Citation
Lanthanide Based Nanoparticles as Multifunctional Agents for Magnetic Resonance Imaging and in Vitro Cancer Therapy

Material Information

Title:
Lanthanide Based Nanoparticles as Multifunctional Agents for Magnetic Resonance Imaging and in Vitro Cancer Therapy
Creator:
Li, Yichen
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
Publication Date:
Language:
english
Physical Description:
1 online resource (175 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Chemistry
Committee Chair:
TALHAM,DANIEL R
Committee Co-Chair:
BUTCHER,REBECCA ANN
Committee Members:
BRUNER,STEVEN DOUGLAS
SMITH,BEN W
WALTER,GLENN A
Graduation Date:
5/2/2015

Subjects

Subjects / Keywords:
Eggshells ( jstor )
Fluorescence ( jstor )
Gadolinium ( jstor )
Imaging ( jstor )
Ions ( jstor )
Lasers ( jstor )
Magnetic resonance imaging ( jstor )
Molecules ( jstor )
Nanoparticles ( jstor )
Polymers ( jstor )
Chemistry -- Dissertations, Academic -- UF
cancer -- gadolinium -- mri -- nanoparticle -- therapy
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Chemistry thesis, Ph.D.

Notes

Abstract:
Magnetic resonance imaging has been widely used in modern biomedicine with the ability to provide anatomical details of internal structures and to detect lesions based on the relaxation time differences between tissues. When there are only small differences between the normal tissues and lesions, MRI contrast agents are used to improve the imaging accuracy and sensitivity. A great deal of efforts has been put into the development of Gd3+ containing nanoparticles as MRI contrast agents. Nanoparticle based MRI contrast agents show a wide range of relaxivity behavior. To begin to understand some of the factors contributing to the relaxivity, different sizes of GdPO4 nanoparticles have been synthesized to investigate the size influence on relaxivity. The results show an increasing relaxivity with decrease of particle size. A correlation between the surface to volume ratio and relaxivity was demonstrated. Further control over particle size and MRI response was achieved using PAMAM and its derivatives. With PAMAM and HyPAM polymers as surface coating molecules, ultrasmall GdPO4 nanoparticles were obtained. A size-dependent relaxivity study gives rise to an optimum particles size, which shows the highest relaxivity for 23 nm particles, with lower values observed for both smaller and larger particles. This behavior is due to the contributing factors that have opposite particle size correlation. One of the factors is surface to volume ratio, which accounts for the most often observed relaxivity increase for smaller particles. On ther other hand, for particles in a certain range of sizes, relaxivity increases for larger particles as tumbling times get long as the hydrodynamic volume of the particle increases. These two opposite contributing factors are expected to give rise to this optimum particle size. Additionally, Eu3+ ions have been doped into the gadolinium phosphate nanoparticles, enabling the nanoparticles to be used for dual-imaging, including MRI and fluorescent imaging. Using a coordination polymer as a host for Gd3+ ions is also reported. Gradient gadolinium ironhexacyanoferrate nanoparticles are synthesized, in which gadolinium ions are introduced and gradiently distributed, presenting less in the particle center and more toward the surface. This gradient strategy can be used for the improvement of particle-based MRI contrast agents. In addition, the Prussian blue host is demonstrated to be an efficient photothermal therapy agent for in vitro cancer cells treatment. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2015.
Local:
Adviser: TALHAM,DANIEL R.
Local:
Co-adviser: BUTCHER,REBECCA ANN.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2016-05-31
Statement of Responsibility:
by Yichen Li.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Embargo Date:
5/31/2016
Classification:
LD1780 2015 ( lcc )

Downloads

This item has the following downloads:


Full Text

PAGE 1

LANTHANIDE BASED NANOPARTICLES AS MULTIFUNCTIONAL AGENTS FOR MAGNETIC RESONAN CE IMAGING AND IN VITRO CANCER THERAPY By YICHEN LI A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULF ILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2015

