MnPB NPs as a function of the manganese concentration (mM)………………………………………………………………....72
−1 Figure 3.25: Transverse relaxation rates (1/T2, s ) of MnPB NPs as a function of the manganese concentration (mM)………………………………………………………………....73
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2+ Figure 3.26: T2-weighted MRI phantoms of NPs with various Mn -concentrations using a 9.4-T scanner…………………………………………………………………………………..74
Figure 3.27: Mn2+ leaching results of MnPB NPs under different conditions……………….....75
- III Figure 3.28: CN leaching results of KMn[Fe (CN)6 under different conditions……………...76
Figure 3.29: Viability of Hela cells after incubation with MnPB NPs for 24 hrs and 48 hrs
(Trypan Blue exclusion viability assay)………………………………………………………....77
Figure 3.30: Confocal microscopic images of Hela cell line (A) Bright field image of cells incubated with dye conjugated nanoparticles for 4 hrs. (B) Fluorescence image of cells incubated with dye conjugated nanoparticles for 4 hrs. (C) Bright field image of untreated cells.
(D) Fluorescence image of untreated cells……………………………………………………....79
Figure 3.31: T2-weighted MRI phantoms of Hela cells incubated in PBS buffer (left),
0.2 mM NPs (central), and 0.5 mM NPs (right) for 6 hours. The images were collected
using a Bruker 9.4-T scanner……………………………………………………………………80
Figure 4.1: TEM image of KBi(H2O)2[Fe(CN)6].H2O nanoparticles………………………...... 84
Figure 4.2: EDX spectrum on a typical PVP-coated nanoparticle………………………….…..85
Figure 4.3: Rietveld refinement plot: difference between observed and calculated patterns is shown at the bottom; reflection positions are shown as vertical lines…………………………..87
Figure 4.4: Crystal structure of KBi(H2O)2[Fe(CN)6].H2O with Bi and Fe shown in yellow and blue polyhedra, respectively. Cyanide ions are shown as thick cylinders (N, blue; C, gray balls). K ions are depicted as large balls………………………………………………..…...…..88
Figure 4.5: The FT-IR spectrum of PVP-coated nanoparticles……………………………...... 89 vii
Figure 4.6: The TGA curve of bulk KBi(H2O)2[Fe(CN)6]·H2O sample…………….……….....90
Figure 4.7: Viability of Hela cells after 24 and 48 hrs of incubation periods with varying concentrations of nanoparticles………………………………………………………………...... 91
Figure 4.8: Leaching levels of free CN-and Bi3+ ions………………………………………...... 92
Figure 4.9: CT intensity values (top) and phantom images (bottom) of nanoparticles with different Bi3+ concentrations………………………………………………………………..……94
Figure 4.10: TEM image of as prepared nanoarticles of Gd@BiPB...... 96
Figure 4.11: EDX spectrum on a nanoparticle……………………………………………..…...97
Figure 4.12: Overlay of the IR spectra of bulk (dark green) and nanoparticles (pink) of
Gd@BiPB and PEG (blue) alone…………………………………………………………..…....97
Figure 4.13: PXRD pattern of bulk compound…………………………………………..……...98
Figure 4.14: Structure of 3D extended polymer network found in Gd@BiPB...... 99
Figure 4.15: CN release test against different conditions……………………………..………..99
Figure 4.16: Viability of Hela cells after incubation with Gd@BiPB nanoparticles for 24 hrs and
48 hrs (MTT assay)…………………………………………………………………..…………100
Figure 4.17: Confocal microscopic images of Hela cells: (upper left) fluorescence image of cells incubated with dye-conjugated NPs for 3 hrs; (upper right) bright field image of cells incubated with dye-conjugated NPs for 3 hrs; (lower left) florescence image of the untreated cells; (lower right) bright field image of the untreated cells……………………………………...………….102
Figure 4.18: The CT attenuation values of different Gd@BiPB concentrations…..………….102
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Figure 4.19: The proton T1 relaxation of PEG-coated Gd@BiPB nanoparticles at different concentrations……………………………………………………………………..……………103
Figure 4.20: T1-weighted MRI phantoms of PEG-coated Gd@BiPB nanoparticles with varying concentrations of gadolinium…………………………………………………………………..104
Figure 5.1: TEM images of as-prepared Au NPs (upper left) and Au@ZnMoS4 NPs (lower left) and histograms of the size distribution for NPs corresponding to each panel on the left……...111
Figure 5.2: EDX spectrum on a typical PVP-coated Au@ZnMoS4 nanoparticle……………..111
Figure 5.3: UV−visible spectra of pure Au NPs and the Au@ZnMoS4 NPs at the different stages of layerby-layer assembling process…………………………………………………….112
Figure 5.4: FT-IR spectra of Au@ZnMoS4 NPs, PEG coated Au@ZnMoS4 NPs and pure
PEG……………………………………………………………………………………………..113
Figure 5.5: Kinetics of copper removal from the aqueous copper solution by Au@ZnMoS4
NPs……………………………………………………………………………………………..114
Figure 5.6: Pseudo first order rate plot for the copper removal by Au@ZnMoS4 NPs…….....116
Figure 5.7: Second order rate plot for the copper removal by Au@ZnMoS4 NPs…….………117
Figure 5.8: Copper removal capacity of Au@ZnMoS4 NPs…………………………………...118
Figure 5.9: Selectivity of several divalent metal ions by Au@ZnMoS4 NPs……………….....119
Figure 5.10: Fluorescence spectra for carboxyfluorescein dye and dye labeled nanoparticles………………………………………………………………………………...... 120
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Figure 5.11: Confocal microscopic images of HepG2 cells: (upper left) fluorescence image of cells incubated with dye-conjugated NPs for 3 hrs; (upper right) bright field image of cells incubated with dye-conjugated NPs for 3 hrs; (lower left) florescence image of the untreated cells; (lower right) bright field image of the untreated cells……………………………………121
Figure 5.12: Effect of Au@ZnMoS4 NPs on viability of HepG2 cells after 12-hr and 24-hr incubation………………………………………………………………………………………122
Figure 5.13: Kinetics of copper uptake in HepG2 cells……………………………………….123
Figure 5.14: Kinetics of copper removal from HepG2 cells…………………………………..124
Figure 5.15: TEM image of as-prepared ZnMoS4 NPs………………………………………..129
Figure 5.16: EDX spectrum on a ZnMoS4 nanoparticle……………………………………….130
Figure 5.17: Overlay of the IR spectra of bulk (blue) and nanoparticles (green) of ZnMoS4 and
PVP (red) alone………………………………………………………..………………………..131
Figure 5.18: PXRD pattern of PVP-coated ZnMoS4 NPs………………………………….…..131
Figure 5.19: PXRD pattern of bulk ZnMoS4 material………………………………………...132
Figure 5.20: Kinetics of copper removal by ZnMoS4 NPs………………………………….....134
Figure 5.21: Pseudo first order rate plot for the copper removal by ZnMoS4 NPs…………....135
Figure 5.22: Second order rate plot for the copper removal by ZnMoS4 NPs…………………136
Figure 5.23: Copper removal capacity of ZnMoS4 NPs……………………………………….137
Figure 5.24: Selectivity of metal removal by ZnMoS4 NPs…………………………………...138
Figure 5.25: Fluorescence spectra of carboxyfluorescein dye and dye labeled nanoparticles..139
x
Figure 5.26: Confocal microscopic images of HepG2 cells: fluorescence image (upper left) and bright field image (upper right) of cells incubated with dye-conjugated NPs for 3
hours; florescence image (lower left) and bright field image (lower right) of the untreated cells……………………………………………………………………………………………..140
Figure 5.27: Confocal images showing endocytosis and exocytosis of nanoparticles………..141
Figure 5.28: Kinetics of copper removal from HepG2 cells………………………………….142
Figure 5.29: Panel 1 bright-field (left) and fluorescent (right) images of calcein-stained HuVEC cells treated with FGF-2(50ng/ml) in basal media showing the tube formation. Panels 2 & 3
FGF-2 induced HuVEC cells treated 4 hrs with 5uM angiogenesis inhibitor Sulforaphane or
50 uM ZnMoS4 NPs, respectively……………………………………………………………...145
Figure 5.30: 20 µM ZnMoS4 gave 50% reduction in tube formation (middle panel) compared to positive control without ZnMoS4 (left panel) and 50 µM ZnMoS4 completely inhibited tube formation (right panel)………………………………………………………………………....146
Figure 5.31: Viability curve of HepG2 cells incubated with ZnMoS4 NPs for 24 hours and 48 hours……………………………………………………………………………………………147
.
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List of schemes
Scheme 1: The magnetic momentum (µ) of the proton………………………………………...... 2
Scheme 2: In the presence of external magnetic field hydrogen nuclei undergo precession….....2
Scheme 3: (A) In the absence of an external magnetic field, spins rotate about their axes in random direction. (B) In the presence of a magnetic field, spins align mainly parallel to the magnetic field, producing, Mz. (C) An RF pulse is applied. (D) Longitudinal magnetization rotates into transverse magnetization, Mxy……………………………………………………..…4
Scheme 4: T1 relaxation. Decay of transverse magnetization and restore of magnetization along the z-axis……………………………………………………………………………………5
* Scheme 5: T2 and T2 relaxation. Dephasing, resulting in the loss of transverse magnetization without energy dissipation………………………………………………………………………...6
Scheme 6: Factors influencing relaxivity…………………………………………………….…..9
Scheme 7: Structures of some commercially available clinically approved CT contrast agents.
Ionic monomers (a), nonionic monomers (b), ionic dimer (c), and nonionic dimer (d)……..….15
Scheme 8: Pathways of copper metabolism in the hepatocyte…………………………………..21
Scheme 9: Chemical structure of 2,3-dimercaptopropanol (British anti-lewisite)….…………..22
Scheme 10: Chemical structure of D- penicillamine………………………………….……..….22
Scheme 11: Clinically approved MRI contrast agents (only the ligands are presented in
the scheme)……………………………………………………………………………………...37
Scheme 12: Synthesis of citrate coated nanoparticles………………………………….……….56
Scheme 13: Schematic diagram showing the synthesis of dye-labeled nanoparticles…..……...57
Scheme 14: Schematic of layer-by-layer self-assembly of Au@ZnMoS4 NPs………...…...... 110
Scheme 15: Basement membrane……………………………………………………………...144
xii
LIST OF TABLES
Table 1: K-shell electron binding energies (K Edge) for selected heavy elements……….…....13
Table 2: Some common commercially available small-molecule iodinated contrast agents.….15
Table 3: Proangiogenic Mediators that Rely on Cu for Expression or Function……………….26
Table 4: Clinically approved gadolinium based MRI contrast agents………………………….36
Table 5: Crystallographic Data for KGd(H2O)2[Fe(CN)6]. H2O………………………………..43
Table 6: Selected interatomic distances (Å)…………………………………………….……....43
Table 7: Comparison of relaxivity data of several selected nanoparticulate Gd3+-based contrast agent………………………………………………………………………………….....47
Table 8: Summary of structure determination for KBi(H2O)2[Fe(CN)6].H2O………….……....86
Table 9: Selected Interatomic Distances (Å)……………………………………………………88
Table 10: The decrease of Cu concentrations vs. time in the ion-exchange reaction…………115
Table 11: The natural log of copper concentration values at each time point for pseudo first order reaction…………………………………………………………………………………...115
Table 12: Values of reciprocal concentration and corresponding time for second order reaction………………………………………………………………………………………....116
Table 13: The decrease of Cu concentrations vs. time in the ion-exchange reaction…………135
xiii
Acknowledgements
First, I would like to express my sincere gratitude and cordial admiration to my advisor
Dr. Songping D. Huang for his continuous help, support, guidance and feedback throughout my
Ph.D. career. I am grateful and sincerely thank him for valuable time and scientific ideas to make my Ph.D. experience highly productive. I also express sincere gratitude to all the members of my doctoral dissertation committee, Dr. Mietek Jaroniec, Dr. Scott D. Bunge, Dr. Qi-Huo Wei and previous committee member Dr. Nicola Brasch for their continuous support throughout my Ph.D. career and valuable comments and suggestions on my research proposal and dissertation. I am grateful to Dr. Gail Fraizer for her assistance with the biological experiments and for her insightful comments, valuable discussions on a collaborative project. Special thanks go to Dr.
Jaroniec and Chris Flask (Case Western Reserve University) for their assistance with the helpful suggestions as co-authors of my publications. I would also like to thank Dr. Mahinda Gangoda for his valuable assistance.
I would like to thank the current and former members of our research group, especially
Murthi Kandanapitiye, Liu Yang, Don Pryor, Ronghui Song, Sriramakrishna Yarabarla and
Yanyi Zang for all their help. I would also like to thank Haiwa Wu from the Biological Sciences.
I would also like to thank Ms. Erin Michael for all her support and assistance. I would also like to thank my family and friends for their support and understanding throughout my whole academic career. I especially thank, my mother (Padma Pathirana), father (Amarasiri Perera), sister (Dulani
Perera) and brother (Ranga Perera) for their inspiration encouragement, continuous guidance and love throughout my life. Last but most importantly, I would like to thank my husband Nilantha
Wickramaratne for his support, encouragement and love in every moment of my ups and downs during all these years. Finally, I would like to thank and express my love to my daughters Saheli
Wickramaratne and Sithumi Wickramaratne.
xiv
I would like to dedicate this dissertation to my family for their support and love throughout my life.
Vindya S Perera
September 25, 2014
xv
Chapter 1: Introduction and background
This dissertation is concerned with the synthesis and characterization of novel inorganic nanoparticles for diagnostic and therapeutic applications. In chapters 3 and 4, the synthesis and characterization of nanoparticles for diagnostic applications are described. Specifically, Chapter 3 reports on the studies of several MRI contrasts agents and Chapter 4 reports on the studies of a new CT contrast agent. The last two chapters, Chapters 5 and 6 of this dissertation concern the therapeutic applications of nanoparticles. In chapter 5, the synthesis and characterization of a copper depleting agent for the potentially more effective treatment of Wilson’s disease is reported.
Finally, in Chapter 6, the study on nanoparticulate antiangiogenesis agents for cancer treatment is described.
1.1. Magnetic resonance imaging (MRI)
Magnetic resonance imaging (MRI) is a prominent noninvasive technique used clinically to diagnose diseases. It provides high-resolution three-dimensional images of anatomic structures as well as functional information about tissues in vivo. The contrast of MRI images depends on three factors. The different distribution of hydrogen nuclei’s spin density and the longitudinal (T1) and transverse (T2) relaxation times of these spins. A hydrogen atom consists of a single proton and a single electron. The proton possesses spin and a positive charge. A magnetic momentum is thus created (µ) and it behaves like a small magnet (Scheme 1). When hydrogen nuclei are exposed to an external magnetic field, B0, the magnetic moments align with the direction of the
1
field and undergo precession as shown in Scheme 2. Precession of the nuclei occurs at a specific speed and is known as the Larmor frequency. The Larmor frequency is directly proportional to the magnetic field strength (B0) and is given by equation 1:
ω0 = γ0 ・ B0 (1)
Where; ω0 is the Larmor frequency in megahertz [MHz], γ0 the gyromagnetic ratio, a constant characteristic to a particular nucleus, and B0 the strength of the magnetic field in tesla [T].
Scheme 1: The magnetic momentum (µ) of the proton.
Scheme 2: In the presence of external magnetic field hydrogen nuclei undergo precession.
2
In magnetic resonance imaging experiment, hydrogen nuclei are exposed to an external magnetic field which results in parallel or anti-parallel arrangement of spins to the magnetic field with parallel alignment being slightly preferred. A radio frequency (RF) known as resonance frequency is then applied to the nuclei. The protons absorb energy and are excited to the antiparallel state and rotate the spins into the transverse plane, Mxy (Scheme 3). After the electromagnetic field is turned off, spin system relaxes releasing the energy absorbed from the RF pulses and settles into a stable state, producing longitudinal magnetization Mz in the z-direction.
During this relaxation, a radio frequency signal is generated, which can be measured with receiver coils. Relaxation takes place via two independent processes that reduce transverse magnetization. They are spin-lattice interaction and spin-spin interaction. These two processes cause T1 relaxation and T2 relaxation, respectively.
T1 relaxation or the longitudinal relaxation is the process of returning nuclei to their ground state (to the equilibrium state along the z axis) by dissipating their excess energy to their surroundings through interactions with neighboring nuclei and molecules (Scheme 4). This is also known as the spin lattice relaxation. The time constant for this process is denoted T1, and defined as the reciprocal of the rate of energy loss. T1 is the time it takes for the longitudinal magnetization to recover approximately 63% [1-(1/e)] of its initial value after applying the RF pulse. T1 depends on the mobility of the molecule in which the nucleus is bound and the type of tissue. For instance, T1 of body fluids is longer than that of the solid tissues. Longitudinal relaxation occurs most efficiently when the molecular tumbling rate is near the Larmor frequency.
The tumbling rate of the medium sized molecules such as lipids and fat is closer to the Larmor frequency. Therefore solid tissues, has a relatively short T1 (on the order of 250 ms at 1.5 T).
Body fluids have free water and hence tumbling rate is much faster than the Larmor frequency which results in a longer T1 (greater than 1 s at similar field strengths). Differences in T1 among tissues are used to generate signal contrast on MR images.
3
A B
B 0 MZ MZ
C D
RF pulse
MZ
MXY
Scheme 3: (A) In the absence of an external magnetic field, spins rotate about their axes in random direction. (B) In the presence of a magnetic field, spins align mainly parallel to the magnetic field, producing, Mz. (C) A RF pulse is applied. (D) Longitudinal magnetization rotates into transverse magnetization, Mxy.
4
Meq MZ MXY
MZ
Meq
63% Meq
T time 1
Scheme 4: T1 relaxation. Decay of transverse magnetization and restore of magnetization along the z-axis.
The magnetization also undergoes transverse relaxation or T2 relaxation due to the loss of phase coherence (dephasing). Spins precess faster or slower according to the magnetic field variations they experience. During the transverse relaxation process spins exchange energy with each other and individual magnetization vectors begin to cancel each other. Therefore this process is called spin-spin relaxation (Scheme 5). Dephasing due to the intrinsic factors such as molecular size and the tissue type is related to the time constant T2 and can be expressed as the time it takes for the magnetic resonance signal to reach 37% (1/e) of its initial value after its generation by tipping the longitudinal magnetization (Mz) towards the magnetic transverse plane (Mxy).
Transverse relaxation also occurs via the process known as the free induction decay due
* to the magnetic field inhomogeneity. The time constant for this process is denoted as T2 . The
5
dephasing caused by free induction decay can be avoided by applying a 180o RF pulse. Therefore the signal we obtain is due to the T2 relaxation.
Mxy
Mxy
37% Mxy
T 2 Time (s)
Scheme 5: T2 relaxation. Dephasing, resulting in the loss of transverse magnetization without energy dissipation.
1.1.1 Contrast agents for MRI
Differences in relaxation times are used to produce signal contrast on MR images. One of the main drawbacks of the technique is that the insufficient contrast. Therefore various magnetic compounds1,2,3 are employed as contrast agents in MR imaging to further enhance the natural contrast between normal and pathological regions. Two types of MRI contrast agents have been
6
developed so far. One is the positive contrast agents or T1 agents which accelerate the longitudinal relaxation of water protons and produce bright spots in regions where the agents localize. For example, nanoparticles of gadolinium oxide4,5,6, manganese oxide7,8,9 and various
10,11,12 gadolinium coordination compounds. The other is the negative contrast agents or T2 agents which accelerate the transverse relaxation of water protons with compared to their natural value, where they accumulate and produce dark spots. For instance, it has been shown that
13,14,3 superparamagnetic iron oxide nanopaticles (SPIOs) can be used to enhance the T2 relaxation of the water protons in its proximity which lead to darker contrast in the images.
The efficiency of a contrast agent is measured in terms of its relaxivity, r1 and r2, which refer to the amount of increase in proton relaxation rate 1/T1 and 1/T2 respectively, per millimole of agent. The r1 and r2 can be expressed as follows (eq.2 and eq.3)
∆(1/T1)/M = r1 (2)
∆(1/T2)/M = r2 (3)
Where T1 = longitudinal relaxation time, T2 = transverse relaxation time and M = concentration of contrast agent in mM.
The observed solvent relaxation rate, 1/Ti,obs , is the sum of a diamagnetic term 1/Ti, d, corresponding to the relaxation rate of the solvent nuclei without the paramagnetic solute, and a paramagnetic term 1/Ti,p which is the relaxation rate enhancement caused by the paramagnetic substance (eq.4).
1/Ti,obs = 1/Ti, d + 1/Ti,p i = 1,2 (4)
This equation can be rearranged in terms of the concentration of the paramagnetic substance, [M]
(eq.5).
1/Ti,obs = 1/Ti, d + ri [M] i = 1,2 (5)
7
The paramagnetic relaxation enhancement is controlled by the processes of the so-called “inner- sphere relaxation” (through interaction with bound water) and “outer-sphere relaxation” (arising from the diffusion of water nearby), eq 6. The Solomon-Bloembergen-Morgan (SBM) equation explains the relaxation of water proton around a paramagnetic metal center (eq 7, Scheme 6).
1/Ti,p = (1/Ti) inner-sphere + (1/Ti) outer-sphere (6)
(7)
Where C = molar concentration of paramagnetic compound, q = number of bound water molecules, τM = the mean residence life-time of the coordinated water molecule and T1M = longitudinal relaxation time of the bound water protons.
In 1973, Paul Lauterbur published the first nuclear magnetic resonance image.
Peter Mansfield, who shared the 2003 Nobel Prize with Lauterbur, also contributed to the development of MRI by discovering a mathematical technique to get images faster. The first human image was taken by Damadian and coworkers, Minkoff and Goldsmith in 1977.15 The discovery of feasibility of using paramagnetic agents for enhancing the contrast in the MR image by Lauterbur and coworkers,16 launched a new era in the MRI field. They used a Mn(II) salt to increase the longitudinal relaxation time of water protons. Young and Clarke were first to introduce paramagnetic ferric chloride solution orally to human body to enhance the contrast in the gastrointestinal tract.17 Extending this work to synthesis first commercial contrast agent Gd(III) diethylenetriaminepentaacetate, [GdDTPA]2- (Magnevist) in 1988 initiated vast research into understanding and modifying a new class of MR contrast agents. Currently, approximately 45% of MRI exams include the use of contrast agents.18
8
O E x c i t e d w at e r p r o t o ns H H O H H H O H
M o le cu l ar t u mb l i ng W a t e r e x c h a n g e r a t e H O H I nn e r s p h e r e c oo r d i na t ed q w at e r m o le c u l e s
C o n t r a s t a g e n t
O H H H H O H O H R e l a x ed w at e r p r o t o ns
Scheme 6: Factors influencing relaxivity.
1.2. X-ray computed tomography (CT)
X-ray computed tomography (CT) is a nondestructive technique for visualizing internal anatomic structures of a patient without surgery. Even with the recent remarkable development of
MRI and other imaging techniques such as ultrasound procedures, CT scanning method remains the backbone of modern radiology. X-ray computed tomography provides three dimensional images with high resolution and high contrast. It has many advantages over the other imaging techniques such as wide availability, high efficiency, cost effectiveness and deep tissue penetration. CT is based on X-ray attenuation by the tissues. It provides superior images of electron-dense materials. In order to differentiate neighboring tissues on a CT image, the tissues
9
must have different densities. These varying densities will result in distinct attenuation coefficients, which produce an image that clearly display the different tissues. Unfortunately, many tissues have quite similar CT numbers, ranging from 0 to 50 Hounsfield units (HU).
Therefore contrast agents are often used to create an artificial density difference between objects.
The discovery of X-ray radiation by Roentgen in 1895, led to the idea of the use of tomography (Greek: tomos = slice, graphein = draw) as a diagnostic imaging tool in medicine.
Thereafter, the search for contrast media and medical imaging became intensively investigated.
The invention of the first successful CT imaging device in 1972 by G. N. Hounsfield, initiated a new era in computed tomography. In 1979, G. N. Hounsfield was awarded the Nobel Prize shared with A. M. Cormack for the invention of computed tomography. In the US alone, there are approximately 68.7 million CT scans were performed in 2007 and it increased to 81.9 million in
2010.19
1.2.1 Contrast agents for X-ray computed tomography (CT)
The major limitation involve in CT technique is its inherent low sensitivity and hence low contrast between different soft tissues. Therefore contrast agents are employed to enhance contrast between different soft tissues. In 2010, CT contrast agents were used in more than 55% of the CT scans performed in USA. The CT contrast agents should have greater X-ray absorption coefficient or attenuation power and hence greater absorbance of X-ray. Greater absorption coefficient value can be achieved, if the incident X-rays have energy similar or slightly greater than the binding energy of K-shell electron of the atom. This energy value is known as the absorption edge (k), and the k value increases with atomic number of the element. Therefore elements with high atomic number and high electron density, such as iodine, barium, gold and bismuth have been used as X-ray contrast agents for CT (see table1). The relationship between
10
the atomic number (Z) of the given element and the X-ray absorption coefficient (µ) can be expressed in the following equation (eq.8).
4 ρZ (8)
µ = 3 AE
Where A is the atomic mass, ρ is the density and E is the X-ray energy.
When an X-ray beam pass through matter reduction in beam intensity due to absorption and deflection of photons will occur. This process is known as the degree of X-ray attenuation and it obeys the following equation.
-µx I = I0 e
Where I is the transmitted X-ray intensity, I0 is the incident intensity, and x is the thickness of the matter (absorber).
The mass attenuation coefficient (µ) is the sum of three interactions between X-ray photons and traversed matter. They are coherent scattering (ω), the photoelectron effect (τ), and
Compton scattering (δ). The mass attenuation coefficient (µ) can be defined using these three terms.
µ = ω+ τ+ δ
In coherent scattering, the energy of the incident X-rays is absorbed by an atom, and an
X-ray photon of the same energy as the primary X-rays is emitted in a random direction. This process contributes to noise on X-ray films. Compton scattering arises from the interaction of an
X-ray photon with an outer-shell electron. After the collision, a portion of the X-ray energy is transferred to the electron as kinetic energy, and the X-ray photon is deflected as scattered radiation in a new direction with a lower energy. Compton scattering is responsible for almost all
11
scattered radiation, which both increases noise and decreases contrast. However Compton scattering can be reduced by increasing the X-ray photon energy.
The photoelectric effect is the most important factor for X-ray attenuation of CT contrast agents. The photoelectron effect comes from the interaction of the X-ray photons with inner-shell
(K shell) electrons. The incident X-ray photon transfers its energy to the inner-shell electrons, which are subsequently ejected from the atom. Since all its energy is given up in this process, the incident photon is absorbed and disappears. The vacancy of the electron shell (typically K or L shell) is filled by outer-shell electrons, producing characteristic X-ray photons with lower energies.
The intensity of CT image is expressed in terms of Hounsfield Units (HU), which can be represented as follows (eq.9).
µx-µwater (eq.9) HU = 1000 × µwater-µair
Where; μwater and μair are the attenuation coefficients of water and air, respectively.
µx is linear attenuation coefficient of the tissue.
Traditional X-ray contrast agents such as water soluble iodinated compounds have been used extensively as contrast media due to the ability of iodine (Z = 53) to attenuate X- rays (effectively absorb X-rays). Table 2 and Scheme 5 show a number of such contrast agents that are clinically approved for medical use. Ionic contrast agents containing iodine such as sodium and lithium iodide are no longer in use because of the toxicity associated with them.20
Small-molecule iodinated contrast agents can be categorized into so called ionic and nonionic contrast agents. Ionic contrast agents are negatively in charge and show tendency to interact with biological structures including peptides and cell membranes. Moreover they are characterized by high osmolality, which leads to renal toxicity. Nonionic iodinated contrast agents have been
12
developed to avoid the problems associated with high osmolality. However, small molecular iodinated aromatic compounds currently using, are suffering from several disadvantages such as nonspecific biodistribution, short blood circulation time and low contrast efficacy, which ultimately lead to side effects due to injection of high concentration of iodine within a short period of time. Further iodine has relatively moderate atomic number and hence relatively moderate absorption edge value (k = 33 keV) with compared to the high atomic number atoms such as 50.2 keV for Gd, 54 keV for Dy, 80.7 keV for Au and 90.5 keV for Bi.21 In addition to iodinated contrast agents, barium sulfate suspension is currently used as a CT contrast agent.
However Ba2+ is toxic and therefore only limited to the gastrointestinal (GI) tract imaging.
Table 1: K-shell electron binding energies (K Edge) for selected heavy elements.
Element Atomic number K edge energy(keV) 20 I 53 33.2 Ba 56 37.4 Ce 58 40.4 Gd 64 50.2 Tb 65 52.0 Dy 66 53.8 Yb 70 61.3 W 74 69.5 Re 75 71.7 Au 79 80.7 Pb 82 88.0 Bi 83 90.5
13
Nanoparticulate contrast agents with high atomic number (high-Z) metal elements (gold, platinum, bismuth, and tantalum) have been developed to overcome the problems of short circulation time and the renal toxicity associated with commercially available iodine contrast agents. These nanoparticulate contrast agents can be easily functionalized with various biomolecules for targeted delivery and multimodal imaging applications. Specifically, larger nanoparticles (greater than 400 nm) are favorably taken up by macrophage cells and thus observe in high concentrations in tissues with high concentrations of phagocytic cells such as liver, spleen, and lymphatic tissues. Nanoparticles varying from 100 to 800 nm favorably accumulate in solid tumors by “enhanced permeation and retention effect”, which is the passive convective transport through leaky endothelium (extravasation) that is caused by pores.22
So far the most studied heavy metals include the gold and bismuth. Au nanoparticles can be easily synthesized and functionalized for targeted delivery. Additionally, it provides about 2.7 times higher contrast than iodine. Gold is an extremely inert material, and no significant cytotoxicity has been reported so far. Thus gold nanoparticles are considered to be biocompatible.
Thus far, various gold nanostructures including spheres, rods, shells, and cages have been reported.23 However the cost of using gold as CT contrast agent is huge. On the other hand bismuth based CT contrast agents has gained much attention due to the low toxicity, cost effectiveness and high X-ray attenuation coefficient. Poly(vinylpyrrollidone) (PVP) coated
24 bismuth sulfide (Bi2S3) nanoparticles as CT contrast agents is recently reported. However, controlling the size of the nanoparticles and lack of effective surface modification remain problems with these nanoparticles for further development as potential CT contrast agents. 25
14
Table 2: Some common commercially available small-molecule iodinated contrast agents.
Common name Commercial name Iohexol Omnipaque Iopromide Ultravist Iodixanol Visipaque Ioxaglate Hexabrix Iothalamate Cysto-Conray II Iopamidol Isovue
Scheme 7: Structures of some commercially available clinically approved CT contrast agents.
Ionic monomers (a), nonionic monomers (b), ionic dimer (c), and nonionic dimer (d).
15
1.3. Contrast agents for dual MRI/CT modalities
As previously discussed, MRI is a noninvasive and extremely versatile imaging technique that uses a magnetic field to measure the relaxation time of water protons in tissues.
This technique provides high resolution images of various structures of our body, such as, arteries and lesions. In addition it gives biochemical and functional information over time as well.
However MR imaging is considered to be an insensitive method and more than 30% of all MRI scans use contrast agents to improve the contrast enhancement between the normal and pathological regions.
Recently there has been a great interest in developing nanoparticulate MRI contrast agents coated with biocompatible polymer because they have longer blood circulation time and higher sensitivity with compared to conventional contrast agents. Moreover, they are used for various targeted drug delivery applications. So far gadolinium is the most frequently used metal in MR imaging due to its high magnetic moment. For example a wide variety of gadolinium
3+ based nanoparticles have been developed such as pegylated Gd2O3, and Gd loaded nanoparticles.
X-ray computed tomography (CT) imaging is another widespread imaging method used for diagnosis of diseases and physiological functions of internal organs. The major advantages of
CT include its ability to make images with high resolution and deep tissue penetration. The contrast between different soft tissues can be improved by employing CT contrast agents. As previously discussed the current commercially available iodine based small molecular contrast agents have disadvantages such as, rapid excretion from the body leading to short imaging time, low contrast efficacy,26 lack of targeted delivery and potential renal toxicity.
X-ray absorption coefficient increases with the atomic number and the electron density of the given element. Therefore elements with high atomic number and high electron density, such as gold and bismuth have been used as X-ray contrast agents for CT. Both of them are considered
16
to be nontoxic. For example bismuth is used in cosmetics and medicine and gold is used as a
FDA approved drug carrier. However Bi is preferred over Au because of its low cost. In addition bismuth has very strong X-ray attenuation power because it is the heaviest nonradioactive element in the periodic table.
During the past few years, development of dual modality imaging contrast agents has gained a great interest since they have a comprehensive imaging capability. They offer superior qualities over the single imaging modality. For example, combinations of single photon emission computed tomography and positron emission tomography (SPECT/PET) show greater sensitivity and MRI/CT provides excellent spatial resolution and deep tissue penetration.27
1.4. Targeting intracellular copper ions for selective removal
Copper is an essential element to all living organisms and available in the Cu(I) and Cu(II) oxidation states. Copper is essential for several cellular processes. Such as mitochondrial respiration, antioxidant defense, neurotransmitter synthesis and connective tissue formation.28 It serves as a cofactor for many enzymes. For example cytochrome c oxidase is a copper containing enzyme that is used in the electron transport chain. Copper is also found in superoxide dismutase, which catalysis the dismutation of superoxide into oxygen and hydrogen peroxide (eq 10).
Further, tyrosinase is copper enzyme involved in pigment formation.
2 HO2 → H2O2 + O2 (10)
17
In healthy people, 85-95% of copper in blood is covalently bound to ceruloplasmin molecules. The remaining 5-15% is loosely bound to albumin and small molecules and is referred to as free copper. The amount of free copper is generally 5-15 µg/dL (concentration of free copper (I) in the body is approximately 10-18 M and total copper concentration is in the micromolar range).29 These free copper in cells undergo Fenton-like reactions to produce reactive oxygen species (ROS).30 They can cause damage to biological molecules including DNA, proteins and lipids. In addition free copper can exchange with the zinc in the zinc-finger proteins and thereby disrupt the DNA binding domain. Therefore free intracellular copper ion concentration should always be tightly regulated. Our body maintains its copper homeostasis by excreting the excess copper by hepetocytes (HepG2) via the bile. Loss of copper homeostasis can lead to several diseases including Wilson’s disease, Menkes disease, Alzheimer’s and related neurodegenerative diseases. Copper-reduction therapy is a plausible option to treat these diseases.
In clinical toxicology, small-molecule chelating agents have been widely used to remove heavy metal ions from the human body in a medical procedure commonly referred to as chelation therapy.31 In addition to providing antidotes to heavy-metal intoxications, chelation therapy has been investigated for its potential in treating a variety of metal homeostasis related diseases and disorders.32,33 Despite its medical importance, the number of available chelators used as either the clinical or investigational drugs is, however, surprisingly small.31,34 Furthermore, such small- molecule-based drugs seldom possess sufficient selectivity towards a specific metal ion from the body.31,34 Neither do they have the ability to cross the cell membrane in order to function as intracellular detoxifying agents, unless there is a receptor-targeting moiety conjugated to the drug.31,34,35 On the other hand, some chelators may mobilize the metal ions that are deposited in the body tissues and reroute them into circulation, thus increasing the risk of secondary exposure of such metal ions to the brain.36 In contrast, the use of solid-state compounds as ion-exchangers to selectively remove the target metal ions from the human body by the well-established
18
thermodynamic and kinetic principles of ion-exchange has been largely unexplored.37 We used the Irving–Williams Series as a guide to design a novel nanoparticulate ion-exchanger that exhibits an extremely high selectivity for copper and can penetrate the cell membrane to act as an intracellular copper detoxifying agent. Specifically, we noticed that the order of relative stability exhibited by the homologous divalent 3d metal complexes shows the trend Mn2+ < Fe2+ < Co2+ <
Ni2+ < Cu2+ > Zn2+ with copper and zinc complexes being the most and the second most stable, regardless of the nature of the ligand.38 Thus, if a highly stable solid-state zinc compound is used as an ion-exchanger, it would preferentially undergo ion-exchange with copper, and consequently, act as a detoxifying agent specifically for copper ions while maintaining homeostasis for other biologically essential metal ions such as Mg2+, Ca2+, Mn2+, Fe2+ and Zn2+ in the body.
1.4.1 Intracellular copper removal as a potentially more effective treatment for the
Wilson’s disease
Wilson’s disease is a genetic metabolic disorder characterized by excess copper accumulation in the liver and other organs. In 1912 Kinnier Wilson discovered this condition and in
1948, it was clarified that the accumulation of copper takes place due to the weakened uptake of Cu (II) into hepetocytes due to the mutation of the gene related to the copper transport protein that transport copper into ceruloplasmin for biliary excretion. In 1993, ATP7B, was identified as the gene that is abnormal in WD and is highly expressed in the liver and kidney.39 ATP7B encodes copper transporting P-type ATPase, which plays a major role in transporting copper into the trans-Golgi compartment, for incorporation into the plasma protein ceruloplasmin, and into the bile, for excretion.
Patients with Wilson’s disease are unable to deliver copper from the liver to the bile and are also characterized by inability to incorporate copper to ceruloplasmin protein due to the mutation in the
19
gene ATP7B. Thus, excretion of the copper is disturbed and results in accumulation of copper in the liver and brain and finally lead to liver failure, tremors and neurological impairments.40 Patients with
WD can have free copper level of 50 µg/dL or higher, which can damage to their liver and brain.41
Dietary copper (which bound to protein) is processed and absorbed by the small intestine.
Copper is taken up into the hepatocyte via copper transporter 1 (CTR1) and transfers copper into cytosol. As discussed earlier, free Cu(I) is toxic to cells. Thus, intracellular Cu is always bound to proteins known as glutathione or metallothioneins proteins. Metallothioneins help to store the copper in the liver and copper is then regulated by the liver. Copper chaperone proteins, CCS and
Atox1 deliver copper to specific cellular destinations. For example, CCS transfers copper to cytosolic Cu, Zn-dependent superoxide dismutase (SOD1) and activates them. On the other hand
Atox1 transfers copper to the Wilson’s disease protein, ATP7B that residing in the trans-Golgi network (TGN).42 Copper is then delivered to the cell periphery by ATP7B to facilitate excretion of copper from the hepatocyte via the bile. Scheme 8 shows the schematic diagram of metabolic pathways of copper in hepetocytes in the liver.
Copper is released to blood by the liver as needed and transport by the proteins called ceruloplasmin (65-70 % of total available copper) and albumin (12-18%) throughout the body.40 Albumin binds with the copper using three amino acids at its N-terminus. Therefore excess copper is not release to the blood. However inorganic copper in drinking water and mineral supplements is not processed by the liver and directly contribute to the free copper concentration in the blood.41
Treatment of WD centers on two phases. The first phase is the promotion of excretion of copper from the body, and the second phase is the reduction of the absorption of copper from the diet. Chelation therapy or the use of chelating reagents to remove the excess copper accomplishes the first aspect and remains the current treatment for the WD.
20
Cp-Cu
ATP7B MT
CTR1
GSH
ATOX1
CTR1
Nucleus
CP
Golgi
Bile canaliculus
Scheme 8: Pathways of copper metabolism in the hepatocyte. Cu = copper, CTR1 = copper transporter 1, MT = metallothioneins, GSH = glutathione, Cp = ceruloplasmin
In 1951, the first chelating agent, dimercaptopropanol (British anti-lewisite, or BAL)
(Scheme 9) was introduced as a treatment for the WD. In 1956, D- penicillamine or (2S)-2- amino-3-methyl-3-sulfanyl-butanoic acid (Scheme 10), a metabolite of penicilin was discovered by John Walsh as orally administered drug.43,44 The main effect of penicillamine in WD is to promote the urinary excretion of copper. However, D-penicillamine is an immunosuppressant also used to treat rheumatoid arthritis. Therefore, the use of penicillamine in WD causes a score of side effects with symptoms ranging from bone marrow and immune suppression, to skin rash, and to deterioration of various neurological functions.45 Trientine or triethylenetetraamine was introduced specifically for patients who developed adverse reactions to penicillamine.46 This also enhances the copper excretion by the kidneys and exhibits several side effects including
21
pancytopenia, hemorrhagic gastritis, loss of taste, and rashes. Moreover chelation of trientine with iron produces a toxic complex. The development of novel copper chelating agents with less or no toxicity remains a challenge in this area of research. Other treatments of WD include the administration of zinc salts to reduce the absorption of copper and liver transplantation. Zinc ions are believed to induce the production of zinc metallothionein and Cu2+ then displaces Zn2+ in metallothionein and thus decreases the intestinal absorption.32
According to the Pearson definition Cu(I) is considered to be a soft metal and hence reacts faster and forms stronger bonds with the ligands having soft donor atoms such as sulfur (in cystein and methionine ) , nitrogen ( aromatic nitrogen in pyridine and histidine ) and phosphorus
(in tertiary phosphine ). Therefore ligands having these soft donor atoms have developed as copper chelating agents.47,48
Scheme 9: Chemical structure of 2,3-dimercaptopropanol (British anti-lewisite).
Scheme 10: Chemical structure of D- penicillamine.
22
1.4.2. Copper depletion as a novel strategy for angiogenesis inhibition
Angiogenesis, also known as neovascularization, is the process of new blood vessel formation from pre-existing vessels. This is a regular physiological incident that takes place during embryonic growth and wound healing. The main steps of angiogenesis include the migration, growth, and differentiation of endothelial cells, which line the inside wall of blood vessels. In healthy adults, both formation and growth of new blood vessels is tightly regulated and coordinated by a variety of angiogenic factors and inhibitors in balance. New blood vessels start to form when angiogenic growth factors are produced in excess of angiogenesis inhibitors.
When inhibitors are present in excess of stimulators, angiogenesis is stopped. The normal, healthy body maintains a perfect balance of angiogenesis modulators. Angiogenesis is important for tumor growth and metastasis. Tumors less than 1 mm3 in size obtain its nutrients and oxygen by diffusion. Once tumors grow greater than 1 mm3, it becomes critical for the tumor to develop its own blood supply system causing a lower partial pressure of oxygen which is defined as hypoxia.
Hypoxic condition leads to an increase in hypoxia-inducible factor 1 (HIF-1) gene expression in different tumor tissues.49 HIF-1 activates the transcription of the vascular endothelial growth factor (VEGF) gene. VEGF, in turn, stimulates the development of new blood vessels from the preexisting vasculature, which contributes to recurrence and metastasis of tumors. Tumors can also stimulate nearby normal cells to produce angiogenesis signaling molecules. The resulting new blood vessels provide oxygen and nutrients to growing tumors and allow the cancer cells to invade nearby tissue, forming new clusters of cancer cells, called metastases. Therefore, angiogenesis or neovascularization is essential for the tumor to grow greater than 1 mm3.50
Angiogenesis is a multistep process controlled by the balance of pro- and antiangiogenic factors. Proliferation and the organization of endothelial cells (ECs) into tube-like structures take place in the latter phases of the angiogenesis. This process is regulated by the expression of a variety of growth factors including vascular endothelial growth factor (VEGF) and fibroblast 23
growth factor (FGF). Initially, VEGF and other endothelial growth factors bind to their receptors on endothelial cells, which results in initiation of signals within these cells that stimulate the growth of new blood vessels. VEGF is considered to be the major stimulator of angiogenesis and increase the number of capillaries in the tumor allowing oxygen and nutrients to diffuse in to the tumor that lead to tumor expansion. On the other hand FGF induces the proliferation and differentiation of endothelial and smooth muscle cells that required for the formation of arterial vessels. Therefore, the use of angiogenesis inhibitors or antiangiogenesis agents to treat cancer has gained much attention in the past three decades.
Antiangiogenesis was first suggested by Judah Folkman in 1971 as a treatment for the cancer.51 Since that time, this antiangiogenic approaches have garnered experimental and clinical support and finally led to the approval of humanized monoclonal antibody called bevacizumab, which is a direct angiogenesis inhibitor that acts against vascular endothelial growth factor
(VEGF). Bevacizumab can specifically bind to VEGF. Therefore, it is unable to activate the
VEGF receptor and angiogenesis is inhibited. Later, two additional antiangiogenic therapies known as sorafenib (Nexavar)52 and sunitinib ((N-[2-(diethylamino)ethyl]-5-[(Z)-(5-fluoro-1,2- dihydro-2-oxo-3H-indol-3-ylidine)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide),53 have been approved by the Food and Drug Administration (FDA). Sunitinib is an orally administered small molecule that targets vascular endothelial growth factor receptors (VEGFR1, VEGFR2, and
VEGFR3) and platelet-derived growth factor receptors (PDGFRα and PDGFRβ), which involve in tumor growth, pathologic angiogenesis, and metastatic progression. On the other hand sorafenib is a type of tyrosine kinase inhibitor. It interferes with molecules that are thought to be involved in chemical messages sent within cancer cells, in the formation of blood vessels that supply tumors, and in cell death. These first-generation antiangiogenic therapeutic approaches have demonstrated prevention of new blood vessel formation. However studies have shown little regression of existing tumors by these anti-angiogenesis agents. Further, their high cost and adverse reactions that have been observed in patients taking these agents have caused alternative
24
research and development efforts in the field of anti-angiogenesis. For example, problems with bleeding, clots in the arteries (with resultant stroke or heart attack), and hypertension were reported as some of the side effects of treatment with angiogenesis inhibitors. Currently there are over 50 antiangiogenic drugs for cancer treatment that are either on the market or at various stages of clinical trials in the US. All of these drugs, once considered very promising in cancer treatment, have failed to live up to the high expectations. The main problems with these angiogenic inhibitors are their limited efficacy, selective but nonspecific effects in different types of cancer, and they engender inherent or acquired resistance. All of these angiogenesis inhibitors prevent angiogenesis by blocking one of the antigenic signaling pathways. However cancers can grow blood vessels using different angiogenic promoters to trigger other signaling pathways.
Therefore development of more effective angiogenesis inhibitors with better efficacy, less toxicity and lower cost remains a challenge.
We intend to approach anti-angiogenesis therapy to cure cancer by targeting the copper ion rather than the many cell-signaling biomolecules. Copper is an essential metal required by all living organisms in several key cellular processes such as mitochondrial respiration, antioxidant defence, neurotransmitter synthesis and connective tissue formation.54 In addition copper is a co- factor for more than a dozen key angiogenic promoters essential for cancer angiogenesis.55 After these promoter molecules are activated, there is a specific requirement for copper in each step of migration, mitosis and differentiation of endothelial cells, and reshaping of matrix proteins into the tubular structure of micro-capillaries.56 Table 3 lists a series of growth-promoting factors whose angiogenic action has been shown to depend on Cu.
In 1980 McAuslan and Reilly found that copper ions induce endothelial cell motility.57
They demonstrated that only micromolar amounts of Cu (10-6 M) is needed to control endothelial cell migration and angiogenesis. Later Cu was found to stimulate the proliferation and migration of endothelial cells in vitro. Further it has been demonstrated that 48-hour exposure to 500 µM
Cu in serum-free medium doubled the number of human endothelial cells in culture. Ziche and
25
coworkers showed that the suppression of blood vessels formation of rabbits who take Cu deficient diet.56 Based on these discoveries, several clinical trials have been carried out to evaluate the anti-angiogenesis therapy to cure cancer, based on copper depletion. For instance, the copper chelator D-penicillamine, is found to suppress the growth of brain tumors in rats.58
Table 3: Proangiogenic mediators that rely on Cu for expression or function.
Growth-promoting factors Reference Fibronectin Ahmed et al.59 Collagenase Lin and Chen60 Gangliosides Gullino61 Prostaglandin E-1 Ziche et al.62 Heparin Alessandri et al.63 Angiogenin Soncin et al.64 S100A13 Mandinov et al.65 FGF-1 (acidic) Landriscina et al.66 FGF-2 (basic) Pan et al.67 FGF receptor-1 Patstone and Maher68 SPARC Lane et al.69 Synaptotagamin Prudovsky et al.70 Vascular endothelial growth Sen et al.71
67 factorTumor necrosis factor-α Pan et al. Ceruloplasmin Raju et al.72 IL-1α Mandinov et al.65 IL-6 Pan et al.67 IL-8 Moriguchi et al.58 Nuclear factor-kB Pan et al.67
FGF - fibroblast growth factor, SPARC - osteonectin/BM40, IL-interleukin.
26
As previously mentioned, importance of copper in angiogenesis has been known for more than two decades. However the mechanism of action of copper has not been discovered until recently. Copper is found to require for the activation of hypoxia-inducible factor-1 (HIF-1) which is the major transcription factor that regulates the expression of VEGF. Since copper play a crucial role in production of VEGF, depletion of copper should prevent angiogenesis.
27
Chapter 2: Materials and Methods
2.1 Materials
All chemicals, unless specified otherwise, were purchased from Sigma Aldrich and used without further purification. All materials used in this dissertation are listed below.