PAGE 2

© 2015 Yichen Li

PAGE 3

To my family

PAGE 4

4 ACKNOWLEDGMENTS I would like to show my deepest gratitude to my supervisor, Dr. Daniel R. Talham, for all his guidance, support, and help, throughout my graduate study . Without his enlightening instruction s , impressive kindness , and patience, I could not have completed my thesis. Each time when I need help, he is always there to support. He not only gives me research direction s , but also trains me as an independent researcher. I would also like to thank all the help and advice provided by my committee members : Dr. Ben W. Smith, Dr. Rebecca A . Butcher, Dr. Steven D. Bruner, and Dr. Gle n n A . Walter. Thank them for agreeing to be my committee members. And I am very grateful for their time spent on my oral qualification exam and dissertation, from which I have learned a lot towards my research . I sincerely acknowledge our collaborators including former group members: Tao Chen and Liping Qiu, current group members: Cuichen Wu, Liqin Zhang, Sena Cansiz, and Carole Champanhac. Thanks for their kindness to let me use all the ins truments from their group. T hey a re all nice, kind, and willing to provide help. I especially would like to thank Tao Chen, who has helped me a lot wi th the experiments and taught me experimental skills. My appreciation is also expressed to Dr. Rebecca A. Butcher and Xinxing Zhang for t he ir generousness, time , and efforts with the experiment with C elegans . Xin xing Zhang is not only a good researcher, but also a good friend and roommate. I would like to thank Dr. Glenn A . Walter and Dr. Parvesh Sharma from Department of Physiology and Func tional Genomics for their help with paramagnetic chemical exchange saturation transfer experiment . I have learned a lot from the discussions with Dr. Parvesh Sharma on experiments.

PAGE 5

5 I would like to say thank you to our collaborators at University of Toulous e, including Dr. Christophe Mingotaud, Dr. Jean Daniel Marty , Camille Frangville, Maylis Gallois, and Hanh Hong Nguyen. I appreciate all the efforts they have put into our collaboration. As members of a world leading polymer group, they have provided profe ssional support in our collaborations with their expertise in the area of polymer science . In addition, t hey gave me tremendous help during my visit to Toulou se in both 2013 and 2014. I am always grateful for all the people from IMRCP lab for their accommo dation and making me feel very welcomed . I would like to thank past and current Talham group members, including former group members Matthieu Dumont, Tyler Russell, Carissa Li, Emily Pollard, Hao Liu, Matthew Andrus, Allison Garnsey, Olivia Risset, Fatimah Al Marzooq, Katherine Somodi, and Vitor Martins, and current group members: Caue Ferreira, Akhil Ahir, Divya Rajan, Corey Gros, Caro l yn Averback, Ashley Felts, Jiamin Liang, James Sternberg, Steve n Anthony , John Cain , EJ Munoz. They are always very suppor tive and provide me a very comfortable working environment. I e specially want to thank Matthieu Dumont, who has taught me a lot on particle synthesis and characterization when I initially joined the group. I also would like to thank Tyler Russell for the h elp with particle synthesis. In addition, I want to express my appreciation for the TEM support fro m Caue Ferreira and Carissa Li. I also want to acknowledge Dr. Huadong Zeng for the MR imaging technique support from Advanced Magnetic Resonance Imaging and Spectroscopy program in McKnight Brain Institute, as well as Nicholas Rudawski and Kerry Siebein from Major Analytical & Particle Analysis Instrumentation Centers for the electron microscope

PAGE 6

6 support. I would like to express my respectful grat itude to Dr. Ben W. Smith and M s. Lori Clark from the graduate office for their help. In addition, thank my teaching supervisor , Dr. James C. Horvath, for the nice teaching experience s . Finally, I am appreciative of my parents and wife , Carissa, for always being there when I need them the most. It is because of them that I am able to complete my degree. They have taught me so many things in my life, also en couraged me when I encounter difficulties.

PAGE 7

7 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ .......... 10 LIST OF FIGURES ................................ ................................ ................................ ........ 11 LIST OF ABBREVIATIONS ................................ ................................ ........................... 16 ABSTRACT ................................ ................................ ................................ ................... 20 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 22 Biomedical Applications of Nanoparticles ................................ ............................... 22 MRI Con trast Agent ................................ ................................ ................................ 26 Study Overview ................................ ................................ ................................ ....... 30 2 EXPERIMENTAL METHODS AND TECHNIQUES ................................ ................ 41 Transmission Electron Microscopy ................................ ................................ ......... 41 Inductively Coupled Plasma Atomic Emission Spectroscopy ................................ .. 42 Ultraviolet Visible Spectros copy ................................ ................................ ............. 42 Fourier Transform Infrared Spectroscopy ................................ ............................... 43 Combustion Analysis ................................ ................................ .............................. 43 Thermogravimetric Analysis ................................ ................................ .................... 44 Dynamic Light Scattering ................................ ................................ ........................ 44 Energy Dispersive X ray Spectroscopy ................................ ................................ .. 45 X ray Powder Diffraction ................................ ................................ ......................... 46 Selected Area Electron Diffraction ................................ ................................ .......... 47 Fluorescence Spectroscopy ................................ ................................ .................... 48 Confocal Laser Scanning Microscopy ................................ ................................ ..... 49 Cell Culture ................................ ................................ ................................ ............. 50 Cytotoxici ty Assay ................................ ................................ ................................ ... 51 Relaxivity Measurement ................................ ................................ .......................... 51 MRI System ................................ ................................ ................................ ............ 53 3 SIZE DEPENDEN T MRI RELAXIVITY AND DUAL IMAGING WITH Eu 0.2 Gd 0.8 PO 4 ·H 2 O NANOPARTICLES ................................ ................................ .. 62 Preface ................................ ................................ ................................ ................... 62 Introduction ................................ ................................ ................................ ............. 62 Materials and Methods ................................ ................................ ............................ 6 4 Synthesis of Eu 0.2 Gd 0.8 PO 4 ·H 2 O Nanoparticles ................................ ................ 64