Potassium ferrocyanide
Potassium ferricyanide
Ferric chloride
Gadolinium chloride
Polyvinyl pyrolidone (MW=40 000, 8000)
Citric acid
Manganese chloride
Zinc acetate
Ammonium tetrathio molybdate
Tetrachloroauric acid
Sodium citrate
3-Mercaptopropionic acid
28
2.2 Experimental design and techniques
Gadolinium based nanoparticles were synthesized using a similar procedure that is developed by a former member of our group, Prof. Yongxiu Li (a visiting scholar from a Chinese university) with a slight modification.
Characterization of synthesized nanoparticles was performed using a number of techniques, including powder x-ray diffraction spectroscopy (XRD), Fourier-transformed infra- red spectroscopy (FT-IR), energy dispersive x-ray spectroscopy (EDX), high- resolution transmission electron microscopy (HR-TEM) and thermogravimetric analysis (TGA). T1 and T2 measurements of nanopartcles were measured at different concentrations using a 1.5 T Siemens
Espree whole-body or a 7.0 T MRI scanner.
2.3 Experimental methodology
Nanoparticles were synthesized by the technique called coprecipitation method.
Coprecipitation reactions involve the simultaneous occurrence of nucleation, growth, coarsening, and/or agglomeration processes. The coprecipitation reaction occurs under conditions of supersaturation and produces a highly insoluble species. A large number of small particles will be formed during the nucleation process and Ostwald ripening and aggregation takes place in a later stage, which result in a dramatic change in the size, morphology, and properties of the products.
Therefore, polymers are often employed as a coating agent to prevent the nanoparticles from aggregation.
29
2.4 Instrumentation
2.4.1 Powder X-ray diffraction spectroscopy (PXRD)
Powder X-ray diffraction spectroscopy (PXRD) is one of the most powerful techniques for the characterization of nanoparticles and can provide a lot of information about the structural properties such as unit cell dimensions of the crystalline materials. The data were collected at room temperature on a Siemens D5000 powder X-ray diffractometer using monochromatic copper Kα radiation. All the XRD patterns were recorded using 0.1 increments and 50 per minutes in the range of 2θ from 100 to 500 using a voltage of 40 kV and current setting of 40 mA. Samples were finely ground to a powder and mounted on a microscope glass slide prior to analysis.
The X-rays produced by a cathode ray tube are subjected to filter to get a monochromatic radiation. This monochromatic radiation is then directed towards the powdered sample and the crystallites in the powder diffract the X-rays according to the Braggs law. The intensities of the diffraction are monitored by a detector and provide diffraction patterns which can be compared to a database of known crystal structures.
2.4.2 Transmission electron microscopy (TEM) and energy dispersive X-ray (EDX)
TEM images provided information on morphology and the size of the nanoparticles. The nanoparticles were first dispersed in water by sonication. Next, one drop of the suspension was placed onto a carbon-coated copper TEM grid (400-mesh) and specimens were then allowed to air-dry prior to analysis. TEM measurements were made using a FEI Tecnai F20 transmission electron microscope (TEM) equipped with a field emission gun and analyzed at accelerated voltage of 200 kV and an emission current of 30 mA. The energy dispersive X-ray spectroscopy
(EDX) results were obtained for a selected area of the sample with the integrated scanning TEM
30
(STEM) unit and attached EDX spectrometer. The spatial resolution is <1 nm through the acquisition of high resolution (~0.2 nm) high-angle angular dark field (HAADF) images, which is sensitive to atomic number (Z) contrast.
2.4.3 Thermogravimetric analysis (TGA)
The thermal analysis was conducted using a TA instrument 2950 high-resolution thermogravimetric analyzer (New Castle, DE, USA) in nitrogen or air from room temperature to
800 °C with a heating rate of 5 °C/min or 10 °C/min. Thermal gravimetric analysis provided information on the average load of the surface coating of the polymer through weight change.
2.4.4 Fourier transform infrared spectroscopy (FTIR)
Fourier transform infrared spectroscopy (FTIR) analysis is one of the most powerful techniques for identification of the functional groups in compounds. IR radiation is passed through the sample and transmitted light reaching the detector is converted to a spectrum. The wavelength absorbed by the sample is characteristic of the chemical bond. Therefore the spectrum of an unknown can be identified by comparison to a library of known compounds. In the experiments described herein, Bruker Vector 33 Fourier Transform Infrared Spectrophotometer is used for product characterization using FTIR spectroscopy.
2.4.5 Confocal microscopy
Confocal florescence imaging of cells was performed with an OLYMPUS FV1000 IX8 confocal fluorescence microscope to confirm the uptake of nanoparticle by cells. Cells were seeded in a 6-well plate with 5 ×104 cells/well and incubated for 24 h. Dye labeled nanoparticles were then introduced to each well with the serum free fresh medium and incubated for 3 h. After
31
3 h incubation time with dye-labelled nanoparticles, cells were washed three times with PBS and fresh culture medium was added before taking images.
2.4.6 T1 and T2 measurements
T1 and T2 measurements of nanoparticles were measured at different concentrations using a 1.5 T Siemens Espree whole-body or a 7.0 T MRI scanner. The concentrations of metal ions in the aqueous solution were measured by inductively coupled plasma optical emission spectroscopy
(ICPOES, Perkin Elmer Optima 3300-DV ICP) and atomic absorption spectroscopy (AAS). For
T1 measurements, an inversion recovery gradient echo sequence with a TE = 4 ms were used. The inversion time can be varied between 30–2000 ms. T2 measurements were performed using a spin–echo sequence of TR of 10000 ms, and TE of 10.6–340 ms. The T1-weighted MR images were acquired using the 7.0 T scanner with a matrix size of 128 × 128, a field of view of 3.0 × 3.0 cm2, a slice thickness of 0.5 mm, TE of 9.4 ms, and TR of 13.9~1500 ms. Data analysis was performed by fitting to relaxivity curves with self-written programs.
2.4.7 Atomic absorption measurements
All measurements were made using a Buck Scientific Model 210 VGP atomic absorption spectrophotometer. The sources were Buck Scientific Mn, K, Fe, Cu, Au, Ca, Mg and Zn hollow cathode lamps, which were operated at 10 mA. Analyiical wavelengths were 279.5 nm, 766.5 nm,
248.3 nm, 324.8 nm, 242.8 nm, 422.7 nm, 285.2 nm and 213.9 nm respectively for each lamp and the slit width was chosen so as to give a spectral band pass of approximately 0.2 nm. An air- acetylene flame was used for all measurements.
32
2.5 Experimental calculations
2.5.1 Relaxivity (r1 and r2 values)
T1 and T2 relaxation times measured from the MRI scanner are sum of the diamagnetic and paramagnetic terms.
(1/Ti)obs = (1/Ti)d + (1/Ti)p i = 1, 2
ri = (1/Ti)p/[C]
(1/Ti)obs = (1/Ti)d + ri[C]
Where T1 = longitudinal relaxation time, T2 = transverse relaxation time, r1 =longitudinal relaxivity , r2 = transverse relaxivity, and C = concentration of contrast agent in mM.
The slope of the graph of (1/Ti)obs Vs concentration of the contrast agent in mM gives the relaxivity value ri.
33
Chapter 3: Contrast agents for magnetic resonance imaging
CONTENTS OF THIS CHAPTER HAVE BEEN PUBLISHED AS AN ARTICLE IN LANGMUIR,
Langmuir 2014, 30, 12018−12026 ALL THE MATERIALS OF THE ARTICLE HAVE BEEN
REPRINTED WITH THE COPYRIGHT (2014) PERMISSION OF THE AMERICAN CHEMICAL
SOCIETY
Magnetic resonance imaging (MRI) is a routine diagnostic tool that provides high resolution and three dimensional images in clinical medicine and biomedical research. They work by altering the longitudinal relaxation time (T1) or transverse relaxation time (T2) of water protons located in close proximity to the contrast agents. Although MRI gives excellent anatomical resolution, it suffers from its intrinsic insensitivity. Therefore, various paramagnetic compounds including magnetic nanoparticles are employed as contrast agents to enhance the contrast between pathological and normal tissues. However relatively high concentration of contrast agents (typically 0.01-0.1mM) is necessary to produce the image contrast. Therefore, increasing the sensitivity of contrast agents remains the main strategy in developing new MR contrast agents.
3.1. Gadolinium based MRI contrast agents
Among various paramagnetic transition metal and lanthanide metal ions, Gd3+ is the most frequently used paramagnetic metal ion in commercial contrast agents. because Gd3+ has seven
34
unpaired electrons and a long electronic relaxation time (10-9 s) which are favorable properties for magnetic resonance imaging (MRI) applications. However, Gd3+ is a very toxic heavy metal ion.
It doesn’t have any function in human body but is known to disrupt the normal function of the
Ca-ion channels because its size is similar to that of the Ca2+. Therefore it is necessary to sequester the Gd3+ ions by chelation before introducing to the human body. For example, complexation of the Gd3+ ion with diethylene triamine pentaacetic acid (DTPA) forms Gd-
DTPA (Magnevist®, Schering AG, Germany) complex with a very high formation constant
23 (KML=10 ). Due to the high thermodynamic stability and kinetic inertness, this complex shows very low toxicity.
The stability of the complex in vivo is a very important parameter to consider for when regarding a contrast agent. The stability or formation constant of the complex is defined by the following equation (1).
ML Mn+ +Ln-
n+ n- KML=[ML]/[M ][L ] (1)
Table 4 and scheme 11 show Gd3+-chelates that have already received approval for clinical use and are currently marketed for clinical MR imaging. These chelates are based on polyaminocarboxylate ligands which use both oxygen and nitrogen atoms for bonding. These ligands occupy eight of the nine available coordination sites of Gd3+ and leave at least one coordination site open for binding water molecules. The Solomon-Bloembergen-Morgan equation predicts that the relaxivity can be significantly increased by increasing the number of inner- sphere water molecules q.73 However increasing the number of water molecules attached to the metal center can drastically reduce the thermodynamic stability of the compound and lead to in vivo release of toxic Gd3+. In addition, central metal ion can be dissociated from the complex due to transmettalation with Zn, Mg or Ca.
GdL + M ↔ ML + Gd3+
35
Where; M= Ca, Mg or Zn and L = ligand
Therefore, increasing the number of water molecules directly coordinated to the metal center of Gd3+ still remains a useful strategy for increasing relaxivity in this field of research as long as such approach does not significantly reduce the stability. Moreover, for the application of these complexes in clinical trials, they should remain intact in the body. Hence these complexes must be kinetically inert under physiological conditions.
Table 4: Clinically approved gadolinium based MRI contrast agents.
Chemical name Brand name Generic name
2- [Gd(DTPA)(H2O)] Magnevist Gadopentetate dimeglumine
- [Gd(DOTA)(H2O)] Dotarem Gadoterate meglumine
[Gd(DTPA-BMA)(H2O)] Omniscan Gadodiamide
[Gd(HP-DO3A)(H2O)] ProHance Gadoteridol
[Gd(DO3A-butrol)(H2O)] Gadovist Gadobutrol
[Gd(DTPA-BMEA)(H2O)] OptiMARK Gadoversetamide
2- [Gd(BOPTA)(H2O)] MultiHance Gadobenate dimeglumine
Gadolinium shows inherent strong oxophilicity. Therefore an oxygen donor atom binds more strongly than a nitrogen donor atom. Hence, gadolinium can form stable chelates with ligand molecules containing COOH, OH (phenolic), OH (hydroxylic), O (carbonyl). In 1995, Xu et al. used this strategy to make very stable new class of MRI contrast agents containing two inner sphere water molecules based on HOPO (hydroxypyridinone) ligand.74 These complexes exhibit
36
-1 -1 high T1 relaxivities (10-13 mM s ) and high thermodynamic complex stabilities (pGd ∼ 17-
18).75
Scheme 11: Clinically approved MRI contrast agents (only the ligands are presented in the scheme).
However, as small molecules, none of these gadolinium chelates can penetrate cells and are developed as extracellular agents. Further, they can be easily excreted from the body and have short life spans in the body, making detailed imaging studies and monitoring of physiological functions impossible. Moreover these small complexes have fast tumbling rates in solution, which acts to lower their relaxivity according to the theoretical model that predicts the relaxivity for various compounds.76 On the other hand, high sensitivities can be achieved by loading Gd based
37
chelates with nano-sized carriers, such as micelles, liposomes, dendrimers, carbon nanotubes, and microemulsions.77,78 In addition to high sensitivity they allow targeted delivery.
Nanoparticulate MRI contrast agents have attracted a great deal of attention in recent years.79,80 The most remarkable feature of these contrast agents is that they have multiple metal centers with high magnetic moments per particle.81 Thus the sensitivity is higher. In addition, they have a long blood circulation time which is desirable if detailed imaging studies and monitoring of physiological functions need to be performed. Furthermore, in vivo metal ion release from the particles would be minimized to reduce the risk of exposing the patient to heavy metal ions.5 In addition, targeted delivery is possible due to the ability of internalization of surface- functionalized nanoparticles by endocytosis.82 For example Weissleder et al. and coworkers have developed iron oxide nanoparticles as target-specific MRI contrast agent.82 Tian and coworkers have designed ultrathin GdF3 nanowires as T1 contrast agents. In addition several other gadolinium based nanoparticles have also been reported.83,84,4
3.2. Biocompatible nanoparticles of KGd(H2O)2[Fe(CN)6]·H2O with extremely high
T1-weighted relaxivity owing to two water molecules directly bound to the Gd(III) center
A great effort has been addressed to the development of nanoparticulate MRI contrast agents in recent years, because they have higher sensitivity and higher water proton relaxivities than the molecular contrast agents.80 Recently, our group has been actively engaged in the synthesis of Prussian Blue (PB) nanoparticles as a MRI contrast agent. However relaxivity is very low for pure PB nanoparticles. In this work, we demonstrate that the incorporation of Gd3+ ions to the PB structure increases the relaxivity dramatically. Prussian blue (PB) exists in two different structures. One form is soluble and known as soluble PB while the other one is insoluble and
38
known as insoluble PB. The insoluble PB, is a mixed-valence iron(III) hexacyanoferrate(II)
III II compound of composition Fe4[Fe(CN)6]3.xH2O. The soluble PB has the form of KFe [Fe (CN)6].
Both structures show face-centered cubic structure with the space group of Fm m. In the structure Fe3+ and Fe2+ centers are bridged by the CN- groups. In the present compound all the
Fe3+ centers with five unpaired electrons are replaced by Gd3+ ions with seven unpaired electrons
II 3+ to give the formula of KGd(H2O)2[Fe (CN)6]H2O. Hence, the incorporation of Gd ions should result in increase in relaxivity of PB. Further, some of these Gd3+ centers are accessible to water coordination which facilitates the inner-sphere mechanism. Hence, the compound
II KGd(H2O)2[Fe (CN)6]H2O, can shorten the longitudinal relaxation times of water protons with compared to the bulk water, resulting in increased relaxivity. Moreover, these nanoparticles are coated with PVP poly(vinylpyrrolidone) to achieve the solubility, stability and the biocompatibility of the nanoparticles. In the present study, we report the synthesis of PVP coated
II nanoparticles of KGd(H2O)2[Fe (CN)6]H2O with a high relaxivity, which make them suitable for
MR imaging.
II Nanoparticles of KGd(H2O)2[Fe (CN)6]H2O (KGdPB NPs) was synthesized by using the following method. Briefly, an aqueous solution of Gd(NO3)3 (1 mM, 10 mL) containing 50 mg of sodium citrate was added dropwise to an aqueous mixture of K4[Fe(CN)6] (1 mM, 10 mL) and
PVP (111 mg, MW=8000) under vigorous stirring for ca. 3 hrs. The reaction product was dialyzed using regenerated cellulose tubular membrane (MWCO is 12000-14000) against distilled water for 2 days. The solid product was collected by lyophilization of the above solution. Finally the solid product was dried for further characterization. Bulk KGdPB was prepared using the same procedure without adding PVP. Briefly, equimolar aqueous solutions of Gd(NO3)3 (0.25 mmol, 20 mL) and K4[Fe(CN)6] (0.25 mmol, 20 mL) were mixed at room temperature under vigorous stirring. The solution turned pale yellow and cloudy after continuous stirring at room temperature for ca. 30 min. The reaction product was dialyzed using regenerated cellulose tubular membrane (MWCO=12000-14000) against distilled water for 8 hours, followed by lyophilization. 39
The solid material obtained from this process was re-dispersed in water by sonication to form slurry and lyophilized one more time to increase its crystallinity. The bulk material was collected, washed with water and acetone, and dried in vacuum.
The nanoparticles were characterized by transmission electron microscopy (TEM), powder X-ray diffraction (XRD), Fourier-transformed infra-red spectroscopy (FT-IR), and thermal gravimetric analysis (TGA). The size and morphology of the nanoparticles were determined by transmission electron microscopy (TEM). The nanoparticles were first dispersed in water. Next, one drop of the suspension was placed onto a carbon-coated copper TEM grid (400- mesh) and specimens were then allowed to air-dry. Figure 3.1 displays TEM image of KGdPB nanoparticles. The image shows irregular shaped nanoparticles with a relatively broad size distribution. The average size of the nanoparticles is approximately 25 ± 10 nm.
Figure 3.1: TEM image of as prepared KGdPB nanoparticles.
40
Energy-dispersive X-ray analysis (EDXA) on a nanoparticle illustrates the composition of nanoparticles. As can be seen in figure 3.2, nanoparticles contained gadolinium, potassium and iron.
Figure 3.2: EDX spectrum on a PVP-coated nanoparticle.
Metal analysis was carried out using inductively coupled plasma optical emission spectroscopy (ICP-OES) with a PerkinElmer Optima 3200 system. A sample of 60 mg of KGdPB bulk material was first decomposed at 620 °C for ca. 6 h to obtain an amorphous oxide powder that was then extracted with 5 mL of concentrated HNO3. After dilution in a volumetric flask, the solution was analyzed by ICP-OES for potassium, gadolinium, and iron. After dilution, the solution was analyzed by ICP-OES and was found to have an atomic ratio of 1:1:1 for K:Gd:Fe.
Fourier transform infrared spectroscopy (FT-IR) data shown in figure 3.3 provides information on the surface of the nanoparticles. The FT-IR spectra of the nanoparticles after 24 hours of dialysis to remove unbound citrate and PVP contained the characteristic IR vibrations attributable to the
41
citrate anion and PVP in addition to the C-N stretching vibration at 2070 cm−1, confirming the presence of both the capping agent and the coating polymer on the surface. The stretching vibration of CN at 2070 cm-1, and the broad band of OH at 3500 cm-1, were observed in the spectra of both bulk and nanoparticles of KGdPB. The absorption bands near 1660 cm-1, 1420 cm-
1 and 1285 cm-1 in the spectrum of nanoparticles, corresponding to the PVP coating on the surface of the nanoparticles. The peak at 1660 cm-1 is due to the contribution from C=O and N-C
-1 stretching vibrations. The peak at 1420 cm can be assigned to the CH deformation of cyclic CH2 groups and the peak at 1285 cm-1 is due to the amide III band (C-N stretch). The PVP is hydrophilic and thus the presence of water is confirmed by the peak at 3465 cm-1 which is assigned to OH stretching vibrations.
1
0.8
0.6 Sodium citrate 0.4 PVP KGdPB bulk
Transmittance (%) Transmittance 0.2 KGdPB NPs 0 750 1750 2750 3750 Wavenumber (cm-1)
Figure 3.3: The FT-IR spectra of sodium citrate, PVP, bulk compound and nanoparticles alone.
42
The crystalline state of the nanoparticles was obtained using a Siemens D5000 powder X- ray diffractometer with a monochromatic copper Kα radiation (40 kV, 40mA). The PXRD pattern of the compound is shown in figure 3.4. Table 5 shows the crystallographic data for KGdPB. The data indicate that the crystal lattice belongs to the orthorhombic crystal system with the unit cell parameters a=12.6098 (4) Å, b=13.6161(4) Å and c=7.2490(3) Å and the space group Pnma. The
2+ 3+ structure of KGd(H2O)2[Fe(CN)6]•H2O consists of Fe octahedra and Gd biface-capped trigonal prisms joined in a 3D framework by CN- groups (see Figure 3.5). The Fe atom is coordinated by six C atoms of the CN- groups while the Gd atom is coordinated by six N atoms and additionally by two O atoms of water molecules O1 and O2 (Table 6). The cavities in the framework are filled up by the K+ ion and water of crystallization (O3) which shows site- occupancy disorder in the same cavity.
Table 5: Crystallographic data for KGd(H2O)2[Fe(CN)6]. H2O
-1 Chemical formula, KGd[Fe(CN)6] fw = 460.25g mol a = 12.6098Å space group Pnma b = 13.6161 Å T = −153 °C c = 7.2490 Å λ = 1.54178 Å -3 α=β = γ=90° ρcalcd = 2.44 g cm
V = 1244.63 Å3 μ = 465.19 cm-1
Table 6: Selected interatomic distances (Å).
Atom1 Atom2 Distance, Å Atom1 Atom2 Distance, Å Gd N1 × 2 2.40112 Fe C1 × 2 1.92208 N2 × 2 2.42436 C2 × 2 1.89469 N3 × 2 2.46226 C3 × 2 1.90203 O1 × 1 2.55421 K/O3 O1 2.85365 O2× 1 2.60647 O2 2.99319 N3 2.99319
43
35,000 34,000 KGdFe(CN)6(H2O)3_Pnma 100.00 % 33,000 32,000 31,000 30,000 29,000 28,000 27,000 26,000 25,000 24,000 23,000 22,000 21,000 20,000 19,000 18,000 17,000 16,000 15,000 14,000 13,000 12,000 11,000 10,000 9,000 8,000 7,000 6,000 5,000 4,000 3,000 2,000 1,000 0 -1,000
15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 2theta (Deg.), CuKa radiation
Figure 3.4: Powder X-ray diffraction spectra for the as-prepared KGd [Fe(CN)6].3H2O.
Powder X-ray diffraction pattern was collected by Peter Y. Zavalij from Department of Chemistry and Biochemistry, University of Maryland.
+ Figure 3.5: The unit-cell packing diagram of KGd(H2O)2[Fe(CN)6]•H2O (left) with the K ion and zeolitic water molecule omitted for clarity. The coordination environment of the Gd3+ ion
(middle and right) showing two water molecules directly bound to the metal center.
44
The thermal analysis was conducted on the bulk sample using a TA instruments 2950 high-resolution thermogravimetric analyzer (New Castle, DE, USA) in nitrogen from room temperature to 600 °C with a heating rate of 10 °C/min. Thermal gravimetric analysis (TGA) on the bulk sample showed a two-step loss of water, i.e. the first step of weight loss was found in the range from room temperature to about 100 oC, corresponding to the loss of one water molecule, and the second step of weight loss was found in the range from 100 to 160 oC, corresponding to the loss of two water molecules (see Figure 3.6). This observation is consistent with the existence of one zeolitic and two coordinative water molecules per formula as revealed by the powder X- ray structure determination.
II Figure 3.6: The TGA curve of bulk KGd(H2O)2[Fe (CN)6] H2O sample.
45
In principle two coordinated water molecules per Gd center should make a significant contribution to the relaxivity. The efficiency of a CA is measured by its ability to enhance the proton relaxation of water, and commonly expressed as proton relaxivity. The latter is defined as the longitudinal or transverse relaxation rate increase of the water protons by unity concentration of the agent in mM. To evaluate the efficacy of our nanoparticles as MRI contrast agents, we performed a series of proton T1 and T2 relaxation measurements in order to determine their longitudinal and transverse relaxivity values, r1 and r2 at both low (1.4 T) and high (7.0 T) magnetic field strength. The 1.4 T relaxometry results were obtained on a Bruker Minispec 60
MHz relaxometer, and the 7.0 T results were obtained on a Bruker Biospec 7.0-T MRI scanner.
Experimentally, the change of relaxation rate with increasing concentration of contrast agent was measured, and the numeric value of relaxivity, r1 or r2, was then extracted from the plot 1/T1 (or
3+ 1/T2 ) vs. concentration of Gd ions in nanoparticles using the following equation:
1/Ti,obs = ri×[Gd] + 1/Ti,d (i=1,2)
Where 1/Ti,d (i=1,2) is the diamagnetic contribution to the relaxation rate and [Gd] is the concentration of Gd3+ ions in nanoparticles.
The relaxation rate of pure water is taken as the diamagnetic contribution in all the experiments. All of the data are reported on a per Gd3+ ion basis at two different magnetic fields in Table 7 and compared to commercial contrast agent Magnevist (i.e., Gd-DTPA) and several known nanoparticle-based Gd(III) MR contrast agents. As shown in Figure 3.7 and Figure 3.8,
−1 −1 −1 −1 the resulting values for these NPs are r1 = 35 mM s and r2 = 38 mM s at 1.4 T and r1= 17
−1 −1 −1 −1 mM s and r2 = 24 mM s at 7.0 T respectively. For comparison, the relaxivity values of Gd-
−1 −1 −1 −1 DTPA are r1 = 3.4 mM s and r2= 3.7 mM s at 1.4 T (Table 7).
46
Table 7: Comparison of relaxivity data of several selected nanoparticulate Gd3+-based contrast agents.
-1 -1 -1 -1 Nanoparticulate Gd-based CA r1(mM × s ) r2(mM ×s ) r2/r1 field Ref ratio (T)
KGd(H2O)2[Fe(CN)6]·H2O 16.8 23.9 1.4 7.0 this work
KGd(H2O)2[Fe(CN)6]·H2O 35.2 38.4 1.1 1.4 this work
a Gd(BDC)1.5(H2O)2 35.8 55.6 1.6 3.0 87
Gd(H2O)2[Fe(CN)6]·2H2O 13.3 20.1 1.5 4.7 88
GdPO4 13.9 15.0 1.1 0.47 89
Gd2O3 4.8 16.9 3.5 7.0 90
aBDC = terephthalic acid
2.5 2.5
2 -1 -1 2
r2 = 38 mM s
) )
1 1 -
1.5 1.5 -
(s (s
1 2
1 1
1/T 1/T
-1 -1 0.5 r1= 35 mM s 0.5
0 0 0 0.01 0.02 0.03 0.04
Gd concentration (mM)
3+ Figure 3.7:The graph of 1/Ti (i=1,2) versus Gd -concentration at the magnetic field strength of
1.4 T.
47
16 16
r = 24 mM-1s-1
12 2 12
)
)
1
1
-
-
(s
(s 1
8 8 2
1/T 1/T 4 4 -1 -1 r1 = 17 mM s 0 0 0 0.1 0.2 0.3 0.4 0.5 Gd concentration (mM)
3+ Figure 3.8: The graph of 1/Ti (i=1,2) versus Gd -concentration at the magnetic field strength of
7.0 T.
To probe if the citrate-coating layer on the surfaces of the nanoparticles would impede water exchange between the bulk and coordinated H2O molecules, we intentionally prepared the nanoparticles under the same synthetic conditions with PVP as the only coating layer of the
-1 -1 nanoparticles. As shown in Figure 3.9, there is a slight increase of r1 value from 35 to 37 mM ×s for the PVP-coated nanoparticles, while the simultaneous change in the r2 value for such nanoparticles from 38 to 47 mM-1×s-1 appears to be more significant. These results suggest that the presence of the citrate-coating layer on the surfaces of the nanoparticles may or may not block the access of the bulk water to the sites of the coordinated H2O because the slight increase of the r1 relaxivity may well be due to the increased outer-sphere contribution as the result of a higher r2 value in the PVP-coated nanoparticles. On the other hand, the opposite may be said about the tight covalently-bound citrate-coating layer, that is, its presence on the surfaces of the nanoparticles can act as a magnetic shielding to reduce the effectiveness of the localized tiny magnetic fields generated by the nanoparticles which in turn would induce the r2 relaxation of the
48
bulk water protons. It is conceivable that the Gd(III) ions on the surface of the nanoparticles have a higher ability to induce the relaxation of the bulk water protons than the contribution to Gd(III) inside the nanoparticles. We therefore prepared PVP and citrate coated nanoparticles with four different sizes by controlling the nucleation rate and coating. The relaxivity measurements showed clear size dependence (Figure 3.10 and 3.11). It is interesting that both r1 and r2 relaxivities decrease with increasing particle size at almost the same rate.
7 7 6 6 r = 47 mM-1s-1
5 2 5
) )
1 1 -
4 4 -
(s (s
1 2
3 3 1/T -1 -1 1/T 2 r1 = 37 mM s 2 1 1 0 0 0 0.05 0.1 0.15 Gd concentration (mM)
3+ Figure 3.9: The plot of 1/Ti (i=1,2) versus Gd -concentration at the magnetic field strength of
1.4 T for PVP coated nanoparticles.
49
50
40
)
1
-
s 1 - 30
(mM 20
1 r 10
0 0 50 100 150 200 Hydrodynamic size (nm)
Figure 3.10: The plot of r1 vs the hydrodynamic particle size for KGdPB nanoparticles.
60
)
1
- s
1 40 -
(mM 20
2 r
0 0 50 100 150 200 Hydrodynamic size (nm)
Figure 3.11: The plot of r2 vs the hydrodynamic particle size for KGdPB nanoparticles.
50
These observed values of r1 relaxivity would place the current NPs among the best
3+ 91,92, 93 nanoparticle-based T1-weighted CAs that contain the Gd ion. Besides the extremely high r1 relaxivity, the small r2/r1 ratios of ca. 1.4 at 7.0 T and 1.1 at 1.4 T, respectively, indicate that these nanoparticles are suitable as a T1-weighted agent to enhance the longitudinal relaxation of the water protons. In contrast, metal oxide-based nanoparticles, particularly iron oxide nanoparticles, have a tendency to exhibit superparamagnetic behavior with an exceedingly large
91 r2/r1 ratio (i.e. r2/r1 > 10). As the result, iron and other metal oxide nanoparticles typically act as
94 highly effective T2-weighted CAs.
According to the SBM theory, the mechanism of inner-sphere relaxation in small molecular
Gd3+-chelates can be understood from the following equation.95
=
Where q is the number of water molecules directly coordinated to the Gd3+ center, [C] is the molar concentration of the contrast agent in mM, T1M is the longitudinal relaxation time of the bound water, and τM is the mean residence life-time of the coordinated water molecule. T1M is in turn a function of several parameters including τR, the rotational correlation time of the complex,
Tie (i=1,2), the longitudinal and transverse electron spin relaxation times, and r, the distance between the metal ion and the protons of the coordinated water molecule:
T1M = f (τM, τR, Tie, r)
The most common approach to enhance the longitudinal relaxivity hinges on the optimization of q, τM and τR. Although a higher number of coordination water can lead to a large increase in relaxivity, the decreased thermodynamic stability and/or kinetic inertness will render the complex highly susceptible to displacement by proteins, biological ligands or endogenous
Zn2+, Ca2+ and Mg2+ ions.10,96 It is tempting to conjecture that the presence of two water molecules directly coordinated to the Gd(III) center as well as the zeolitic water in the structural
51
cavities plays an important role to contribute to the inner-sphere longitudinal relaxation of the water protons. Furthermore, the structural rigidity resulting from the 3D extended polymeric network and the reduced tumbling rate of such particulate contrast agents also favor high relaxivity. Recently, Perrier and co-workers reported the synthesis and NMR relaxivity studies of nanoparticles based on a similar coordination polymer with the formula Gd(H2O)2[Fe(CN)6]•H2O where the Gd3+ contains two coordinated water molecules as well, although the cytotoxicity and cellular penetrating ability of these nanoparticles remained to be seen.97 As expected, the
PEGylated nanoparticles of the coordination polymer also exhibit high T1-weighted relaxivity
3- with a low r2/r1 ratio. However, the [Fe(CN)6] anion in Gd(H2O)2[Fe(CN)6]•H2O contains the low-spin Fe(III) center with S = ½ that may be magnetically coupled with the Gd(III) center with
S = =7/2 to give rise to a significant contribution of the outer-sphere relaxation to the overall T1- weighted relaxivity, thus making the distinction of the coordinated water molecules to the inner- sphere longitudinal relaxation from the out-sphere relaxation impossible. On the other hand,
4- because the [Fe(CN)6] anion in our KGd(H2O)2[Fe(CN)6]•H2O contains the low-spin Fe(II) center with S = 0, the Gd(III) ions can be viewed as isolated paramagnetic centers, the rendering the description of their T1-weighted relaxivity using the SBM model meaningful.
To further confirm the efficacy and evaluate their performance as effective MR contrast agents, we obtained both T1- and T2-weighted MRI phantom images of KGdPB nanoparticles in aqueous solution with various concentrations on a 7.0-T scanner (Figure 3.12 and 3.13). The T1- weighted images rapidly become brighter with increasing concentrations of nanoparticles, while the T2-weighted images respond to the increase of the concentration by slightly darkening the contrast. These results suggest that the current nanoparticles can act as an effective T1-weighted contrast agent at the high magnetic field.
52
3+ Figure 3.12: T1-weighted MR phantom images of KGdPB nanoparticles with various Gd - concentrations using a 7.0-T scanner.
T1-weighted MR phantom images was collected by B.O. Erokwu from Case Center for Imaging Research, Case Western Reserve University.
The ability of nanoparticles to cross the cell membrane is a critical pre-requisite for cellular MR imaging applications to be realized. We studied cellular uptake of our nanoparticles in HeLa cells using the fluorescent confocal microscopic imaging technique. Nanoparticles were synthesized according to the above procedure with a slight modification. Scheme12 and 13 show the synthesis of dye-labeled nanoparticles. Briefly, 200 mg of PVP (Mw=40000) was added to 50
53
ml of 1 mM K4[Fe(CN)6] solution while stirring. Then 50 ml of 1 mM GdCl3 solution containing
100 mg of citric acid was added to it under vigorous stirring. The resultant product was stirred for
Ca. 3 hrs. Next the product was dialyzed and lyaphalized. After that it was resuspended in 2 ml of
PVP solution (100 mg of PVP in 1 ml). 1.5 equivalents of ethylenediamine were then added to it in the presence of 1.2 eq of EDC reagent under vigorous stirring. The resultant mixture was stirred for another 24 hrs and excess ethylenediamine was removed by dialysis against distilled water for two days. Then 5 ml of carboxyfluorescein dye (CbF) (0.35 mg/ml) was reacted with
1.2 eq of EDC (≈1.5 mg) for 24 hrs. The ethylenediamine coated nanoparticles were then added to the 2 mL of above reaction mixture and stirred for Ca. 24 hrs. Finally the product was dialyzed to remove excess dye. The fluorescence spectrum of the carboxyfluorescein dye and the nanoparticles after attaching the dye was obtained to confirm the conjugation of the dye to the nanoparticle surface. The fluorescence spectra shown in figure 3.14 clearly show that the presence of dye on the surface of the nanoparticles. The fluorescent dye-labeled nanoparticles were then incubated with Hela cells to visualize the cellular uptake of these nanoparticles by confocal microscopy. Briefly, Hela cells were seeded in an eight well chamber at a density of approximately 1.5×105 cells per well in complete medium in the absence of antibiotics and incubated for 24 hrs at 37 oC. The cells were then incubated with dye-labeled nanoparticles for 3 hrs at 37 oC. The cells were washed with PBS three times and then imaged using a confocal microscope with 488 nm excitation wavelength.
54
3+ Figure 3.13: T2-weighted MR phantom images of KGdPB nanoparticles with various Gd - concentrations using a 7.0-T scanner.
T2-weighted MR phantom images was collected by B.O. Erokwu from Case Center for Imaging Research, Case Western Reserve University.
55
700
600
500 Carboxyfluorescein dye 400 Dye-labeled NPs 300
Intensity (A.U.) 200
100
0 500 550 600 650 700 750 Wavelength (nm)
Figure 3.14: Fluorescence spectra of carboxyfluorescein dye and dye labeled nanoparticles.
PVP (Mw, 40K) +
Scheme 12: Synthesis of citrate coated nanoparticles.
56
1. 1.2 eq EDC, 24 hrs stirring 2. Dyalysis
Scheme: 13 Schematic diagram showing the synthesis of dye-labeled nanoparticles.
The advantage of using CbF attached to the nanoparticles as the fluorescent probe is that due to its high anionic negative charge, the CbF dye molecule itself is membrane impermeable.98
57
The images of confocal microscopy showed the presence of bright green fluorescent signals inside the cells that were incubated with the dye-labeled nanoparticles for 3 hours. The untreated
HeLa cells were used as the negative control. Figure 3.15 shows the typical confocal fluorescent images of HeLa cells treated with the dye-labeled nanoparticles and the control cells. The uniform fluorescent emission in the perinuclear region of the cell indicates an untargeted cytoplasmic distribution of nanoparticles, i.e. there is no specific binding of nanoparticles to any small organelle in the region. These observations are consistent with the notion that internalization of such nanoparticles is most likely to occur via endocytosis.
A B
C D
Figure 3.15: Confocal microscopic fluorescence image of HeLa cells incubated with dye- conjugated nanoparticles for 3 hours (A), the bright field image of the cells from Panel A (B), the fluorescence image of untreated HeLa cells as the negative control (C), and the bright field image of the cells from Panel C (D).
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To evaluate the cytotoxicity of PVP coated nanoparticles, cell viability assay was performed as follows. Hela cells were grown in DMEM low glucose media containing 10% FBS
(fatal bovine serum) plus penicillin-streptomycin and was incubated at 37 °C with 5% CO2. The effect of nanoparticles on the viability of the cells was assessed using a Trypan Blue solution. The
96-well plates were seeded at 1 × 104 cells per well and incubated for 5 h to allow cells to attach to the surface. Cells were then be exposed to varying concentrations of KGdPB nanoparticles, followed by incubation for 24 or 48 h. The cells were then be trypsinized and re-suspended in 100
µL full media which was then added to 100 µL of 0.4% Trypan Blue solution. Viable and non- viable cells were counted using a hemocytometer. The assay was run in triplicate. Results are presented as percent viable cells (Figure 3.16).
24 hrs 48 hrs 100
80
60
40
Cellviability(%) 20
0 0.00 0.05 0.25 0.50 0.75 1.00 Gd(III) concentration/mM
Figure 3.16: Viability of Hela cells after incubation with KGdPB nanoparticles for 24 hrs and 48 hrs (Trypan Blue exclusion assay).
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As shown in Figure 3.16, more than 90% of the cells were still viable after 48 hours of
II incubation with 1.00 mM KGd(H2O)2[Fe (CN)6]H2O nanoparticles, indicating the nontoxic nature of such nanoparticles. We attribute the low cytotoxicity of the nanoparticles to the proper surface coating and the high structural integrity of the coordination polymer itself. To further confirm the stability of such nanoparticles against the leaching of toxic CN- and Gd3+ ions, we carried out a series of leaching experiments under the physiologically relevant conditions. About
200 mg nanoparticles were sealed in a membrane dialysis bag (MWCO=3,000). This dialysis bag was gently stirred in 25 mL solutions of distilled water for 24 hours. The same procedure was followed for the solutions of saline, acidic water (pH=1), Zn2+, Ca2+ and Mg2+. The resulting solutions were analyzed for free CN- ions released from the nanoparticles by a fluorometric method using the cyanide test kit developed by LaMotte Co. (Chestertown, Maryland; Code
7387-01).
Figure 3.17 shows the concentrations of the CN- ions released from the nanoparticles under different conditions. The highest cyanide concentration was found in an extremely acidic, albeit physiologically less relevant, solution of pH=1 to be approximately 3.5 ppm (this value approximately equal to the 0.12% of total CN present in the sample), while the cyanide concentration released from the nanoparticles was found to be ~0.8 ppm in a saline solution, and
~0.5 ppm in distilled water and other media containing a biologically relevant divalent metal ion.
In comparison, the maximum allowable level of cyanide in drinking water is 0.2 ppm as set by the environmental protection agency (EPA).99 It should be noted that in cigarette smokers, the cyanide concentration in blood can become as high as 35-65 ppm right after a cigarette is smoked.100
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4
3 /ppm
- 2 CN 1
0 pH=1 Saline Zn(II) Ca(II) Dis.water Mg(II)
Figure 3.17: CN- releasing test for different conditions.
The analysis of leached Gd3+ ions was carried out by the ICP-OES method. As shown in figure 3.18, under all the tested conditions, the concentrations of the Gd3+ ions released from the nanoparticles were all below 1 ppm (or <6 µM), except in the acidic solution of pH=1 where the concentration of the Gd3+ ions was found to be approximately 3 ppm (i.e. ~ 20 µM). This remarkable stability of the coordination polymer stems from the fact that as a ligand, the
4- hexacyanoferrate(II) anion, [Fe(CN)6] , possesses the highest-possible ligand field stabilization energy (LFSE) for any complex containing the low-spin Fe(II) center and that the formation of an extended 3D coordination network structure results in high lattice energy for the coordination polymer. Consequently, both the CN- group and the Gd3+ ion are completely locked in their corresponding lattice positions and cannot be released from the structure by self-dissociation or ion-exchange, confirming that the nanoparticles are extremely stable and resistant to the displacement of gadolinium in the presence of the biologically relevant divalent metal ions. In sharp contrast, some small molecule-based contrast agents are susceptible to in vivo transmetallation reactions with endogenous Zn2+ and Ca2+ to release Gd3+ ions. The latter was 61
linked to the development of nephrogenic systemic fibrosis (NSF) in some renal impaired patients,101 which prompted the U.S. Food and Drug Administration (FDA) in 2007 to issue a public health advisory regarding the use of gadolinium-containing contrast agents. Currently, the manufacturers of such contrast agents are required by the FDA to include a new Boxed Warning and new Warnings section in the labels to describe the possible link between the use of such contrast agents and the development of NSF.102
4
3
2 [Gd(III)]/ppm 1
0 pH=1 Saline Zn(II) Ca(II) Mg(II) Dis.water
Figure 3.18: Gd3+ releasing test for different conditions.
II To determine whether the internalized KGd(H2O)2[Fe (CN)6]H2O nanoparticles could enhance the T1-weighted MRI contrast of cells, we incubated PC3 cells with various
II concentrations of KGd(H2O)2[Fe (CN)6]H2O nanoparticles and examined the T1-weighted image for each sample at 37 °C using a spin-echo saturation recovery sequence on a Bruker 9.4-T MRI scanner. Approximately 1×105 PC3 cells were placed in T25 flasks and incubated for 48 hours.
The cells were then rinsed with serum free medium and incubated with varying concentrations of
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nanoparticles for 6 hours. The treated cells were washed with PBS for 3 times, and harvested by trypsinisation. Cells were then pelleted in 0.6 mL tubes for imaging studies. MR imaging of cell pellets was performed on a Bruker 9.4-T MRI scanner at 37 °C using a conventional gradient echo acquisition with an inversion recovery preparation. The other acquisition parameters were field of view = 3.0×3.0 cm2, matrix = 128×128, slice thickness = 3.0 mm.
As shown in Figure 3.19, there is a considerable change of image brightness in the pellets of the PC3 cells incubated with the nanoparticles. Particularly, the cells treated with the nanoparticles at the concentration equivalent to 0.25 mM Gd3+ ions prior to imaging showed a strong MRI signal brightening effect. These results demonstrate that KGdPB nanoparticles have the potential to be used as an effective T1-weighted contrast agent for cellular imaging at a high magnetic field.
Figure 3.19: T1-weighted MRI phantoms of PC3 cells incubated in PBS buffer (left), 0.13 mM nanoparticles (central), and 0.25 mM nanoparticles (right) for 6 hours. The images were collected using a Bruker 9.4-T scanner.
T1-weighted MR phantom images was collected by B.O. Erokwu from Case Center for Imaging Research, Case Western Reserve University.
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We have developed a simple one-step method for preparing extremely stable and biocompatible nanoparticles of the gadolinium ferrocyanide coordination polymer. Such nanoparticles can readily penetrate the cell membrane and exhibit no cytotoxicity. Furthermore, we have demonstrated that such nanoparticles exhibit extremely high T1-weighted relaxivity, suggesting the potential of this coordination-polymer structural platform in the development of the new-generation T1-weighted cellular MR probes for biological receptors or markers within the cell to study molecular events as well as for in vivo MR imaging in biomedical research and clinical applications.
3.3. Manganese-based MRI contrast agents
As previously discussed, gadolinium-based chelates are the most commonly used contrast agents so far. However, gadolinium-based contrast agents have been shown to be associated with nephrogenic systemic fibrosis (NSF), especially in the patients with impaired renal functions who are at risk for developing NSF.85 Therefore, development of new effective non-gadolinium(III)- based MRI contrast agents is imperative for the continued clinical use of MRI as a diagnostic tool. After Gd(III), high-spin Fe(III) and high-spin Mn(II) have the next highest number of unpaired electrons, and hence the second highest spin (S=5/2). Between these two transition metal ions, Fe(III) is better studied and its biochemistry is well known. Therefore, some iron- containing compounds seem to be potential candidates as MRI contrast agents. For example,
Fe(III) compounds such as ferric ammonium citrate and iron(III) chloride were investigated as potential contrast agents for MRI.188,189
However, Fe(III) is considered to be more toxic to cells than manganese because free ferric or ferrous ions can catalyze the production of reactive oxygen species via the Fenton reactions (eq.2 and 3).
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2+ 3+ − Fe + H2O2 Fe + OH· + OH (2) 3+ 2+ + Fe + H2O2 Fe + OOH· + H (3)
These reactive oxygen species can cause damage to liver, heart and other organs in the body. In order to prevent Fe(II) and Fe(III) from leaching out from the compounds, extremely stable and insoluble nanoparticulate compounds such as superparamagnetic iron oxide nanoparticles (SPIOS) were developed. So far, all the commercially available T2 contrast agents are based on SPIOS. They are highly sensitive and have a longer blood circulation time than Gd- based small molecule contrast agents. Moreover, they can penetrate the cells, and hence the cellular imaging is possible with these iron oxide nanoparticles. However, these contrast agents have several drawbacks that limit their widespread clinical applications. These contrast agents shorten the transverse relaxation time (T2) of bulk water more than that of longitudinal relaxation
(T1), and thus producing a negative contrast, i.e. darkening the image. As the result, the resulting darken signals make it difficult to distinguish other hypointense areas. For example areas where bleeding, calcinations or metal deposits occur.
Manganese(II) is an essential element of cells and serve as a cofactor for a number of enzymes with various biological functions. Thus, toxicity of Mn(II)-based contrast agents is generally regarded as lower than that of Gd(III)-based contrast agents. In addition, Mn(II) compounds are promising candidates for clinical use as MRI contrast agents because of their relatively high electronic spin and relatively fast water exchange rate.89 As the result, various
Mn(II)-based contrast agents have been investigated as novel agents over the last three decades.103,104 So far FDA has approved a solution of manganese(II) chloride (LumenHance®) an oral agent, and manganese(II) dipyridoxaldiphosphate (Mn-DPDP; Teslascan®) a liver imaging agent for clinical applications in the USA.105 However, it is reported that Mn(II) is released from the first agent due to ionization and from the second agent due to the transmetallation with
Zn(II).106,107 It has been shown that the elevated level of Mn(II) in the body can cause
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neurological disorder (Manganism), although the adverse toxicity caused by Mn(II) is usually less severe than that caused by Gd(III). We envisage that this problem may be solved by incorporating multiple Mn(II) centers into nanomaterials with a stable inorganic core structure.
3.4. PVP-coated KMn[Fe(CN)6] nanoparticles as a potential contrast agent for MRI
As previously discussed, nanoparticulate MRI contrast agents have attracted a great deal of attention in the recent years.108,6 The most remarkable feature of these contrast agents is that they have multiple metal centers with high magnetic moments per particle.103 Thus their sensitivity is higher. In addition, they have a long blood circulation time which is desirable if detailed imaging studies and monitoring of physiological functions need to be performed.109
Furthermore, in vivo metal-ion release from the particles would be minimized to reduce the risk of exposing the patient to heavy metal ions. In addition, targeted delivery is possible due to the ability of internalization of surface-functionalized nanoparticles by endocytosis. In the present work, Prussian blue (PB) was employed as such a nanoparticle platform to develop high- relaxivity, cell penetrating and nontoxic MRI contrast agents. Here we present the synthesis and potential use of poly(vinylpyrrolidone) (PVP) coated manganese-containing PB analog nanoparticles of KMn[Fe(CN)6] as contrast agents in magnetic resonance imaging. PVP coating was used to prevent the particles from agglomeration. Furthermore, PVP is used extensively as a protecting agent to improve the stability, biocompatibility110 and the solubility111 of colloidal dispersions. We demonstrate that these nanoparticles act as powerful negative contrast agents and are even stronger than the representative commercial T2 agent feridex®. Numerous other manganese containing compounds have found usefulness in the application of magnetic resonance imaging. For instance, mesoporous silica-coated hollow manganese oxide
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nanoparticles and superparamagnetic MnFe2O4 nanocrystals were reported as a novel magnetic resonance imaging (MRI) contrast agent.8,112
In this study, we have synthesized KMn[Fe(CN)6] nanoparticles (MnPB NPs) stabilized by PVP with a particle size distribution in the range of 10- 25 nm and characterized them by X- ray powder diffraction (XRD), transmission electron microscopy (TEM) , UV-vis spectroscopy and FT-IR spectroscopy.
PVP coated nanoparticles were prepared using the following procedure. First, 0.222 g of
PVP (Average MW=40000) was dissolved in 20 ml of K3[Fe(CN)6] (1 mM) solution. Then 20 ml of 1 mM MnCl2 solution was added dropwise to the above mixture under vigorous stirring. A pale yellow dispersion formed immediately. The reaction mixture was then stirred for another ca.