PAGE 8

8 Sample 1 ................................ ................................ ................................ .... 64 Sample 2 ................................ ................................ ................................ .... 65 Sample 3 ................................ ................................ ................................ .... 66 In Vitro and I n V ivo Fluorescent Imaging ................................ .......................... 66 Results and Discussion ................................ ................................ ........................... 67 Particle Characterization ................................ ................................ .................. 67 Size Dependent MRI Rel axivity ................................ ................................ ........ 68 Fluorescence Imaging ................................ ................................ ...................... 71 Cytotoxicity Measurements ................................ ................................ .............. 72 Con clusions ................................ ................................ ................................ ............ 73 4 POLYAMIDOAMINE AND ITS DERIVATIVES MEDIATED SIZE CONTROLLED SYNTHESIS OF GADOLINIUM PHOSPHATE NANOPARTICLES: COLLOIDAL PROPERTIES AND MRI RELAXIVITY STUDY ................................ ...................... 83 Preface ................................ ................................ ................................ ................... 83 Introduction ................................ ................................ ................................ ............. 83 Materials and Methods ................................ ................................ ............................ 84 Materials ................................ ................................ ................................ ........... 84 Polymer Synthesis ................................ ................................ ............................ 84 Preparation of hyperbranched polyamidoamine core ................................ . 84 Double and single shell synthesis ................................ .............................. 85 Grafting of PEG 750 C 17 COOH or PEG 750 COOH onto the HyPAM core .... 85 Gd 3+ /HyPAM Interactions ................................ ................................ ................. 86 GdPO 4 /Polymer Nanowire Synthesis and Analysis ................................ .......... 86 NaCl Stability Study ................................ ................................ .......................... 87 Drug Loading Capacity ................................ ................................ ..................... 87 Results and Discussion ................................ ................................ ........................... 88 Particle Characterization ................................ ................................ .................. 88 Size Dependent Relaxivity Study ................................ ................................ ..... 89 Core Shell HyPAM Polymer ................................ ................................ ............. 90 Co nclusions ................................ ................................ ................................ ............ 92 5 ONE STEP SYNTHESIS OF GRADIENT GADOLINIUM IRONHEXACYANOFERRATE NANOPARTICLES: A NEW PARTICLE DESIGN EASILY COMBINING MRI CONTRAST AND PHOTOTHERMAL THERAPY ...... 102 Preface ................................ ................................ ................................ ................. 102 Introduction ................................ ................................ ................................ ........... 102 Materials and Methods ................................ ................................ .......................... 104 Materials ................................ ................................ ................................ ......... 104 Concentration Gradient K 0.3 Gd 0. 2 Fe[Fe(CN) 6 ] 4.9 H 2 O, g Gd PB, Synthesis .. 105 Measureme nt of P hotothermal P erformance ................................ .................. 105 Photothermal Ablation of Cancer Cells ................................ ........................... 106 Cell Viability Assay with and without Laser Treatment ................................ ... 107 Gd 3+ Release from g Gd PB Nanoparticles ................................ .................... 107 Results and Discussion ................................ ................................ ......................... 107