3 hrs. The reaction product was dialyzed using regenerated cellulose tubular membrane (MWCO is 3.5kDa) against distilled water for 2 days. PVP-coated nanoparticles, denoted as MnPB NPs.
Bulk MnPB were prepared by the same method, but without the addition of PVP into the solution.
Briefly, 110 mg of K3[Fe(CN)6] in 10 mL of deionized water was added to the aqueous solution
(10 mL) of MnCl2.4H2O (66 mg) while stirring. The precipitate was separated by centrifugation.
Figure 3.20 shows the transmission electron microscopy (TEM) image of nanoparticles.
The TEM studies revealed that these nanoparticles have well-defined cubic shape with the size distribution in the range of 10 - 25 nm. Furthermore, these nanoparticles are well separated from each other, suggesting that PVP coating stabilized the nanoparticles from aggregation.
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Figure 3.20: TEM images of PVP-coated KMn[Fe(CN)6 nanoparticles.
The crystal structure of the NPs was examined using powder X-ray diffraction pattern
(PXRD). As can be seen from figure 3.21 all peaks can be indexed to face-centered-cubic structure (space group fm3̅m) with a calculated lattice constant a = 10.82 A °. Crystal structure of the bulk compound is shown in figure 3.22.
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Figure 3.21: PXRD pattern of bulk MnPB material.
The elemental analysis was performed by inductively coupled plasma optical emission spectroscopy (ICP-OES) to establish the stoichiometry of the compound synthesized.
Before measuring the metal contents, samples of nanoparticles were heated in the oven at 600 oC for 8 hours and were extracted with concentrated nitric acid. The samples were then diluted with deionized water to a suitable volume so that all Fe, Mn and K concentrations fall into the linear dynamic range of the instrument. Calibration curves for iron, manganese and potassium were each established with the respective standard solutions. Our analysis indicates that the K:Fe:Mn ratio is 1:1:1.
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Figure 3.22: Crystal structure of bulk MnPB, color code: red=K, dark yellow=Fe and Mn, yellow=C, blue=N.
The FTIR spectra of MnPB NPs, bulk MnPB and PVP are shown in figure 3.23. The IR spectra of bulk and nanoparticles show strong characteristic CN stretching vibrations at ~2073 cm-1 and 2082 cm-1 respectively. Moreover, the IR spectrum of the nanoparticles shows additional bands at 1650 cm-1, 1421 cm-1 and 1280 cm-1 compared to the bulk compound, which indicates the presence of PVP at the surface of the nanoparticles. The peak at 1650 cm-1 is due to the contribution from C=O and N-C stretching vibrations. The peak at 1421 cm-1 can be assigned to
-1 the CH deformation of cyclic CH2 groups and the peak at 1280 cm is due to the amide III band
(C-N stretch). The PVP is hydrophilic and thus the presence of water is confirmed by the peak at
3465 cm-1 which is assigned to OH stretching vibrations.
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1
0.8
0.6 PVP 0.4 Bulk MnPB 0.2 Transmittance (%) Transmittance MnPB NPs 0 1000 2000 3000 4000 -1 Wavenumber (cm )
Figure 3.23: Overlay of the IR spectra of bulk (red) and nanoparticles (blue) of MnPB and PVP
(green) alone.
The relaxivities of the nanoparticles were measured using a 7.0 T medical MR scanner.
The particles exhibited very large longitudinal (r1) and transverse (r2) relaxivities. The relaxivity values of the MnPB NPs were calculated using the following equation:
r1=(1/T1-1/Td)/[Mn]
r2=(1/T2-1/Td)/[Mn]
Where T1 = longitudinal relaxation time, T2 = transverse relaxation time and [Mn] = concentration of manganese measured by ICP-OES and Td is the intrinsic diamagnetic solvent relaxation time.
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Figure 3.24 and figure 3.25 show the inverse relaxation times (1/T1 and 1/T2) as a function of the manganese millimolar concentration [Mn] respectively. The r1 and r2 values per millimole of manganese were found to be 5.22 s-1mM-1 and 170.7 s-1mM-1 respectively. The ratio between r2 and r1 is used to express the efficiency of a T2 contrast agent. The higher the ratio the better the efficiency of the T2 contrast agent. In the present work, r2/r1 was calculated to be about
32, which is way better than commercial Resovist (Schering AG, Germany).
-1 -1 113 The r2 and r1 relaxivity values for Resovist are 164.0 and 25.4 mM s respectively at 0.47 T.
The higher r2 value attributed to the superparamagnetism of these nanoparticles.
1.0
-1
(0), s 1
0.5 -1/T
1 1/T r1=5.22
0.0 0.00 0.05 0.10 0.15 0.20
[Mn2+], mM
−1 Figure 3.24: Longitudinal relaxation rates (1/T1, s ) of MnPB NPs as a function of the manganese concentration (mM).
Longitudinal relaxation times were collected by Dr. Anatoly Khitrin from the Department of Chemistry, Kent State University.
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40
30
-1
(0), s 20
2
-1/T 2 s-1 -1 1/T 10 r =170.7 mM 2
0 0.00 0.05 0.10 0.15 0.20
[Mn(II)], mM
−1 Figure 3.25: Transverse relaxation rates (1/T2, s ) of MnPB NPs as a function of the manganese concentration (mM).
Transverse relaxation times were collected by Dr. Anatoly Khitrin from the Department of Chemistry, Kent State University.
To further confirm the efficacy and evaluate their performance as effective MR contrast agents, we obtained both T2-weighted MRI phantom images of MnPB nanoparticles in aqueous solution with various concentrations on a 9.4-T scanner (Figure 3.26). The T2-weighted images rapidly become darkening with increasing concentrations of NPs. These results suggest that the current nanoparticles can act as an effective T2-weighted contrast agent at the high magnetic field.
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2+ Figure 3.26: T2-weighted MRI phantoms of NPs with various Mn -concentrations using a 9.4-T scanner.
T1-weighted MR phantom images was collected by B.O. Erokwu from Case Center for Imaging Research, Case Western Reserve University.
To demonstrate that our MnPB NPs are more stable than typical small molecular Mn complexes (e.g. Mn-DPDP) against leaching of Mn(II) in solution, we performed metal leaching studies. The potential leaching of MnPB NPs was evaluated in 0.9% NaCl solution, distilled water and in 0.1 M HCl solution. The nanoparticles were also tested for possible transmetallation reactions with 0.1 M Ca2+, 0.1 M Zn2+ and 0.1 M Mg2+ solutions. A solution of 5 ml (10 mM) nanoparticle solution of MnPB was placed in a dialysis bag (3500 molecular weight cutoff regenerated cellulose membrane) and soaked in 10 ml of 0.9% NaCl solution. The container was tightly stopped and incubated at 37 0C for 48 hrs. The above experiment was repeated for all the other solutions mentioned in the above. The leachates were analyzed for manganese by atomic absorption spectroscopy. The results are shown in figure 3.27. The potential leaching of CN- by nanoparticles was evaluated in all the above mentioned solutions. For that pH of the leachates were adjusted to a range of 5.5-6.0 and analyzed for CN- by using the cyanide test kit developed by LaMotte Co. (Chestertown, Maryland; Code 7387-01) (figure 3.28).
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5 4
3
2 [Mn]/ppm
1
0 pH=1 Saline Ca(II) K(I) Zn(II) Dis.water
Figure 3.27: Mn2+ leaching results of MnPB NPs under different conditions.
Results obtained after 48 hrs of incubation of nanoparticles in different solutions showed that these nanoparticles are not releasing CN- and Mn2+ ions of the toxic amounts. The highest
Mn(II) concentration leached was found to be approximately 4 ppm or 0.07 mM, which is less than the minimal toxic level of 0.1 mM. In the crystal structure, the cyanide groups are strongly coordinated to both iron(III) and manganese(II) ions and completely locked in the lattice positions due to the strong ligand field stabilization energy. As the result, leaching of cyanide and metal ions is minimized.
In order to find out the cytotoxicity of these nanoparticles, samples of MnPB NPs with five different concentrations, ranging from 0.1 to 0.5 mM (by manganese concentration) were incubated with Hela cells for 24 and 48 h and Trypan Blue exclusion viability assay was performed. Briefly, Hela cells were maintained in DMEM (Dulbecco’s Modified Eagle’s Medium) low glucose medium containing 10% FBS (fatal bovine serum) and 1% penicillin-streptomycin and grown at 37 °C in an atmosphere of 5% CO2 and 95% air. Hela cells were seeded in a 96-well plate at a density of 1 × 104 cells per well with the DMEM (Dulbecco’s Modified Eagle’s
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Medium) low glucose medium containing 10% FBS (fatal bovine serum) plus 1% penicillin- streptomycin and incubated for 5 hours at 37 °C in an atmosphere of 5% CO2 and 95% air to allow cells to attach to the surface. Cells in each well were then incubated with 100 µL of fresh medium (without antibiotics) containing various concentrations of the nanoparticles for 24 hours and 48 hours. Control wells contained the same medium without nanoparticles. The cells were then trypsinized and re-suspended in 100 µL medium without serum, and then 10 µL of this cell suspension was added to 10 µL of 0.4% Trypan Blue solution. Viable and non-viable cells were counted using a hemocytometer. Each concentration was tested in replicates of three. The assay results were presented as percent viable cells. Data clearly indicates that the nanoparticles are not toxic to cells. More than 90% of the cells were viable even after incubation of nanoparticles with concentration of 0.5 mM for 24 hrs. More than 88% cells were viable after incubating with 0.5 mM nanoparticle solution for 48 hrs. (Figure 3.29)
Zn(II) Dis.water K(I) Ca(II) saline pH=1
0 0.1 0.2 0.3 0.4 0.5 [CN-]/ppm
- Figure 3.28: CN leaching results of MnPB NPs under different conditions.
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24 hrs 48 hrs 100
80
60
40
Cellviability(%) 20
0 0.0 0.1 0.2 0.3 0.4 0.5 [Mn]/mM
Figure 3.29: Viability of hela cells after incubation with MnPB NPs for 24 hrs and 48 hrs
(Trypan Blue exclusion viability assay).
We have also prepared fluorescent labeled nanoparticles to study the cell uptake ability of these nanoparticles. MnPB NPs were synthesized according to the method discussed above, except citric acid and ethylenediamine were added to the reaction mixture to allow the conjugation of nanoparticles to carboxyflourescene dye. Briefly, 100 mg of PVP (Mw=40000) was added to 25 ml of 1 mM K3[Fe(CN)6] solution while stirring. Then 25 ml of 1 mM MnCl2 solution containing 100 mg of citric acid was added to it under vigorous stirring. The resultant product was stirred for Ca. 3 hrs. Next the product was dialyzed and lyaphalized. After that it was resuspended in 2 ml of distilled water. 1.5 equivalents of ethylenediamine were then added to it in the presence of 1.2 eq of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) reagent under vigorous stirring. The resultant mixture was stirred for another 24 hrs and excess ethylenediamine were removed by dialysis against deionized water. In a separate vial, 2 ml of carboxyfluoroscene dye (0.35 mg/mL) was activated by reacting it with 1.2 eq (0.427 mg) of EDC at room temperature for 24 hrs. This reaction was performed in the dark. The ehylenediamine coated
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nanoparticles were then added to the above reaction mixture and stirred for Ca. 24 hrs. Finally the product (nanoparticle-ethylenediamine-dye conjugate) was dialyzed against deionized water
(MWCO=3500) to remove excess dye. The fluorescent dye-labeled nanoparticles were then used to visualize the cellular uptake of these nanoparticles by confocal microscopy. Hela cells were seeded in a 8 well chamber at a density of approximately 1.0 ×105 cells per well in complete medium in the absence of antibiotics and incubated for 24 hrs at 37 oC. The cells were then incubated with dye-labelled nanoparticles for 3 hrs at 37 oC. The nanoparticle internalization into the cells was examined by confocal microscope using a 488 nm excitation wavelength.
The dye-labeled nanoparticles show green fluorescence. The fluorescence is clearly visible in the confocal fluorescence microscopic image (figure 3.30) of Hela cells incubated with fluorescent labeled nanoparticles for 4 hrs, confirming the efficient uptake of nanoparticles by
Hela cells.
To determine whether the internalized MnPB nanoparticles could enhance the T2- weighted MRI contrast of cells, we incubated Hela cells with various concentrations of MnPB nanoparticles and examined the T2-weighted image for each sample at 37 °C using a spin-echo saturation recovery sequence on a Bruker 9.4-T MRI scanner. Approximately 1×105 Hela cells were placed in T25 flasks and incubated for 48 hours. The cells were then rinsed with serum free medium and incubated with varying concentrations of nanoparticles for 6 hours. The treated cells were washed with PBS for 3 times, and harvested by trypsinisation. Cells were then pelleted in
0.6 mL tubes for imaging studies. MR imaging of cell pellets was performed on a Bruker 9.4-T
MRI scanner at 37 °C using a conventional gradient echo acquisition with an inversion recovery preparation. The other acquisition parameters were field of view = 3.0×3.0 cm2, matrix =
128×128, slice thickness = 3.0 mm.
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A B
C D
Figure 3.30: Confocal microscopic images of Hela cell line (A) Bright field image of cells incubated with dye conjugated nanoparticles for 4 hrs. (B) Fluorescence image of cells incubated with dye conjugated nanoparticles for 4 hrs. (C) Bright field image of untreated cells. (D)
Fluorescence image of untreated cells.
As shown in figure 3.31, there is a considerable change of image darkening in the pellets of the Hela cells incubated with the nanoparticles. Particularly, the cells treated with the nanoparticles at the concentration equivalent to 0.5 mM Mn2+ ions prior to imaging showed a strong MRI signal darkening effect. These results demonstrate that MnPB nanoparticles have the potential to be used as an effective T2-weighted contrast agent for cellular imaging at a high magnetic field.
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Figure 3.31: T2-weighted MRI phantoms of Hela cells incubated in PBS buffer (left), 0.2 mM
NPs (central), and 0.5 mM NPs (right) for 6 hours. The images were collected using a Bruker 9.4-
T scanner.
T2-weighted MR phantom images was collected by B.O. Erokwu from Case Center for Imaging Research, Case Western Reserve University.
In summary, using a one-step procedure we have prepared PVP coated MnPB NPs. These nanoparticles have the cubic rigid 3D coordination network structure. The Fe(III) and Mn(II) ions are completely locked in their lattice positions of the rigid network due to the strong coordination to the strong-field CN- ligand, making it very stable and nontoxic by not releasing metal ions to the surroundings. Moreover, PVP coating makes the nanoparticles biocompatible and nontoxic to the cells. These nanoparticles shorten T2 more than T1 due to their large magnetic moment.
Therefore, they act as a negative contrast agent.
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Chapter 4: Contrast agents for X-ray computed tomography.
CONTENTS OF THIS CHAPTER HAVE BEEN PUBLISHED AS AN ARTICLE IN
INORGANIC CHEMISTRY, Inorg. Chem., 2011, 50 (17), 7910–7912 ALL THE
MATERIALS OF THE ARTICLE HAVE BEEN REPRINTED WITH THE COPYRIGHT
PERMISSION OF THE AMERICAN CHEMICAL SOCIETY
X-ray computed tomography (CT) is one of the most useful and extremely versatile imaging techniques that used for diagnosis of diseases and physiological functions of internal organs. This technique provides high resolution images of various structures of the human body based on the differential X-ray absorption of the tissues. In addition, it shows deep tissue penetration ability as well. However, CT imaging is considered to be an insensitive method.114
Therefore CT contrast agents are often administered intravenously to increase the contrast between different soft tissues. Elements with high atomic number and high electron density have high X-ray absorption coefficient. Therefore, compounds containing heavy elements such as iodine, gold and bismuth are suitable as X-ray contrast agents for CT.
The intensity of CT image is expressed in terms of Hounsfield Units (HU), which can be represented as follows.
µx-µwater HU = 1000 ×
µwater-µair
Where; μwater and μair are the linear attenuation coefficients of water and air, respectively.
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Traditional X-ray contrast agents such as water soluble iodinated compounds have been used extensively as contrast media due to the ability of iodine to attenuate X-rays. However, they suffer from several disadvantages such as short blood circulation time and low contrast efficacy, which ultimately lead to side effects due to injection of high concentration of iodine within short period of time.
The use of nanoparticles in different imaging modalities have become an important area of research in the last decade.115,116,117 Nanoparticle-based contrast agents have several advantages over the conventional molecular counterparts. For example they can have a greater blood circulation half-life and may be concomitantly detected by multiple imaging modalities.118,119 In addition, nanoparticles can penetrate cells, and hence are suitable for cellular and molecular imaging applications. Particularly relevant to the development of CT contrast agents drawn on the nanoplatform is the ability to derive water dispersible nanoparticles from elements of higher atomic numbers than iodine (Z = 53).
Bismuth-based nanoparticles are promising candidates for use as X-ray contrast agents because of their high contrast, biocompatibility, stability and low cost. Moreover, bismuth has strong X-ray attenuation power (Z = 83). Typical bismuth complexes have a low solubility in aqueous solution or organic solvents.120 Therefore, none of the small molecular bismuth contrast
20 agents have been approved for clinical trials. Recently, polymer-coated Bi2S3 nanoparticles have been developed as an injectable CT contrast agent.121 Although these nanoparticles exhibit high
X-ray adsorption, long circulation times, and excellent imaging efficacy, the in vivo hydrolytic stability of such nanoparticles remains unclear.
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4.1 Nanoparticles of the novel coordination polymer KBi(H2O)2[Fe(CN)6].H2O as a potential contrast agent for computed tomography
Herein, we describe a simple one-step aqueous solution route for preparing biocompatible
KBi(H2O)2[Fe(CN)6].H2O nanoparticles. Polyvinylpyrrolidone (PVP) was used as a surface coating agent to control the size and prevent agglomeration of nanoparticles. PVP is a water- soluble and biocompatible polymer that has been used as a blood plasma expander for trauma victims. Various other applications of this polymer are also known in pharmaceuticals and cosmetics.122
PVP coated KBi(H2O)2[Fe(CN)6].H2O nanoparticles were synthesized according to the following procedure. In a typical synthesis, 100 mg of sodium citrate was first added to a 100-mL
1.0 mM aqueous Bi(NO3)3 solution under stirring at room temperature. To this solution was added a 100-mL 1.0 mM aqueous K4[Fe(CN)6] solution containing 250 mg PVP (average
MW=8000). A clear colorless to pale yellow dispersion formed immediately. After stirring for 30 minutes at room temperature, an aliquot of 10 mL was added with an equal volume of acetone, and centrifuged at 10,000 rpm for ~ 15 min., which resulted in the formation of a pellet of nanoparticles. The latter was re-dispersed in ~10 mL deionized water by sonication and separated again by the addition of equal volume of acetone and centrifugation. The purification process was repeated for two more times.
Bulk compound was synthesized by mixing a solution of 85 mg K4[Fe(CN)6] ( 20 mL) with a solution of 97 mg Bi(NO3)3.5H2O ( 50 mL) under vigorous stirring at room temperature to give a pale yellow precipitate. After stirring for ~15 min. at room temperature, the product was filtered, and washed with water two times and acetone two times. The product was dried in air at room temperature for 24 hours.
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Size and the morphology of the nanoparticles were determined by transmission electron microscope (TEM). The samples were first suspended in water, and then placed as the droplet onto a carbon-coated copper TEM grid (400-mesh). Specimens were allowed to air-dry and analyzed at 200 KV using a FEI Tecnai F20 transmission electron microscope (TEM) equipped with a field emission gun. TEM images of the nanoparticles showed a wide distribution of size from ca. 10 to over 30 nm (Figure 4.1).
The energy dispersive X-ray spectroscopy (EDX) results were obtained with the integrated scanning TEM (STEM) unit and attached EDAX spectrometer. The spatial resolution is <1 nm through the acquisition of high resolution (~0.2 nm) high-angle angular dark field
(HAADF) images, which is sensitive to atomic number (Z) contrast. Distinctive signals for K, Fe, and Bi were detected by (EDX) analysis (Figure 4.2), confirming the presence of each element in the nanoparticles.
Figure 4.1: TEM image of KBi(H2O)2[Fe(CN)6].H2O nanoparticles.
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Figure 4.2: EDX spectrum on a typical PVP-coated nanoparticle.
The elemental analysis was performed by inductively coupled plasma atomic emission spectroscopy (ICP-OES) to establish the stoichiometry of the compound synthesized. A sample of
50 mg KBi(H2O)2[Fe(CN)6]·H2O bulk sample was decomposed at 600 °C to amorphous oxide powder which was extracted with 2 mL concentrated HNO3. After dilution, the solution was analyzed by ICP-OES and was found to have an atomic ratio of 1:1:1 for K:Fe:Bi.
The crystal structure of the compound was established using X-ray powder diffraction on the bulk sample and found to be isostructural to gadolinium compound of the same stoichiometry.123 Atomic parameters of the latter structure were used as the initial ones in the
Rietveld refinement performed using TOPAS software from Bruker. The final refinement (Figure
4.3) in space group Pnma yielded a = 12.57141(13) Å, b = 13.56839(10) Å, c = 7.26925(9) Å, V
3 = 1239.94(2) Å3, and Fcalc = 2.72142(5) g/cm and converged at RBragg = 0.76%, Rwp = 7.04%, and
Rp = 7.35%.25. The refinement included 36 atomic coordinate and isotropic displacement parameters (Table 8) refined against 837 reflections within angular range 10-110o 2θ.
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Table 8: Summary of structure determination for KBi(H2O)2[Fe(CN)6].H2O.
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Figure 4.3: Rietveld refinement plot: difference between observed and calculated patterns is shown at the bottom; reflection positions are shown as vertical lines.
Powder X-ray diffraction pattern was collected by Peter Y. Zavalij from Department of Chemistry and Biochemistry, University of Maryland.
2+ 3+ The structure of KBi(H2O)2[Fe(CN)6].H2O consists of Fe octahedra and Bi biface- capped trigonal prisms joined in a 3D framework by CN groups (Figure 4.4). The Fe atom is coordinated by six C atoms of the CN groups, while Bi is coordinated by six N atoms and additionally by two O atoms of water molecules O1 andO2 (Table 9). The cavities in the framework are filled up by a K+ ion and water of crystallization (O3), which shows occupation disorder residing in the same cavity. Both K and O3 are slightly shifted from each other by 0.4 Å so that K distances to nearby O and N atoms are in the range of 2.9-3.2 Å, while the O3 water molecule forms two H bonds with O1 and O2 (Table 9)
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Table 9: Selected Interatomic Distances (Å).
Atom1 Atom2 Distance, Å Atom1 Atom2 Distance, Å
Bi N1 × 2 2.359(13) Fe C1 × 2 1.88(2)
N2 × 2 2.440(14) C2 × 2 1.97(3)
N3 × 2 2.503(13) C3 × 2 2.02(2)
O1 2.610(14) O3 O1 2.66(4)
O2 2.736(15) O2 2.72(4)
Figure 4.4: Crystal structure of KBi(H2O)2[Fe(CN)6].H2O with Bi and Fe shown in yellow and blue polyhedra, respectively. Cyanide ions are shown as thick cylinders (N, blue; C, gray balls).
K ions are depicted as large balls.
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Fourier transform infrared spectroscopy (FTIR) data were obtained on a Bruker Tensor
27 Instrument using KBr matrix. The FT-IR spectra of the nanoparticles exhibit a strong characteristic C≡N stretching vibration at 2063 cm-1, in addition to the other characteristic stretching and bending vibrations attributable to PVP (Figure 4.5).
Figure 4.5: The FT-IR spectrum of PVP-coated nanoparticles.
Thermal gravimetric analysis (TGA) was conducted on a KBi(H2O)2[Fe(CN)6]•H2O bulk sample using a TA instruments 2950 high-resolution thermogravimetric analyzer (New Castle,
DE, USA) in air from room temperature to 600 °C with a heating rate of 10 °C/min (Figure
4.6).The results from thermal gravimetric analysis (TGA) on the bulk sample showed a two-step loss of water before 150 oC, which is consistent with the existence of one zeolitic and two
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coordinative water molecules per formula (Figure 4.6). We have found that the PVP-coated
KBi(H2O)2[Fe(CN)6]•H2O nanoparticles are stable in solution for over six months.
Figure 4.6: The TGA curve of bulk KBi(H2O)2[Fe(CN)6]·H2O sample.
The effect of PVP-coated KBi(H2O)2[Fe(CN)6]·H2O nanoparticles on the viability of cells was assessed using a Trypan Blue exclusion viability assay. Hela cells were seeded in a 96- well plate at a density of 1 × 104 cells per well with the DMEM (Dulbecco’s Modified Eagle’s
Medium) low glucose medium containing 10% FBS (fatal bovine serum) plus penicillin- streptomycin and incubated for 5 hours at 37 °C in an atmosphere of 5% CO2 and 95% air to allow cells to attach to the surface. Cells in each well were then incubated with 100 µL of fresh medium containing various concentrations of the nanoparticles for either 24 hours or 48 hours.
Control wells contained the same medium without nanoparticles. The cells were then trypsinized
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and re-suspended in 100 μL full medium, then added to 100 μL of 0.4% Trypan Blue solution.
Viable and non-viable cells were counted using a hemocytometer. Each concentration was tested in replicates of three. The assay results were presented as percent viable cells. After 48 h of incubation with a concentration of 400 µM, the cell viability was found to be ca. 90.4% (Figure
4.7), indicating that the PVP coated KBi(H2O)2[Fe(CN)6]•H2O nanoparticles exhibit no significant cytotoxicity.
24 hrs 48 hrs 100 80 60 40
Cellviability (%) 20 0 0 100 200 300 400 Bi(III) concentration (µM)
Figure 4.7: Viability of Hela cells after 24 and 48 hrs of incubation periods with varying concentrations of nanoparticles.
We determined the release of free cyanide ions from nanoparticles into solutions using a fluorometric method using the Konig reaction.124 Approximately 200 mg nanoparticles were sealed in a membrane dialysis bag (MWCO=3,000). This dialysis bag was gently stirred in 25 mL distilled water solutions of different pH values for 24 hours. The resulting solutions were analyzed for free CN- ions released from the nanoparticles by a fluorometric method using the
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cyanide test kit developed by LaMotte Co. (Chestertown, Maryland; Code 7387-01). The calibration curve was established using the standard KCN solutions with the concentrations at the ppm level. The analysis of leached Bi3+ ions was carried out by the ICP-OES method. We found that the cyanide concentration detected after 24 h of incubation with nanoparticles was below
∼0.5 ± 0.2 ppm at neutral pH and 1.2 ± 0.2 ppm at pH = 1 (Figure 4.8). These levels of free cyanide ions are comparable to those found in drinking water or certain plants and fruit seeds or stones.100 The intake of small amounts of cyanide by humans can be rapidly detoxified by the mitochondrial enzyme Rhodanese that converts cyanide into thiocyanate.125 The elemental analysis using ICP showed that the levels of free Bi3+ ions released to the same solutions were below 2 ±1 ppm at neutral pH and about 7 ± 1 ppm at pH = 1 (Figure 4.8), while the oral LD50
126 value of Bi(NO3)3 .5H2O in the rat was found to be 4042 mg/kg. The stability of these nanoparticles at such a low pH value is remarkable and suggests the potential of oral delivery of such contrast agents.
Figure 4.8: Leaching levels of free CN-and Bi3+ ions.
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X-ray attenuation coefficients of nanoparticle solutions at different concentrations, distilled water and air were measured using a Gamma Medica Xspect. The CT phantom imaging studies were carried out with the following parameters: 512 slices/360o rotation; 75 kVp, 110 μA; field of view, 39.47, resolution, 150 micron. Absorption measurements were obtained as density values in Hounsfield Units (HU). By definition, the density value of water and air is assigned as 0 and-1000 HU, respectively. As shown in Figure 4.9, a solution of 600 mM PVP-coated
KBi(H2O)2[Fe(CN)6]•H2O nanoparticles has a CT value of 3450 HU. This value is an equivalent
X-ray absorption to that of about 1596 mM iodinated contrast agent, indicating that the linear attenuation coefficient of our nanoparticles is ca. 2.7 times of the typical iodine-based CT contrast
121 agent but ca. 62% of the Bi2S3 nanoparticles at this concentration. The CT value starts to plateau at a concentration of 500-600 mM Bi3+. We noticed that the samples of such high concentrations appear slightly cloudy due to the saturation of dispersibility, which causes the data to deviate from a linear fit in this range. These results suggest that the PVP-coated
KBi(H2O)2[Fe(CN)6]•H2O nanoparticles possess potential as a novel CT contrast agent.
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Figure 4.9: CT intensity values (top) and phantom images (bottom) of nanoparticles with different Bi3+ concentrations.
CT intensity values were collected by Jihua Hao from Department of Radiology, Case Western Reserve University.
In summary, we have discovered an extremely stable and biocompatible nanoplatform to deliver bismuth as a CT contrast agent. Currently, CT is not considered a cellular imaging modality due to the lack of contrast agents that are either cell-permeable or surface-modified with suitable targeting agents that can selectively bind to certain receptors of the cell exterior.127 Our preliminary studies showed that KBi(H2O)2[Fe(CN)6]•H2O nanoparticles can be internalized by cells via endocytosis.
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4.2. Nanoparticles of KBiXGd(1-X)[Fe(CN)6] as a potential bimodal contrast agent for
MRI and CT
Nanoparticulate imaging agents for magnetic resonance imaging (MRI), fluorescent imaging (FI), X-ray computed tomography (CT), positron emission tomography (PET), and single photon emission computed tomography (SPECT) have been the focus of extensive research over the past decade in the biomedical imaging field.128,129 This is mostly because of their superior imaging properties compared with those of small molecular imaging agents. As previously mentioned, they have longer vascular circulation times than molecular agents and thus provide longer imaging times. Nanoparticles are also ideal for synthesizing dual or multimodal imaging agents. Multimodal imaging or the combination of different imaging modalities can be generally used to improve the accuracy of disease diagnoses. Furthermore, it provides more comprehensive diagnostic information.
In effort to develop MRI/CT bimodal contrast agent, we first synthesized polyvinylpyrrolidone (PVP) coated KBi(H2O)2[Fe(CN)6].H2O nanoparticles by using the method described previously in section 4.1, and examined their application as a CT contrast agent. Herein, we report the development of the modified nanoparticles based on the same structural platform for MRI/CT dual modality imaging. KBi(H2O)2[Fe(CN)6].H2O nanoparticles were modified by doping with gadolinium ions to get nanoparticles of KBiXGd(1-X)[Fe(CN)6]( Gd@BiPB). They form a solid solution because Gd3+ ion has an ionic radius of 1.07 A0 which is compatible with the ionic radius of Bi3+ which is 1.03 A0. They act as contrast agent for both CT and MRI because of the presence of Bi and Gd respectively.
In a typical synthesis, 2 ml of 1 mM Gd(NO3)3 was first added to 18 ml of Bi(NO3)3 (1 mM) solution in 100 mg of sodium citrate while stirring. Then 0.100 g of PEG (average
MW=8000) was dissolved in 20 ml of K4[Fe(CN)6] (1 mM) solution and was added to the above
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reaction mixture dropwise under vigorous stirring. A pale yellow dispersion formed immediately.
The reaction mixture was stirred for about another 3 hrs. The reaction product was dialyzed using regenerated cellulose tubular membrane (MWCO is 3.5kDa) against distilled water for 2 days.
The size and morphology of the nanoparticles were determined by transmission electron microscopy (TEM) studies. As can be seen in the figure 4.10, these nanoparticles are approximately 50 nm in size and they have narrow size distribution. Distinctive signals for K, Fe,
Bi and Gd were detected by energy dispersive X-ray spectroscopy (EDX) analysis (figure 4.11).
Figure 4.10: TEM image of as prepared nanoarticles of Gd@BiPB.
The Gd@BiPB nanoparticles were coated with PEG (polyethylene glycol) to make the nanoparticles biocompatible and water soluble. Further, surface coating helps to protect nanoparticles from aggregation. The presence of the surface coating was confirmed by comparing the FTIR spectra obtained for bulk, PEG and nanoparticles separately (Figure 4.12).
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Figure 4.11: EDX spectrum on a nanoparticle.
Bulk Na Citrate 3 PEG
Nano Transmittance(%)
1000 2000 3000 4000 -1 Wavenumber (Cm )
Figure 4.12: Overlay of the IR spectra of bulk (dark green) and nanoparticles (pink) of
Gd@BiPB and PEG (blue) alone.
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The crystal structure of the bulk compound was confirmed by powder X-ray diffraction pattern (PXRD). As shown in figure 4.13 all the peaks can be indexed to space group Pnma with the cell dimension parameters a=12.5714(1) Å, b=13.5684(1) Å, c=7.2693(1) Å, V=1239.9(1) Å3.
In the crystal structure both Gd3+ and Bi3+ ions occupy the same crystallographic positions and are bound by six N-donor atoms from the C≡N ligands and two coordinative water molecules, resulting in distorted bi-capped trigonal prism geometry, while the Fe(II) center has a regular octahedral geometry. Both compounds possess a 3D extended polymer network structure as depicted in figure 4.14.
420
320
220
Intensity (A.U) Intensity 120
20 10 30 50 70 2theta (Deg.)
Figure 4.13: PXRD pattern of bulk compound.
The potential leaching of CN from Gd@BiPB was evaluated in 0.9% NaCl solution, distilled water and in 0.1 M HCl solution. The sample was also challenged by 0.1 M
Ca2+, 0.1 M Zn2+, 0.1 M K+ and 0.1 M Mg2+ solutions. 5 ml of 10 mM nanoparticle solution of
Gd@BiPB was placed in a dialysis bag (3500 molecular weight cutoff regenerated cellulose membrane) and soaked in 10 ml of 0.9% NaCl solution. The container was tightly stopped and incubated at 37 0C for 48 hrs. The above experiment was repeated for all the other solutions also.
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The leachates were analyzed for cyanide ions using a fluorometric method using the Konig reaction. Figure 4.15 shows no obvious leaching of cyanide even after a 48 hrs dialysis in pH=1 solution. According to Environmental Protection Agency (EPA), the maximum allowed CN‾ contamination level in drinking water is 0.2 ppm. Further, no leaching of free Gd3+ ions could be detected by inductively plasma mass spectrometry. Thus, nanoparticles of Gd@BiPB have a great potential for in vivo use.
Figure 4.14: Structure of 3D extended polymer network found in Gd@BiPB.
0.3
0.2
]/ppm -
[CN 0.1
0 0.1 M Saline Ca(II) K(I) Mg(II) Zn(II) Dis. HCl Water
Figure 4.15: CN- release test against different conditions.
The cytotoxicity of the nanoparticles was tested using the (3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide) (MTT) MTT assay. Approximately, 1 × 104 cells of Hela
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(human cervical carcinoma) were seeded in a 96-well plate at a density of 1 × 104 cells per well with the DMEM (Dulbecco’s Modified Eagle’s Medium) low glucose medium containing 10%
FBS (fatal bovine serum) plus 1% penicillin-streptomycin and incubated for 5 hours at 37 °C in an atmosphere of 5% CO2 and 95% air to allow cells to attach to the surface. Cells in each well were then incubated with 100 µL of fresh medium containing various concentrations of the nanoparticles for 24 hours and 48 hours. Control wells contained the same medium without nanoparticles. Each concentration was tested in replicates of three. At the end of the incubation period, 10 μL of 5 mgmL-1 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well and incubated for another 3 hrs. Then 100 µl of detergent reagent was added to each well and incubation was continued for another 4 hrs at 37 oC. Finally the absorbance was determined at 570 nm using BIORAD ELISA plate reader. The assay results were presented as percent viable cells. Viability percentage was determined from the ratio of the absorbance of the treated cells to the untreated controls. The viability of the Hela cells treated with different concentrations of nanoparticles, is shown in figure 4.16. Nanoparticles exhibited no cytotoxicity at concentrations up to 2 mg/mL, even after incubation with 48 hrs, indicating the nontoxic nature of these polymer coated nanoparticles.
24 hrs 48 hrs
100
80
60
40
Cellviability(%) 20
0 0.0 0.1 0.2 0.3 0.4 0.5 Bi(III) concentration (mg Bi/mL)
Figure 4.16: Viability of hela cells after incubation with Gd@BiPB nanoparticles for 24 hrs and
48 hrs (MTT assay).
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Confocal scanning microscopy was used to investigate the cellular uptake studies.
Nanoparticles were conjugated with the carboxyfluorescein dye before incubation with cells. First,
1.5 equivalents of ethylenediamine were added to 5 mM nanoparticle solution (2 mL) in the presence of 1.2 eq of EDC reagent under vigorous stirring. The resultant mixture was stirred for
24 hrs and excess ethylenediamine was removed by dialysis against distilled water for two days.
Then 4 ml of carboxyfluoroscene dye (0.38 mg/ml) was reacted with 1.2 eq of EDC (1 mg) for 24 hrs. The ethylenediamine coated nanoparticles were then added to the above reaction mixture and stirred for Ca. 24 hrs. Finally the product was dialyzed to remove excess dye. The fluorescent dye-label nanoparticles were then incubated with Hela cells to visualize the cellular uptake of these nanoparticles by confocal microscopy. Briefly, Hela cells were seeded in an 8 well chamber at a density of approximately 1.5×105 cells per well in complete medium in the absence of antibiotics and incubated for 24 hrs at 37 oC. The cells were then incubated with dye-labelled nanoparticles for 3 hrs at 37 oC. The cells were then washed with PBS and then imaged using a confocal microscope with 488 nm excitation wavelength (figure 4.17).
The X-ray absorption of nanoparticles was measured using a Gamma Medica Xspect instrument and the results were expressed in Hounsfield Units (HU). Figure 4.18 shows that a solution of 560 mM PEG-coated Gd@BiPB nanoparticles has the CT value of 3150 HU, indicating that the linear attenuation coefficient of such nanoparticles is ca. 2.4 times of the typical iodine-based CT contrast agent.
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Figure 4.17: Confocal microscopic images of Hela cells: (upper left) fluorescence image of cells incubated with dye-conjugated NPs for 3 hrs; (upper right) bright field image of cells incubated with dye-conjugated NPs for 3 hrs; (lower left) florescence image of the untreated cells; (lower right) bright field image of the untreated cells.
Figure 4.18: The CT attenuation values of different Gd@BiPB concentrations.
CT intensity values were collected by Jihua Hao from Department of Radiology, Case Western
Reserve University
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The proton T1 relaxation of PEG-coated Gd@BiPB nanoparticles were made using a 1.5-
T clinical Siemens Espree MRI system as shown in figure 4.19. The longitudinal relaxivity value,
-1 -1 r1, as determined from the slope of the plot of 1/T1 versus sample concentration is 37 mM s .
This value is approximately 8.6 times of that for the clinical MRI contrast agent, Magnevist®.
The presence of two water molecules directly bound to the paramagnetic Gd metal center, may be responsible for the exceptionally high relaxivity value compared to the commercially available contrast agents. The phantom images were obtained with varying concentrations of Gd3+ show increasing contrast with the increasing concentration (figure 4.20). The concentrations of each metal ion in the aqueous solutions were measured by inductively coupled plasma optical emission spectroscopy (ICPOES, Perkin Elmer Optima 3300-DV ICP).
Figure 4.19: The proton T1 relaxation of PEG-coated Gd@BiPB nanoparticles at different concentrations.
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Figure 4.20 T1-weighted MRI phantoms of PEG-coated Gd@BiPB nanoparticles with varying concentrations of gadolinium.
T1-weighted MRI phantoms were collected by Jihua Hao from Department of Radiology, Case Western Reserve University
In summary, we described a method to synthesize a highly stable, polymer coated, biocompatible Gd@BiPB nanoparticles. These nanoparticles displayed low toxicity and provided higher contrast value than commercially available iodinated contrast agents. Furthermore, incorporation of Gd metal ions into the structure, allowed us to modify these nanoparticles as
MRI contrast agent and the relaxivity values were significantly higher compared to the current contrast agents. Thus, PEG coated Gd@BiPB nanoparticles have great potential for using as a bimodal contrast agent for MRI and CT.
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Chapter 5: Nanoparticles for therapeutic applications
In the past decades, nanoparticles based therapeutics has emerged as a most promising treatment for cancer, diabetes, infections, and Inflammation. Moreover researchers have demonstrated that, nanoparticles can be used as a treatment of various neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease.130 In addition, various types of nanoparticles such as polymeric nanoparticles131 and ceramic nanoparticles132 have been used as carriers of insulin.
In the last decade, the synthesis and development of nanoparticles for the treatment of cancer have become an important area of research. One aspect of using nanoparticles is to deliver chemotherapy drugs to cancer cells. Specifically, controlled and targeted drug delivery is required.
For instance, Lee et al. have designed doxorubicin loaded poly-(lactic-co-glycolic acid) (PLGA)– gold half-shell NPs. These gold nanoparticles release doxorubicin, which is a drug used in cancer chemotherapy, upon irradiation with near IR due the heat generation by NIR resonance of the Au nanoparticles.133 Another aspect of nanoparticles in cancer treatment is to use them in thermotherapy. Here magnetic nanoparticles will be directed to cancer cells using a magnetic field and heated by being exposed to an alternative magnetic field. The heat produced, increases the tumor temperature which result in the destruction of the tumor. For example, superparamagnetic iron oxide nanoparticles (SPION) have been used for thermotherapy.134,135
Another important area of cancer research is photo-based nanomedicine. All of the nanoparticles used in photo-based applications are made up of noble metals such as gold and silver, because of their tunable optical properties which are highly desirable in photo-based nanomedicine. For example, gold has very unique optical and physical properties due to the surface plasmon
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resonance (SPR). SPR is often found in noble metal nanoparticles due to their high surface area to volume ratio.136 Several other nanomaterials such as single-walled carbon nanotubes and multi-walled carbon nanotubes have been developed and examined for photo- based nanomedicine.137,138
However, toxicity of nanoparticles remains a debate. Because, it is found that some of the nanoparticles are accumulated in some organs such as liver, spleen and kidney and become toxic over the time. However, it is reported that the surface modification of nanoparticles by biocompatible polymers like polyethylene glycol (PEG) and Polyvinylpyrrolidone (PVP) reduces the cytotoxicity of such nanoparticles. Moreover coating of nanoparticles by polymers such as polyethylene glycol (PEG) was shown to increase blood circulation time of these nanoparticles.139
5.1. Cell permeable Au@ZnMoS4 core-shell nanoparticles: Towards a novel cellular copper detoxifying drug for Wilson’s disease
CONTENTS OF THIS CHAPTER HAVE BEEN PUBLISHED AS AN ARTICLE IN CHEMISTRY
OF MATERIALS, Chem. Mater., 2013, 25 (23), 4703–4709. ALL THE MATERIALS OF THE
ARTICLE HAVE BEEN REPRINTED WITH THE COPYRIGHT (2013) PERMISSION OF THE
AMERICAN CHEMICAL SOCIETY
Progressive hepatolenticular degeneration, also known as Wilson’s disease (WD), is a genetic disorder characterized by excess copper accumulation in the liver and other vital organs.140, 141 There are wide clinically presenting symptoms of WD including a variety of hepatic, neurological, ophthalmic and psychiatric symptoms.142 If untreated, WD can lead to severe disability, a need for liver transplantation and death.143 The current treatment of WD is based on
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the use of copper chelating agents to block the adsorption of this ion in the stomach and to promote its urinary excretion from the body.144 Before chelation therapy was introduced by John
Walshe in 1956, WD had been almost invariably progressive and fatal.145 D-penicillamine or
(2S)-2-amino-3-methyl-3-sulfanyl-butanoic acid, a metabolite of penicillin, possesses a combination of soft, intermediate and hard donor atoms including S, N and O for forming a stable chelate with the copper ion. This metabolite has been used as an orally active drug for WD in the clinic.146 However; D-penicillamine is an immunosuppressant that is also used to treat rheumatoid arthritis. Therefore, its use in WD causes a score of side effects with symptoms ranging from bone marrow and immune suppression, to skin rash, and to deterioration of various neurological functions, to name but a few.146,147 For patients who show intolerance to D-penicillamine, another oral drug triethylenetetraamine (trientine) is used, but it is less effective as a copper chelating agent.148 There is clear evidence to suggest that both D-penicillamine and trientine can mobilize copper ions stored in the body tissues and reroute them into circulation, thus increasing the concentrations of copper in the brain.149 It has been estimated that 50% of the WD patients treated with these drugs can suffer neurologic deterioration, and half of them or 25% of the original patients would never recover.150 Despite these undesirable and severe side effects, D- penicillamine will remain the treatment of choice for WD until a safer and more effective drug is developed.151
Recently, an investigational oral drug, the ammonium salt of tetrathiomolybdate (TTM),
152 i.e. (NH4)2MoS4, for WD has completed several clinical trials with promising results. TTM forms a non-bioabsorbable form of certain ternary complexes with copper and food proteins in the gastrointestinal (GI) tract to block the intestinal absorption of copper from the diet.153 Such a slow-acting mechanism renders incremental of improvement of treatment efficacy by TTM for
WD incremental. Yet TTM is known to be susceptible to hydrolysis leading to release of hydrogen sulfide (H2S) and forms various polynuclear thiomolybdate species under the acidic
154,155 conditions of the stomach. Although a small amount of H2S is constantly produced in the
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human digestive tract from the anaerobic digestion of food, it can normally be detoxified by several enzymes. Unfortunately, H2S is considered more toxic than hydrogen cyanide (HCN) to the neural and circulating system.156 These facts suggest that the manufacture, storage and clinical use of TTM will be a safety concern. On the other hand, in 1997 the US Food and Drug
Administration (FDA) approved the use of zinc acetate as a clinical drug for WD.157 Zinc ions from this drug can stimulate the production of metallothionein in gut cells. This protein in turn can bind copper ions and prevent their absorption and transport to the liver. However, zinc acetate is only effective as a maintenance therapy for WD.158 It should be noted that as small molecules, none of the above drugs can penetrate cells to help remove excess free copper ions in the vital organs for the WD patients, particularly those who have progressed to a stage characterized by a significant amount of hepatic copper deposition. In the latter case, the only option for the patient is liver transplantation.159 Clearly, there is an unmet clinical need for a safer and more effective treatment of WD, particularly one that can function at the cellular level.
The work here is aimed to develop a novel drug delivery system that can penetrate the cell membrane to selectively remove excess free copper ions from the cell. Gold nanoparticles
(Au NPs) are chosen as the drug carrier for the investigation due to its many desirable properties such as the high surface area to volume ratio, and multivalent surface architecture that enables the incorporation of multiple therapeutic agents and targeting molecules on the surface to improve the delivery efficiency of therapeutic payloads.160-164 Additionally, the thiophilic properties of
2- gold allow for a facile functionalization of the ligand molecule [MoS4] to the Au nanoparticle
(NP) surface. We prepared the citrate-coated Au NP cores with the average size of 16±1 nm and
2- 2+ treated the NPs with the [MoS4] and Zn ions alternately to form a ZnMoS4 shell which was then PEGylated to impart hydrophilicity to the core-shell NPs. We examined the kinetics, capacity and selectivity of the ion-exchange of such NPs towards copper ions in aqueous solution.
We also studied the cellular uptake, cytotoxicity and intracellular copper removal of these NPs, thus demonstrating their potential as a novel cell-permeable copper detoxifying agent. To the best
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of our knowledge, this study is the first example to use a nanoparticulate composite to remove cellular copper ions via ion-exchange rather than chelation. This work provides a new paradigm for the design and development of the next-generation therapy for WD.
The citrate-coated AuNP cores were synthesized using the modified Turkevich
165 method. Specifically, an aqueous solution of HAuCl4 (0.25 mM, 50 mL) was first heated to boiling point with rigorous stirring, to which an aliquot of sodium citrate solution (1%, 1.75 mL) was added. In less than a minute, the solution turned from pale yellow to wine red, indicating the formation of AuNPs. After boiling and stirring for another 30 min, the resulting solution was cooled to room temperature.
The ZnMoS4 shell was grown on the surface of Au NPs by a layer-by-layer self-assembly technique as shown in scheme12. The as-synthesized citrate-coated Au NPs in aqueous solution was sealed in a dialysis bag and immersed in a water-formamide (FM) solution of (NH4)2MoS4
2- (the ratio of H2O:FM was 1:5 in volume) to form the first layer of the [MoS4] ions anchored on the Au surface via the thiophilic interaction of gold.166,167 Such an anchoring process was shown
2- to produce robust attachment of the [MoS4] ions on the surface of Au NPs as the immediate
2- dialysis against distilled water did not cause the loss of the [MoS4] anions from the Au NPs.
2- While still remaining in the dialysis bag, the [MoS4] plated Au NPs were then treated with an aqueous solution of zinc acetate followed by another dialysis against distilled water. The
2+ 2- coordination of the Zn ions to the S atoms from the [MoS4] anions displaced the
+ noncoordinating NH4 counter ions. This was confirmed by the detection of the latter from the solution outside the dialysis bag, thus completing the assembly of the first layer of ZnMoS4 on the Au NP surface.168,169 This process was repeated for the assembly of the subsequent layers of
ZnMoS4. The maximum number of cycles we could carry out for the deposition without triggering the core-shell NPs to aggregate was eleven. We noticed that by now the original citrate coating on the Au NPs was completely displaced. To increase the surface stability and impart
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water dispersability, we immediately coated the core-shell NPs with polyethylene glycol (PEG;
MW= 8000) after eleven cycles of deposition were completed.