PAGE 9

9 Particle Synthesis and Characterization ................................ ......................... 107 MRI Relaxivity Study ................................ ................................ ...................... 109 Photothermal Therapy ................................ ................................ .................... 110 Conclusions ................................ ................................ ................................ .......... 112 6 CONCLUSIONS ................................ ................................ ................................ ... 126 APPENDIX A HIGHLY POROUS NANOPARTICLES FOR MRI CONTRAST ENHA NCEMENT AND ANTICANCER DRUG DELIVERY ................................ ................................ 130 Preface ................................ ................................ ................................ ................. 130 Introduction ................................ ................................ ................................ ........... 130 Materials and Methods ................................ ................................ .......................... 133 Materials ................................ ................................ ................................ ......... 133 HPGd Nanoparticle Synthesis ................................ ................................ ........ 133 Anticancer Drug Loading ................................ ................................ ................ 133 In Vitro Drug Delivery ................................ ................................ ..................... 134 Cell Viability Assay with and without Drug Loading HPGd Nanoparticl es ...... 134 Results and Discussion ................................ ................................ ......................... 134 Particle Synthesis and Characterization ................................ ......................... 134 Relaxivity Measurements and M R Imaging ................................ .................... 137 In Vitro Drug Delivery ................................ ................................ ..................... 138 Cytotoxicity Assay ................................ ................................ .......................... 138 Conclusions ................................ ................................ ................................ .......... 139 B COPYRIGHT CLEARANCE FORMS ................................ ................................ .... 153 LIST OF REFERENCES ................................ ................................ ............................. 166 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 175

PAGE 10

10 LIST OF TABLES Table page 1 1 Some nanoparticle compositions and biomedical applica tions. .......................... 40 3 1 Size dependent relaxivity data for samples 1 3 . ................................ ................ 81 3 2 Chemical and physical properties of GdPO 4 based contrast agen ts. ................. 82 4 1 Comparison between the experimentally obtained diffraction spacing and the values reported in JCPDS no. 39 232. ................................ ............................. 101 4 2 Comparison between the experimentally obtained diffraction spacing and the values reported in JCPDS no. 21 337. ................................ ............................. 101 4 3 Relaxivity comparison of GdPO 4 coated with HyPAM and functionalized HyPA M polymers. ................................ ................................ ............................. 101 5 1 ICP results of the g Gd PB and pure PB. ................................ ......................... 125

PAGE 11

11 LIST OF FIGURES Figure page 1 1 Nanoparticles synthesized with different structures. ................................ ........... 33 1 2 Typical sized CQDs optical images illuminated under white (left; daylight lamp) and UV light (right; 365 nm). ................................ ................................ ..... 33 1 3 QD resistance to photobleaching and multicolour labelling. ............................... 34 1 4 Room temperature upconversion emission spectra of NaYF 4 :Yb/Er , NaYF 4 :Yb/Tm, and NaYF 4 :Yb/Tm/Er particles in ethanol solutions. ................... 35 1 5 Whole animal imaging of a BALB/c mouse injected via tail vein with the HA (NaYbF 4 :0.5% Tm 3+ )/CaF 2 core/shell nanoparticles . ........................... 36 1 6 Breast cancer cells were selectively enhanced in T 1 weighted MRI by the Herceptin functionalized MnO nan oparticles. ................................ ..................... 36 1 7 In vivo CT coronal view images of a rat after intravenous injection of 1 mL PEG 1 ) solution at timed intervals. ................................ .. 37 1 8 Nanoparticles accumulate in tumor tissue due to the EPR effect. ...................... 37 1 9 Proposed mechanism for targeted cancer chemotherapy. ................................ . 38 1 10 After injection, these nanoparticles accumulate into targeted tumor. By absorbing NIR radiation, nanoparticles cause photothermal ablation of tumor. .. 38 1 11 MRI ima ges showing 3 orientations and the 3D view of a brain tumor . .............. 39 1 12 Structure of gadolinium chelate based T 1 MRI contrast agents. ......................... 39 2 1 Illustration of a transmission electron microscope. ................................ ............. 54 2 2 Scheme representing the essential components of an inductively coupled plasma atomic emission spectrometer. ................................ .............................. 55 2 3 Schematic illustration of the ultraviolet visible spectrometer. ............................. 55 2 4 Scheme representation of the principal parts of a Fourier transform in frared spectrometer. ................................ ................................ ................................ ...... 56 2 5 Block diagram illustrating the essential components of a CHN elemental analyzer. ................................ ................................ ................................ ............. 56 2 6 Schematic diag ram of thermogravimetric analysis technique. ............................ 57