Scheme 14: Schematic of layer-by-layer self-assembly of Au@ZnMoS4 NPs.
Figure 5.1 shows the TEM images of both the citrate-coated Au NPs and the
Au@ZnMoS4 NPs formed from eleven cycles of layer-by-layer deposition followed by
PEGylation. The particle size and size distribution for each batch of samples were obtained from measuring and averaging the size of 120 NPs. The average size of the Au@ZnMoS4 NPs is 22 ± 3 nm, while the average size of the Au cores is 16 ± 1 nm. The TEM images show that the shape is nearly spherical for both the citrate-coated Au NPs and the PEGylated Au@ZnMoS4 NPs.
Furthermore, the energy-dispersive X-ray spectroscopy (EDX) analysis on individual PEGylated
Au@ZnMoS4 NPs clearly showed the presence of Zn, Mo and S besides Au, suggesting that the sample consists of surface-coating ZnMoS4 shell on the Au core rather than the presence of a mechanical mixture of Au and ZnMoS4 NPs (figure 5.2).
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Figure 5.1: TEM images of as-prepared Au NPs (upper left) and Au@ZnMoS4 NPs (lower left) and histograms of the size distribution for NPs corresponding to each panel on the left.
Figure 5.2: EDX spectrum on a typical PVP-coated Au@ZnMoS4 nanoparticle
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As shown in figure 5.3, the UV-vis spectra of the sample aliquots taken from the reaction mixture after each cycle showed a red-shift of the plasmon resonance of Au NPs, indicating the altered surface characteristics of the Au NPs after the layer-by-layer assembly of the ZnMoS4 shell.170 This red-shift of the plasmon resonance is accompanied with a color change of the solution from purple to burgundy.
Figure 5.3: UV−visible spectra of pure Au NPs and the Au@ZnMoS4 NPs at the different stages of layer by-layer assembling process.
The FT-IR spectrum of the Au@ZnMoS4 NPs is shown in figure 5.4 in comparison to that of PEG. This IR spectrum exhibits bands attributable to PEG, clearly indicating the presence of PEG coating on the surface of the Au@ZnMoS4 assembly.
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0.8 PEG
Au@ZnMoS4-NPs coated with 0.4 PEG Au@ZnMoS4-NPs without Transmittance (%) Transmittance coating 0 750 1500 2250 3000 3750 4500 Wavenumber (Cm-1)
Figure 5.4: FT-IR spectra of Au@ZnMoS4 NPs, PEG coated Au@ZnMoS4 NPs and pure PEG.
Although Cu(II) is more stable than Cu(I) in aqueous solution where oxygen is present, in the biological system Cu(II) is first reduced to Cu(I) by various metalloreductases and then transported by Ctr1 into cells where this oxidation is maintained.171 Therefore, we carried out all of the ion-exchange studies of Au@ZnMoS4 NPs using Cu(I) ions in aqueous solution. Because
Cu(I) disproportionate in aqueous solution to give Cu(II) and Cu(0), copper(I) chloride was dissolved in 80% acetonitrile solution to form a stable Cu(I)-acetonitrile complex.172 The kinetics of the ion-exchange between Au@ZnMoS4 NPs and Cu(I) ions were determined by placing a dialysis bag containing 100 mg of NPs into an acetonitrile-water solution containing Cu(I) ions.
The initial copper concentration was 500 µM. The decrease of the copper concentration in this solution was monitored by taking aliquots of outside copper solution at different time intervals.
The aliquots were then diluted with 2% HNO3 acid and copper content was analyzed with atomic absorption spectrometry (AA). Figure 5.5 and table 10 show the copper removal kinetics by NPs.
The measured kinetic raw data were found to obey two separate rate laws. It was found that between zero second to 3600 seconds, the reaction rate obeys a pseudo first-order reaction
-4 -1 (equation 5.1) to give a rate constant of k1= 3.0×10 s or a half-life of t1/2 = 38 min (see table 11 and figure 5.6). The remaining part of data can be fitted into the second order rate law (equation
-1 -1 -1 5.2) with k2 = 1.45× 10 M s (see table 10 and figure 5.7). 113
ln[Cu] = - kt + ln[Cu]0 (5.1)
1/[Cu]t = 1/[Cu]0 + kt (5.2)
Table 11 shows the natural log of copper concentration values at each time point for the determination of pseudo first order reaction. The slope of the plot of ln[Cu] vs. time shown in figure 5.6 gives the pseudo first order rate constant. After this time point, the ion-exchange
-1 -1 -1 suddenly switched to a slower and second-order reaction with a rate constant k2= 1.45× 10 M s , indicating the need for Cu(I) ions now to penetrate into the inner layers of ZnMoS4 to react with the Zn(II) ions after the Zn(II) ions are consumed (figure 5.6 and table 10). Overall, these rate measurements suggested that such NPs are kinetically suitable for depleting intracellular copper ions.
500
400
300
200 [Cu] µM [Cu]
100
0 0 200 400 600 800 1000 Time (Min)
Figure 5.5: Kinetics of copper removal from the aqueous copper solution by Au@ZnMoS4 NPs.
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Table 10: The decrease of Cu concentrations vs. time in the ion-exchange reaction.
Time/ sec Cu concentration/µM Standard deviation
0 510.6 12.9 5 436.9 13.4 10 385.5 12.8 15 334.1 16.2 20 302.0 19.6 40 237.8 17.0 60 192.9 17.0 120 180.1 7.4 240 147.9 16.2 480 122.3 13.4 600 103.0 9.8 680 96.6 6.4 720 90.2 16.2 900 90.2 11.1
Table 11: The natural log of copper concentration values at each time point for pseudo first order reaction.
ln[Cu] Time (s)
6.24 0 6.08 300 5.95 600 5.81 900 5.71 1200 5.47 2400 5.26 3600
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6.4
6.2
6
5.8 ln[Cu] 5.6
5.4
5.2 0 1000 2000 3000 4000 Time (s)
Figure 5.6: Pseudo first order rate plot for the copper removal by Au@ZnMoS4 NPs.
Table 12: Values of reciprocal concentration and corresponding time for second order reaction.
1/[Cu](M-1) Time (s)
4204.6 3600 5017.3 7200 6759.0 14400 8178.7 28800 9707.9 36000 10353.2 40800 11090.4 43200
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Figure 5.7: Second order rate plot for the copper removal by Au@ZnMoS4 NPs.
The ability of a substance to remove metal ions from a solution is often expressed in terms of metal removal capacity. This parameter specifies the maximum amount of metal in milligrams that can be removed by one gram of the given substance. Metal removal capacity (q, mg/g) can be determined using the mass balance equation, q = (Ci-C)/(V/W), where Ci (mg/L) is the initial metal concentration, C (mg/L) is the metal concentration in solution after time t (min),
V(L) is the volume of metal solution, and W(g) is the weight of NPs. To evaluate the metal removal capacity, we performed the metal removal experiments for our NPs using a batch reaction method. The copper removal capacity was found to be 107 mg/g as shown in figure 5.8.
In a comparison study, we observed that bulk ZnMoS4 reacting with excess CuCl gave Cu2MoS4 and ZnCl2 by ion-exchange. The structure of Cu2MoS4 was previously determined by the X-ray powder data to be a layered compound.173,174 It is tempting to conjecture that the ion-exchange
+ between Cu ions and the surface-bound Cu2MoS4 layers proceeds via a similar mechanism.
117
120
100
80
60
Copper removal Copper 40
20 (mg copper/ g nanoparticles) nanoparticles) g copper/ (mg
0 0 500 1000 1500 Time (min)
Figure 5.8: Copper removal capacity of Au@ZnMoS4 NPs.
Based on the so-called Irving-Williams Series, the stability of the first-transition divalent
MMoS4 compounds should follow the trend CrMoS4 < MnMoS4 < FeMoS4 < CoMoS4 < NiMoS4
< CuMoS4 > ZnMoS4. Therefore, we expected the ion-exchange between ZnMoS4 and the above divalent ions to mostly favor the Cu2+ ion. To quantitatively evaluate the selectivity of the Cu(I) removal by Au@ZnMoS4 NPs in the presence of other biologically relevant divalent metal ions including Mg2+, Ca2+, Fe2+ and Mn2+, we studied the ion-exchange competition among all these ions. In the typical experiment, 5-mL NPs (10 mM) were sealed in a dialysis bag and placed in a solution (20 mL) containing copper, magnesium, calcium, iron and manganese ions each at the
100-ppm level. The solution concentration for each of the above ions was analyzed by atomic absorption spectrometry after 24-hr incubation at 22 ºC. The selectivity for each ion was normalized against the removal of Cu+ ions being set as 100%, and expressed as the percent removal for each ion. Figure 5.9 clearly shows that, Au@ZnMoS4 NPs are most selective towards the Cu+ ion in the presence of all the other divalent metal ions tested. The observed selectivity
118
towards the Cu+ ion is also consistent with the expected outcomes based on the HSAB principle.
Copper (I) is a soft Lewis acid and should strongly bind to a soft Lewis base, i.e. the S atoms
2- from the [MoS4] ligand in this case.
Figure 5.9: Selectivity of several divalent metal ions by Au@ZnMoS4 NPs.
We studied cellular uptake of Au@ZnMoS4 NPs in HepG2 cells using the fluorescent confocal microscopic imaging technique. Due to their morphological and functional differentiation, HepG2 cells are often employed as an in vivo model for the study of intracellular trafficking and dynamics, liver metabolism, toxicity and drug targeting in human hepatocytes.117
To prepare dye-labeled Au@ZnMoS4 nanoparticles, 45 μL of 0.25 mM ethylenediamine solution was added to a 200-μL Au@ZnMoS4 nanoparticle solution (250 μM) under vigorous stirring. The resulting reaction mixture was continuously stirred for 24 hrs. The product was purified by dialysis to remove unbound ethylenediamine molecules. Next 10 mL of carboxyfluorescene dye (0.5 mM) was allowed to react with 1.2 eq. of 1-ethyl-3-(3- dimethylaminopropyl)-carbodiimide (EDC) (1.15 mg) for 24 hrs in a separate container. Finally,
119
ethylenediamine coated Au@ZnMoS4 nanoparticle solution was added to 100 μL of the above- mentioned dye solution and stirred for another 24 hrs. To remove the un-conjugated dye molecules, the resulting product was dialyzed against distilled water for two days and analyzed by fluorescence spectroscopy (Figure 5.10 ).
800
600 Carboxyfluorescein
400 Dye-labeled NPs
Intensity(A.U.) 200
0 500 550 600 650 700 Wavelength (nm)
Figure 5.10: Fluorescence spectra for carboxyfluorescein dye and dye labeled nanoparticles.
5 For cellular uptake study of Au@ZnMoS4 nanoparticles, first 1.2× 10 HepG2 cells per well were seeded in a 8-well chamber slide and incubated for 24 h. The culture medium was then replaced with a medium containing dye-labeled nanoparticles at the concentration of 150 μM and incubated for another 3 hrs. For live cell imaging, after 3 hrs of incubation with the dye-labeled
Au@ZnMoS4 NPs, cell cultures were washed with PBS three times and then directly imaged using a laser scanning confocal microscope without fixation. The fluorescent images of confocal microscopy showed the presence of bright green fluorescent signals inside the cells that were incubated with the dye-labeled Au@ZnMoS4 NPs for 3 hrs while the untreated HepG2 cells were used as the negative control. Figure 5.11 shows the typical confocal fluorescent images of HepG2 cells treated with the dye-labeled Au@ZnMoS4 NPs and the control cells. The uniform
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fluorescent emission in the perinuclear region of the cell indicates an untargeted cytoplasmic distribution of NPs without specific binding to any small organelle in the region, which is consistent with cellular uptake via endocytosis. It should be noted that the nuclear uptake of NPs is negligible as can be seen from the much weaker fluorescent signals inside the nuclei.
Figure 5.11: Confocal microscopic images of HepG2 cells: (upper left) fluorescence image of cells incubated with dye-conjugated NPs for 3 hrs; (upper right) bright field image of cells incubated with dye-conjugated NPs for 3 hrs; (lower left) florescence image of the untreated cells;
(lower right) bright field image of the untreated cells
To assess the cytotoxicity, we performed cell viability assays in HepG2 cells. Trypan blue was used as a marker to assess cell membrane integrity for cell viability HepG2 cells were seeded in a 96-well plate at a density of 2 × 104 cells per well with the Dulbecco’s Modified
Eagle Medium (DMEM) low glucose medium and incubated for 5 hrs at 37 °C in an atmosphere of 5% CO2 and 95% air to allow cells to attach to the surface. Cells in each well were then treated with 100 μL of fresh medium containing varying concentrations of the nanoparticles and then
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incubated for 12 hrs or 24 hrs. Control wells contained the same medium without nanoparticles.
The cells were then trypsinized and re-suspended in 100 μL medium without serum, then added to 100 μL of 0.4% trypan blue solution. Viable and non-viable cells were counted using a hemocytometer. Three independent trials were conducted, and the averages and standard deviations were reported. The reported percent cell survival values are relative to the control cells.
Figure 5.12 shows the viability of HepG2 cells treated with Au@ZnMoS4 NPs. The results clearly indicate that the NPs are nontoxic to cells. More than 92% of the cells were viable even after incubation of NPs with concentration of 100 μM for 12 hrs. More than 89% of the cells were viable after incubating with 100 μM nanoparticle solution for 24 hrs.
12 hour 24 hour 100 80 60 40
20 Cellviability (%) 0 0 10 25 50 100 Au concentration (µM)
Figure 5.12: Effect of Au@ZnMoS4 NPs on viability of HepG2 cells after 12-hr and 24-hr incubation.
To evaluate the cellular copper detoxification ability by Au@ZnMoS4 nanoparticles,
HepG2 cells were induced with elevated levels of copper. The elevated copper concentrations inside the HepG2 cells were artificially induced by incubating cells with the medium
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supplemented with a Cu(II) salt (i.e. CuSO4) because Cu(II) is more stable in the aqueous culture medium, but can be reduced to Cu(I) upon taking up by cells.176 The intracellular copper concentrations were determined from the cell lysates as a function of incubation time using AA.
As can be seen from the curve in figure 5.13, it took about 12 hrs of incubation for the cells to become saturated with copper ions, while the cells remained healthy and thriving.
1600
)
15 -
1200
800
400 Amount of Cu per cell/g cell/g per of (10 Amount Cu 0 0 5 10 15 20 25 30 Time/hrs
Figure 5.13: Kinetics of copper uptake in HepG2 cells.
In order to evaluate the Au@ZnMoS4 NPs as potential copper depleting agents, we incubated the HepG2 cells containing elevated copper ions with Au@ZnMoS4 NPs. For that cells with elevated levels of copper were washed three times with PBS and incubated with the culture medium containing nanoparticles (200 µg/ mL). After 4 hours of incubation with nanoparticles at
37 oC, cells were washed three times with PBS to remove the uninternalized nanoparticles and further incubated with the fresh culture medium. To quantify the concentration of copper in cells in each cell culture flask (T25 flask), the cells were washed three times with PBS at different time intervals and lysed using concentrated nitric acid. After dilution with deionized water, the
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concentration of each lysate was determined by AA. As the control experiment, HepG2 cells with elevated copper ions were incubated with fresh culture medium, but not treated with
Au@ZnMoS4 NPs. The concentrations of copper in these cell lysates were determined side-by- side with those treated with Au@ZnMoS4 NPs. The results were normalized by the number of cells in order to determine the intracellular amount of copper per cell.
1200 Untreated cells
)
15 - 1000 NP-treated cells
800
600
400
200 Amount of Cu per cell/ cell/ g(10per of Amount Cu 0 1 2 3 4 5 Time (hrs)
Figure 5.14: Kinetics of copper removal from HepG2 cells.
As shown in figure 5.14, the cells treated with Au@ZnMoS4 NPs showed a substantial decrease in the cellular copper level. Specifically, the cellular concentration of copper dropped from the highly elevated level of 1.1 pg/cell to 0.6 pg/cell after 1 h incubation with Au@ZnMoS4
NPs, and to 0.4 pg/cell after 6 h incubation with NPs. In comparison, the cells incubated with the regular medium (i.e. the control cells) had a small efflux of copper ions from 1.1pg/cell to 0.8 pg/cell within the first two hours, and then kept the copper concentration steady at this level.
Therefore, it is tempting to conjecture that the ion-exchange had led to the formation of
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Au@CuMoS4 NPs and the faster and continuous decrease of copper concentrations in these cells might have occurred via exocytosis of such NPs.
` In summary, we have developed a stable and biocompatible nanoplatform for delivering
ZnMoS4 as a cellular copper detoxifying agent. Currently, the treatment of choice for WD relies on the use of D-penicillamine, a slow-acting orally active chelating agent with numerous adverse side-effects. This clinical drug also lacks the ability to cross the cell membrane in order to remove excess copper ions deposited intracellularly. Our studies have shown that Au@ZnMoS4 NPs are readily internalized by cells via endocytosis and can selectively remove copper ions in the presence of other divalent ions from the cell. The novel approach may provide a real possibility of developing a safer and more effective treatment to reverse the progression in the late onset WD often characterized by liver cirrhosis, psychosis and organ failure.
5.2. Nanoparticles of ZnMoS4 as novel inhibitors of angiogenesis
CONTENTS OF THIS CHAPTER HAVE BEEN PUBLISHED AS AN ARTICLE IN JOURNAL OF
MATERIAL CHEMISTRY B, J. Mater. Chem. B, 2014,2, 257-261. ALL THE MATERIALS OF
THE ARTICLE HAVE BEEN REPRODUCED WITH PERMISSION FROM THE ROYAL
SOCIETY OF CHEMISTRY.
The growth of new blood vessels from the preexisting blood vessels is known as the angiogenesis.177 Angiogenesis is needed when tumors grow larger than 2 mm in size. Because oxygen and the nutrients that is required for the tumor cells is supplied by the new blood vessels formed. Therefore inhibition of the angiogenesis became an important approach to treatment of the cancer in the past few decades. Vascular endothelial cells involve the formation of new blood
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vessels. Hence, control of the growth of the vascular endothelial cells remains a useful strategy to control the tumor growth. As previously discussed, copper has been found to stimulate the proliferation and migration of endothelial cells which play a crucial role in the angiogenesis process.177 The fundamental hypothesis of antiangiogenesis using copper-depletion therapy is that the level of copper required for angiogenesis is higher than that required for essential copper- dependent cellular functions. Therefore researchers consider lowering of copper levels in the body to suppress the angiogenesis but to keep other cellular functions that required copper not affected. For instance, Brem et al. demonstrated that inhibition of the tumor development in the rats and rabbits with implanted intracerebral tumors by supplementation of Cu-chelator penicillamine and a low-Cu diet.178 Penicillamine is an FDA-approved drug used to lower the excess copper accumulation in the liver of the patient with Wilson’s disease (WD), an autosomal recessive disorder that leads to abnormal copper accumulation. Penicillamine works by binding with copper and increasing renal excretion. However, later extended penicillamine treatment to patients with brain tumor did not find improvement in survival due partly to the fact that as a small molecule penicillamine is unable to cross the blood brain barrier (BBB) as well as its modest binding ability to copper ions. Moreover it has been reported that up to 20% of patients who take penicillamine can suffer neurological symptoms. About half develop other adverse effects such as fever, rash, or joint pains. These people are usually changed to trientine another chelating compound which removes excess copper from the body. It has been reported that trientine, suppress tumor development without causing any serious side effects.58,179
Ammonium tetrathiomolybdate (TM) is a strong copper chelating agent used to treat
Wilson’s disease. Molybdenum is a nontoxic and essential trace mineral that is needed for the proper function of certain enzyme-dependent processes, including the metabolism of iron.
Therefore TM is mostly useful to patients who suffer potential adverse reactions to the standard chelating agents, penicillamine and trientine. TM lowers the body's copper level by chelating the copper and protein, making a stable compound that cannot be used by the tumor cells or any part
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of the body. Brewer and collaborators conducted several Phase I clinical trials using a ammonium
180 tetrathiomolybdate ([NH4]2MoS4). TM can form a tripartite complex with many types of proteins. As an oral drug TM is almost insoluble in water and has a very low bioavailability, it only binds copper with food proteins and therefore prevents the absorption of copper. Both food derived copper and endogenously secreted copper in saliva and gastric juice are bound in this manner, putting the patient into a negative copper balance. As found in one of these trials, 120 milligrams of TM per day can bring copper levels to the target of 20% of baseline. Five of six patients whose copper levels were kept at these levels for more than 90 days had no growth of existing tumors or formation of new ones. However, the foul-smelling and water-insoluble TM is very unstable in water and can decompose to give poisonous H2S gas in less than four hours via
2- 2- the reaction MoS4 + 4H2O → MoO4 + 4H2S. In the human body, a small amount of H2S is produced in the digestive tract (GI) from anaerobic digestion of food and can be detoxified by several enzymes. However, H2S is considered more toxic than hydrogen cyanide to the neural and circulating system. This fact suggests that the free TM itself is unsuitable for the i.v. delivery as a drug. Furthermore, the manufacture, storage and use of this compound as a drug will be a general safety concern. On the other hand, TM is an oral drug that suffers from its low bioavailability, slow action and potential toxicity. As the result, it requires continued use of the drug for six weeks in order to reach the targeted 20% of baseline copper levels. In a recent clinical trial involving 100 patients, some patients did not benefit from TM treatment because the action of the drug is too slow to bring the copper levels down.
All of these drugs are orally active and can reduce the intestinal absorption of copper, but as small molecules, none of them can penetrate cells to reduce the intracellular copper concentration. In summary, initial successes with the clinical trials of TM would have provided a strong impetus for researchers to seek out other copper chelating agents for a similar role in anti- angiogenic cancer treatment. We propose to solve the problem by developing nanoparticle-based copper chelating agents that can function at the cellular level.
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Small-molecule chelating agents have been widely used to remove heavy metal ions from the human body in a medical procedure commonly referred to as chelation therapy.31 The main disadvantage of using small-molecule-based drugs is that they suffer from less-selectivity towards a specific metal ion from the body.181 Further they are unable to cross the cell membrane in order to function as intracellular detoxifying agents, unless there is a receptor-targeting moiety conjugated to the drug.35 On the other hand, some chelators may mobilize the metal ions that are deposited in the body tissues and reroute them into circulation, thus increasing the risk of secondary exposure of such metal ions to the brain. In contrast, the use of solid-state compounds as ion-exchangers to selectively remove the target metal ions from the human body by the well- established thermodynamic and kinetic principles of ion-exchange has been largely unexplored.182
We used the Irving-Williams Series as a guide to design a novel nanoparticulate ion-exchanger that exhibits an extremely high selectivity towards copper and can penetrate the cell membrane to act as an intracellular copper detoxifying agent. Specifically, we noticed that the order of relative
2+ 2+ stability exhibited by the homologous divalent 3d metal complexes shows the trend Mn ˂Fe
˂Co2+ ˂Ni2+ ˂ Cu2+ ˃Zn2+ with copper and zinc complexes being the most and the second most stable, regardless of the nature of ligand.38 We therefore rationalized that if a highly stable solid- state zinc compound is used as an ion-exchanger, it would preferentially undergo ion-exchange with copper, thus making it possible to act as a detoxifying agent specifically for copper ions while maintaining homeostasis for other biologically essential metal ions such as Mg2+, Ca2+,
Mn2+, Fe2+ and Zn2+ in the body.
In this section we report the synthesis and characterization of nanoparticles (NPs) of zinc tetrathiomolybdate ZnMoS4. Importantly, we demonstrate that such novel NPs are water- dispersible, biocompatible and able to penetrate cells by receptor-independent endocytosis to selectively remove intracellular copper via ion-exchange rather than chelation. The reaction of
(NH4)2MoS4 in formamide (FM) with Zn(O2CCH3)2 in an aqueous solution containing 3- mercaptopropionic acid, NH4OH and polyvinylpyrrolidone (PVP) led to the formation of PVP-
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coated ZnMoS4 NPs. In a typical reaction, ZnMoS4 nanoparticles were synthesized according to the following procedure. 111 mg of Zn(O2CCH3)2 in 6 mL of deionized water was added to a mixture of 3-mercaptopropionic acid (25 mL) and 1N NH4OH (10 mL) solution dropwise. Next
130 mg of (NH4)2MoS4 in 20 mL of formamide (FM) solution was added into the above mixture under stirring. Finally, polyvinylpyrrolidone (PVP; 3 g, MW= 8000) was added as a surface- coating agent. The sample was dialyzed using regenerated cellulose tubular membrane (MWCO is 3500) against distilled water for two days. The solid product was collected by lyophilization.
As previously mentioned, PVP is a polymer commonly used as a surface-coating agent to enhance biocompatibility and water dispersibility in a variety of inorganic nanoparticle preparations.183 Transmission electron microscopic (TEM) studies revealed that the PVP-coated
ZnMoS4 NPs are near spherical in shape and have an average size of 16±2 nm, as determined by counting and averaging the size of 23 particles in this TEM picture frame (Figure 5.15). The energy dispersive X-ray spectroscopic (EDX) measurements showed distinctive signals of Zn,
Mo and S from several individual NPs randomly selected from the TEM sample (Figure 5.16).
Figure 5.15: TEM image of as-prepared ZnMoS4 NPs.
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Figure 5.16: EDX spectrum on a nanoparticle.
The Fourier transform infrared (FT-IR) spectra of PVP, nanoparticles and the bulk compound are presented in Figure 5.17. As can be seen from the figure, these NPs showed the presence of the surface-coated PVP. The bands at approximately 1660 cm-1, 1420 cm-1 and 1285 cm-1 are the characteristic IR bands of PVP. The X-ray powder diffraction (XRD) patterns of both the NPs and bulk materials of ZnMoS4 match those of the ternary solid-state phase of the same composition previously reported in the literature (Figure 5.18).184 Although the structure determination for this compound has not been carried out yet, it is tempting to conjecture that both the Mo and Zn centers have tetrahedral coordination while the S atoms are engaged in a double-bridging mode of bonding, which is reminiscent of the coordination environment in the
185 GeS2 structure.
The reaction between (NH4)2MoS4 and a copper(II) salt failed to give the anticipated
CuMoS4. Instead, a solid-state compound containing the copper(I) center was formed,
2+ 2- presumably via a redox reaction between the Cu ion and the MoS4 ligand. The direct reaction of (NH4)2MoS4 with a copper(I) salt gave the same product. The XRD pattern of this compound matches that of Cu2MoS4 whose crystal structure was determined from the X-ray powder data to 130
be a layered compound with both the Mo and Cu centers in tetrahedral coordination environment with each S atom coordinating to two Cu atoms and one Mo atom within a layer.173,174
1
0.5 ZnMoS4-Bulk PVP
ZnMoS4-NPs Transemittance (%) Transemittance
0 750 1750 2750 3750 Wavenumber (cm-1)
Figure 5.17: Overlay of the IR spectra of bulk (blue) and nanoparticles (green) of ZnMoS4 and
PVP (red) alone.
10 20 30 40 50 60 70 80 o 2 Theta/
Figure 5.18: PXRD pattern of PVP-coated ZnMoS4 NPs
PXRD pattern was collected by Nilantha P. Wickramaratne from Department of Chemistry, Kent State University
The X-ray diffraction (XRD) measurements were recorded for the samples using a
PANanalytical, Inc. X’Pert Pro (MPD) Multi-Purpose Diffractometer with Cu Kα radiation 131
(1.5406 A) at an operating voltage of 45 kV. The ZnMoS4 nanoparticles prepared at room temperature using the above described procedure gave a product with the elemental composition of Zn:Mo =1:1, but with poor crystallinity. In order to increase the crystallinity, the reaction mixture was charged into a Teflon-lined autoclave and heated at 120 ºC for 1 hour. After dialysis and lyapholization, the isolated ZnMoS4 nanoparticles were found to be crystalline although the size of nanoparticles had grown to larger than 100 nm. Similarly, the bulk ZnMoS4 material was prepared under solvothermal conditions at temperature of 150 ºC. In a typical reaction, 87 mg of
(NH4)2MoS4 and 85 mg of zinc acetate were reacted in 1 : 1 mixture of ethanol and formamide solution (2 mL) in a sealed Teflon-lined hydrothermal autoclave for 12 hrs. The final dark brown powder was separated by centrifugation with acetone and water mixture (1:1 volume ratio). The
X-ray diffraction (XRD) patterns of both the bulk ZnMoS4 (Figure 5.19) and nanoparticle samples showed three main diffraction lines at 28.70°, 47.65°, 56.51°, with d values of 3.10781,
184 1.90663, 1.62704 respectively, which matched the previously reported values for ZnMoS4.
300
200
100 Intensity(a.u)
0 10 30 50 70
2Theta /o
Figure 5.19: PXRD pattern of bulk ZnMoS4 material.
PXRD pattern was collected by Nilantha P. Wickramaratne from Department of Chemistry, Kent State University
ZnMoS4 and Cu2MoS4 are sparingly soluble salts and are in equilibrium with its constituent ions in solution. Hence the solubility equilibrium can be expressed as
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2+ 2- ZnMoS4(S) ↔ Zn (aq) + [MoS4] (aq)
+ 2- Cu2MoS4(s) ↔ 2Cu (aq) + [MoS4] (aq)
Solubility product constants, Ksp for each of the compounds, are expressed by the following equations.
2+ 2- Ksp = [Zn ] [MoS4 ]
+ 2 2- Ksp = [Cu ] [MoS4 ]
-11 2 -2 -19 3 -3 The Ksp values for ZnMoS4 and Cu2MoS4 are 2.7×10 mol L and 9.1×10 mol L , respectively, as determined through a static method in which the bulk materials of each compound were allowed to equilibrate with deionized water at room temperature, and the metal concentrations were then analyzed by chemical analysis. In brief, about 300 mg of each compound was weighed into separate 1 L beakers. 500 mL of deionized water was then added to each beaker, and the solutions were stirred at 25 oC for 48 hrs. The solutions were kept undisturbed for another 24 hrs to allow any suspended particles to settle. 25 mL aliquot from each supernatant solution was pipetted into 50 mL beakers and heated to dryness. Next 1 mL of concentrated nitric acid was added to each beaker and diluted the solutions by adding 2 mL of deionized water. The metal ion concentration was determined using AA for each of the solutions. We expected the difference in the equilibrium concentrations of [Zn2+]=5.218×10-6 M and [Cu+]=6.102×10-7 M to provide a
2+ + driving force for the ion-exchange between ZnMoS4 NPs and Cu /Cu ions to occur. The kinetics
+ of the ion-exchange of ZnMoS4 NPs with Cu ions in aqueos medium was determined only because the intracellular free copper is most likely to exist in this oxidation state.186,176
Since Cu(I) ions are prone to the disproportionation reaction in aqueous solution to form
Cu(II) ions and the Cu(0) metal, acetonitrile was used to stabilize this oxidation state in water by forming a Cu(I)-acetonitrile complex. The kinetics of copper removal was done by using 50 mL of 500 µM copper(I) solution. Furthermore, Cu(I) is more soluble in acetonitrile than in water.172
Therefore, copper(I) chloride was dissolved in 80% acetonitrile solution to prepare Cu(I) solution.
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100 mg of ZnMoS4 nanoparticles was placed in a dialysis bag followed by adding copper solution while stirring. Then aliquots of outside solution were taken out at different time intervals. The aliquots were then evaporated to dryness and re-dissolved in 2% HNO3 acid and diluted with deionized water before analyzing the copper content with AA. Kinetics of copper removal by
ZnMoS4 NPs is shown in Figure 5.20 and Table 13..
Figure 5.20: Kinetics of copper removal by ZnMoS4 NPs.
The kinetic data of the ion-exchange reaction can be fitted into two separate rate laws. A pseudo first-order up to the reaction time point of ~60 min with a rate constant of
-4 -1 k1=3.0×10 s with a half-life of t1/2 = 38.5 min (Figure 5.21). The second part can be fitted to the
-1 -1 -1 second order rate law with a rate constant of k2= 1.8 × 10 mM s (Figure 5.22). The shift to the slower and second-order reaction is presumably attributable the need for Cu+ ions to penetrate into the NPs to react with the inner Zn2+ ions after the surface layer is consumed.
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Table 13: The decrease of Cu concentrations vs. time in the ion-exchange reaction.
Time (min) [Cu] (µM)
0 505.4 5 366.8 10 361.6 15 341.1 20 330.8 40 228.1 60 212.7 120 151.0 240 115.1 350 104.8 480 84.3 600 84.3 720 79.1 900 74.0
6.4 6.2 6
5.8 ln[Cu] 5.6 5.4 5.2 0 10 20 30 40 50
Time (min)
Figure 5.21: Pseudo first order rate plot for the copper removal by ZnMoS4 NPs.
135
16000
12000
)
1 -
8000 1/[Cu](M 4000
0 0 20000 40000 60000 Time/s
Figure 5.22: Second order rate plot for the copper removal by ZnMoS4 NPs.
The copper removal capacity of the NPs in aqueous solution was determined. As previously mentioned, the capacity of a solid-state material to remove metal ions from a solution is expressed as the maximum amount of metal in milligrams that can be removed by one gram of the given material. The metal removal capacity (q, mg/g) at different time intervals can be calculated using the mass balance equation q = (Ci - C)/(V/W), where Ci (mg/L) is the initial metal concentration, C (mg/L) is the metal concentration remaining in solution after adsorption at given time t (min), V(L) is the volume of metal solution, and W(g) is the weight of NPs.187 The copper removal capacity for the PVP-coated ZnMoS4 NPs was found to be 130 mg/g (Figure
5.23), indicating that these NPs can effectively remove copper from the aqueous solution. On the other hand, good selectivity towards the intended metal ion is probably a more important criterion for evaluating the performance of copper detoxifying drugs because other endogenous metal ions, being both extracellular and intracellular, will compete for coordination sites in the same agent.
Such a competition not only lowers the efficiency, but also disturbs the homeostasis of other essential metals. The typical approach towards designing a selective therapeutic metal detoxifying ligand relies mostly on the hard-soft acid-base theory132 with other considerations such as the chelating effect and pro-ligand approaches.133 Thus far the design and synthesis of novel small-
136
molecule chelating agents with high metal binding selectivity has remained a challenge.121,34,34In contrast, our ZnMoS4 NPs should possess intrinsically high selectivity towards copper as dictated by the the Irving-Williams Series. To evaluate such selectivity, the 5 mL of NPs (10 mM) sealed in a dialysis bag was submerged in an aqueous solution containing Cu+, Fe2+, Mn2+, Ca2+ and K+ ions, each at the ~100-pm level for 48 hours. The solution concentration of each ion was analyzed before and after the competition reaction. An aliquot of solution was taken out and diluted with
2 % HNO3 acid and analyzed for each metal ion using AA. The results were normalized against the removal of the Cu+ ion from the solution, and expressed as the percent removal. As can be seen in Figure 5.24, the NPs showed excellent selectivity towards copper. Such a high selectivity is probably unachievable by use of small chelating agents.
160 140 120 100 80
60 Cu removal removal Cu 40
(mg of Cu per g of NPs) of g per Cu of (mg 20 0 0 200 400 600 800 Time (Min)
Figure 5.23: Copper removal capacity of ZnMoS4 NPs.
137
100
80
60
40
Metal removal(%) 20
0 Cu(II) K(I) Ca(II) Mn(II) Fe(II) Zn(II)
Figure 5.24: Selectivity of metal removal by ZnMoS4 NPs.
To visualize the cellar uptake of the PVP-coated ZnMoS4 NPs, a fluorophore was conjugated to the surface of NPs by the EDC coupling reaction to permit the optical detection of the fluorescently labeled NPs inside cells. Nanoparticles were resuspended in 2 mL of PVP solution (100 mg of PVP in 1 mL). 1.5 equivalents of ethylenediamine were then added to it under vigorous stirring. The resultant mixture was stirred for another 24 hrs and excess ethylenediamine was removed by dialysis against distilled water for two days. Then 5 mL of carboxyfluorescein dye (0.35 mg/mL) was reacted with 1.2 eq of EDC (≈1.5 mg) for 24 hrs. The ethylenediamine coated nanoparticles were then added to the 2 mL of above reaction mixture and stirred for Ca. 24 hrs. Finally the product was dialyzed to remove excess dye. The fluorescence spectra of the carboxyfluorescein dye and the nanoparticles after attaching the dye were obtained to confirm the conjugation of the dye to the nanoparticle surface. The fluorescence spectra shown in Figure 5.25 clearly show that the presence of dye on the surface of the nanoparticles. The fluorescent dye-labeled nanoparticles were then incubated with HepG2 cells to visualize the cellular uptake of these nanoparticles by confocal microscopy. Briefly, HepG2 cells were seeded in an 8 well chamber at a density of approximately 1.3×105 cells per well in complete medium in
138
the absence of antibiotics and incubated for 24 hrs at 37 oC. The cells were then incubated with dye-labeled nanoparticles for 3 hrs at 37 oC. The cells were washed with PBS three times and then imaged using a confocal microscope with 488 nm excitation wavelength.
Figure 5.25: Fluorescence spectra of carboxyfluorescein dye and dye labeled nanoparticles.
The fluorescent images of the live HepG2 cells treated with dye-labeled NPs showed strong fluorescent signals that are uniformly distributed in the entire cytoplasm without specific binding to any small organelles, while the untreated HepG2 cells gave almost no fluorescent signal (Figure 5.26). This observation seems to suggest that the cellular uptake of these NPs is via endocytosis. Cellular uptake and intracellular localization of nanoparticles have been studied by many research groups. Cells use endocytosis for uptake of nanoparticles and nonpolar molecules through hydrophobic cell membrane.
139
Figure 5.26: Confocal microscopic images of HepG2 cells: fluorescence image (upper left) and bright field image (upper right) of cells incubated with dye-conjugated NPs for 3 hours; florescence image (lower left) and bright field image (lower right) of the untreated cells.
Endocytosis and exocytosis of nanoparticles were studied using confocal microscopy.
Briefly, HepG2 cells were seeded in 8 well chambers at a density of approximately 1.4×105 cells per well in complete medium in the absence of antibiotics and incubated for 24 hrs at 37 oC. The cells were then incubated with nanoparticles for 2 hrs at 37 oC. The cells were washed with PBS three times and incubated with fresh medium (full medium). The cells in each chamber were washed again with PBS at different time intervals and imaged using a confocal microscope with
488 nm excitation wavelength.
140
1 hr 2 hr
7.5 hrs
5 hr 5 hr 7.5 hr
Figure 5.27: Confocal images showing endocytosis and exocytosis of nanoparticles.
In order to study the intracellular copper remoal by these NPs, we first induced an elevated copper level in HepG2 cells by incubating the cells with a medium supplemented with
CuSO4 to reach the saturation of copper uptake in these cells. The elevated copper level in HepG2 cells was induced by incubating them with the DMEM medium supplemented with 200 uM of copper(II) sulfate solution. Copper uptake kinetics by HepG2 cells were determined by measuring the copper levels of the cells at different time intervals. For that, the cells were washed three times with PBS and resuspended in serum free medium and counted using hemocytometer.
Atomic absorption measurements were used to determine the final copper concentration of the
HepG2 cells after incubation of different time periods with copper sulfate solution. The cells were 141
lysed by concentrated nitric acid and diluted with deionized water before taking the atomic absorption measurements. When the copper concentration becomes saturated, cells were washed three times with PBS and further incubated with the culture medium containing nanoparticles
(200 µg/ mL). After 4 hours of incubation with nanoparticles at 37 oC, cells were washed three times with PBS to remove the uninternalized nanoparticles and further incubated with the fresh culture medium. To quantify the concentration of copper in cells in each cell culture flask (T25 flask), the cells were washed three times with PBS at different time intervals and lysed using concentrated nitric acid. After dilution with deionized water, the concentration of each lysate was determined by AA. The copper-saturated HepG2 cells were then incubated with ZnMoS4 NPs at
37 oC for different times. As can be seen in Figure 5.27, such copper-saturated cells treated with
NPs showed a significant decrease in the cellular copper level after 1.5 hour of incubation. An overall substantial deacrese in copper concentration lasted as long as 6 hours of incubation.
Longer incubation than 6 hours did not cause the intracellualr copper level to go down any further, presumably because the remaining ZnMoS4 NPs along with the absorbed copper ions had exited the cells by now via exocytosis.
Figure 5.27: Kinetics of copper removal from HepG2 cells.
142
In an effort to study the antiangiogenic effect of nanoparticles of ZnMoS4, we performed in vitro tube formation assay using HUVEC (human umbilical vein endothelial cells) cell line.
The principle cell line involed in angiogenesis is endotheliel cells, which line the interior surface of all blood vessels. As previously mentioned, angiogenesis involves several steps. The first step is the leakage of endothelial cells from their stable location by breaking through the basement membrane, which is a thin sheet of fibers that lie beneath the epithelial tissue. Basement membranes not only support cells and cell layers, but also play an essential role in tissue organization that affects cell adhesion, migration, proliferation and differentiation (see scheme
15). After breaking basement membrane, endothelial cells migrate toward an angiogenic stimulus, which is released by tumor cells. Simultaneously endothelial cells proliferate to supply enough number of cells for making a new vessel. Subsequently, endothelial cells form a three dimentionally tubular structute. In vitro tube formation assay test the ability of vascular endothelial cells to form capillary-like structures when cultured on a supportive matrix (basement membrane), and scientists use this assay to identify proangiogenic factors and inhibitors.
We used Trevigen’s In Vitro angiogenesis assay kit, to perform the tube formation assay.
Cultrex® Reduced Growth Factor (RGF) Basement Membrane Extract (BME) is used as a soluble form of basement membrane, which gels at room temperature to form a reconstituted protein matrix. Sulforaphane [1-isothiocyanato-(4R)-methylsulfinyl)-butane], is used as a control for inhibition of in vitro endothelial cell tube formation on Cultrex® RGF BME. FGF-2 growth factor serves as a positive control. Calcein AM is used to stain cells for visualizing the cells under confocal microscope. Calcein AM is a non-fluorescent, hydrophobic compound that easily permeates intact, live cells. The hydrolysis of Calcein AM by intracellular esterases produces calcein, a hydrophilic and strongly fluorescent compound that is well-retained in the cell cytoplasm. Therefore cell viability can be measured simultaneously. Tube formation is quantified by measuring the length or number of branch points of these capillary-like structures in two- dimensional microscope images of the culture dish.
143
Basement membrane
Epithelial cells
Capillary
Scheme 15: Basement membrane.
Tube formation assay was done according to the following procedure. 50 µL aliquots of
Cultrex® Reduced Growth Factor (RGF) Basement Membrane Extract (BME) were added to wells in a 96 well plate and allowed to gel at 37 °C and 5% CO2 for 30 min. HUVEC cells were seeded on to the gel at 1.5 ×104 per well in EGM medium containing either FGF-2 growth factor
(50 ng/ml), 50 µM ZnMoS4 NPs or 5µM angiogenesis inhibitor sulforaphane and incubated at
37 °C and 5% CO2 for 6 h. After 6 hrs of incubation medium was removed and each well was washed with 150 µL of PBS. Then 100 µL of 2 μM Calcein AM solution in PBS was added to each well and incubate for 30 minutes at 37 oC. Finally Calcein AM solution was carefully aspirated and 100 µL of PBS was added before taking images. Endothelial tube formation was visualized using confocal microscope.
Figure 5.29 shows the confocal images obtained for tube formation assay. As can be seen in the figure tube-like structures were well established after incubation for 6 h in the well treated with the positive control, FGF-2. Treatment with HUVEC with either angiogenesis inhibitor sulforaphane or 50 µM ZnMoS4 NPs inhibited the formation of tubular structures. 144
FGF induced tube formation 2
ZnMoS NPs inhibited tube 4 formation
Sulforaphane inhibited
Figure 5.29: Panel 1 bright-field (left) and fluorescent (right) images of calcein-stained HuVEC cells treated with FGF-2(50ng/ml) in basal media showing the tube formation. Panels 2 & 3
FGF-2 induced HuVEC cells treated 4 hrs with 5uM angiogenesis inhibitor sulforaphane or 50 uM ZnMoS4 NPs, respectively.
Confocal fluorescent images were obtained by Haiwa Wu from the Department of Biological Sciences, Kent State University
In order to determine whether ZnMoS4 NPs could inhibit the tube formation in a concentration-dependent manner, HUVEC cells were exposed to varing concentration of nanoparticles. Briefly, 50 µL aliquots of BME were added to wells in a 96 well plate and allowed to gel at 37 °C and 5% CO2 for 30 min. HUVEC cells were seeded on to the gel at 1.5 ×104 per well in EGM medium containing varying concentration of ZnMoS4 NPs. As shown in Figure 5.30,
ZnMoS4 significantly inhibited the tube formation in a concentration-dependent manner. 20 µM
ZnMoS4 gave 50% reduction in tube formation (middle panel) compared to positive control without ZnMoS4 (left panel) and 50 µM ZnMoS4 completely inhibited tube formation (right panel).
145
FGF w/o ZnMoS NPs 20 µM ZnMoS NPs 50 µM ZnMoS NPs 2 4 4 4
Figure 5.30: 20 µM ZnMoS4 gave 50% reduction in tube formation (middle panel) compared to positive control without ZnMoS4 (left panel) and 50 µM ZnMoS4 completely inhibited tube formation (right panel).
Confocal images were obtained by Haiwa Wu from the Department of Biological Sciences, Kent State University
To confirm that ZnMoS4 NPs has an effect on the inhibition of the tube formation while maintaining the cell survival, a cell viability assay was performed. HUVEC cells were treated with the varying concentrations of NPs and the viability of the cells was tested. Cytotoxicity studies were performed using the trypan blue exclusion viability assay. HepG2 cells were seeded in a 96-well plate at a density of 2 × 104 cells per well with the DMEM low glucose medium and incubated for 5 hrs at 37 °C in an atmosphere of 5% CO2 and 95% air to allow cells to attach to the surface. Cells in each well were then treated with 100 μL of fresh medium containing varying concentrations of the nanoparticles and then incubated for 24 hrs or 48 hrs. Control wells contained the same medium without nanoparticles. The cells were then trypsinized and re- suspended in 100 μL medium without serum, then added to 100 μL of 0.4% trypan blue solution.
Viable and non-viable cells were counted using a hemocytometer. Each concentration was tested in replicates of three. The assay results were presented as percent viable cells. After 24-h incubation with a concentration of 50 µM, the cell viability was found to be ca. 95±4% while the cell viability became 81±8% after 48-h incubation with the same concentration (Figure 5.31).
146
24 Hrs 48 Hrs 100
80
60
40
20
Cellviability (%) 0 0 10 20 30 50 ZnMoS4 NPs concentration/µM
Figure 5.31: Viability curve of HepG2 cells incubated with ZnMoS4 NPs for 24 hours and 48 hours.
Although the NPs reduce copper levels, none of the tested doses showed significant toxicity for tumor cell lines, as measured by the MTT assay. We have developed novel copper depleting agents that can penetrate the endothelial cell membrane, exhibit no cytotoxicity and inhibit tumor angiogenesis. We expect this approach to significantly impact cancer therapy in the future.
147
Chapter 6: Conclusions
Recently, much attention has been given to the development of biomedical applications of nanotechnology which known as nanomedicine. Nanomedicine utilizes nanoparticles such as liposomes, polymeric nanoparticles, carbon nanotubes, nanowires, quantum dots and inorganic nanoparticles to diagnose and treat various diseases. This dissertation research has focused on the development of inorganic nanoparticles for diagnostic and therapeutic applications.
Targeted nanoparticulate magnetic resonance imaging (MRI) contrast agents discussed in chapter 3 showed high MRI signal and will be invaluable for future tissue specific imaging and investigation of molecular and cellular events. Specifically, this study highlights that simple one- step method for preparing extremely stable and biocompatible NPs of the gadolinium ferrocyanide coordination polymer. These NPs exhibit no cytotoxicity. Furthermore, we have demonstrated that such NPs possess extremely high T1-weighted relaxivity, suggesting the potential of this coordination-polymer structural platform in the development of new-generation
T1-weighted cellular MR probes for biological receptors or markers within the cell to study molecular events as well as for in vivo MR imaging in biomedical research and clinical applications.
In chapter 4 synthesis and characterization of nanoparticulate contrast agent for X-ray computed tomography is reported. These nanoparticles offer a much higher contrast efficacy compared to clinical iodinated agents at 120 kVp. Together with long circulation time and low toxicity, these nanoparticles can act as a high-performance CT contrast agent for in vivo applications.
148
The remaining chapters of this dissertation concern the synthesis of therapeutic nanoparticles. The first part of the chapter 5 describes the synthesis and characterization of
Au@ZnMoS4 nanoparticles as copper depleting agent for the treatment of Wilson’s disease. Their utility as copper depleting agent has been clearly demonstrated in vitro.
In the second part of Chapter 5, the synthesis and characterization of therapeutic nanoparticles for angiogenesis inhibition is reported. In this dissertation project, anti-angiogenic function and mechanism of ZnMoS4 NPs in primary HUVECs was investigated in detail.
149
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