PAGE 12

12 2 7 Block diagram illustrating the DLS instrument. ................................ ................... 57 2 8 Schematic diagram of ene rgy dispersive X ray spectroscopy. ........................... 58 2 9 Schematic illustration of the X ray diffraction with the sample. ........................... 58 2 10 Scheme illustra ting X ray powder diffractometer. It consists of X ray source, sample holder, and a detector to detect the diffracted X rays. ............................ 59 2 11 Block diagram illustrating the essential components of a fl uorescence spectrometer. ................................ ................................ ................................ ...... 59 2 12 Schematic drawing of the main parts of a confocal laser scanning microscope and the light path. ................................ ................................ ............ 60 2 13 Schematic diagram of a MRI scanner. ................................ ................................ 61 3 1 TEM images of PMIDA modified Eu 0.2 Gd 0.8 PO 4 ·H 2 O, samples 1 3 . ................... 74 3 2 XRD patterns of Eu 0.2 Gd 0.8 PO 4 ·H 2 O samples 1 3 and EDS maps across a Eu 0.2 Gd 0.8 PO 4 ·H 2 O nanoparticle. ................................ ................................ ........ 75 3 3 F rom left to right: Eu 0.2 Gd 0.8 PO 4 ·H 2 O nanoparticles; Eu 0.2 Gd 0.8 PO 4 ·H 2 O nanoparticles after modific ation with PMIDA only; Eu 0.2 Gd 0.8 PO 4 ·H 2 O nanoparticles after modification with both Gd 3+ and PMIDA. .............................. 76 3 4 FT IR spectra of Eu 0.2 Gd 0.8 PO 4 ·H 2 O nanoparticles before (red) and after (blue) modific ation with PMIDA. ................................ ................................ .......... 76 3 5 Plot of the proton relaxation rate (1/T 1 ) of water suspensions of Eu 0.2 Gd 0.8 PO 4 ·H 2 O nanoparticles and the corresponding relaxivites. ................. 77 3 6 Molar relaxivity, r 1 , as a function of the surface to volume ratio and r elaxation enhancement per particle as a function of nanoparticle size. ............................. 78 3 7 T 1 weigh ted MR images (4.7 T) of Eu 0.2 Gd 0.8 PO 4 ·H 2 O particles from sample 1 at various Gd 3+ concentrations. ................................ ................................ ....... 78 3 8 Emission spectra of Eu 0.2 Gd 0.8 PO 4 ·H 2 O nanoparticles; The inset shows an aqueous sample solution before and after irradiation with a UV lamp. ............... 79 3 9 Confocal laser scanning microscopy images of Hela cells incubated with and without sample 1 nanoparticles. ................................ ................................ ......... 80 3 10 Confocal laser scanning microscopy images of C. elegans fed with and without nanoparticles. ................................ ................................ ......................... 80 3 11 In vitro cell viability of Hela cells and A549 cells incubated with Eu 0.2 Gd 0.8 PO 4 ·H 2 O sample 1 at different concentrations ................................ .... 81

PAGE 13

13 4 1 TEM pictures of GdPO 4 /HyPAM nanoaprticles and s elected area electron diffraction pattern of the GdPO 4 /HyPAM na noaprticles. ................................ ..... 93 4 2 JCPDS file of GdPO 4 ·H 2 O (JCPDS no. 39 232) and JCPDS file of GdPO 4 ·1.5H 2 O (JCPDS no. 21 337) ................................ ................................ .. 94 4 3 TEM images of GdPO 4 nanoparticles synthesized with increasing amounts of PAMAM. ................................ ................................ ................................ ............. 9 5 4 4 TEM images of GdPO 4 nanoparticles synthesized with increasing amounts of HyPAM. ................................ ................................ ................................ .............. 95 4 5 Average lengths of GdPO 4 nanoparticles, determined from TEM analysis, versus concentration of HyPAM, HyPAM PEG, and HyPAM C 18 PEG. ............. 96 4 6 1 H NMR study for [HyP AM]=1 × 10 4 M and increasing Gd 3+ concentration in D 2 O. ................................ ................................ ................................ .................... 97 4 7 GdPO 4 /HyPAM size dependence on r 1 and r 2 relaxivities (1.41 T). .................... 98 4 8 TEM pictures of GdPO 4 nanowires synthesized with incr easing amount of HyPAM PEG and HyPAM C 18 PEG. ................................ ................................ .. 99 4 9 DLS measurements of the GdPO 4 /hyperbranched polymers upon NaCl addition. ................................ ................................ ................................ ............ 100 4 10 Normalized emission fluorescence spectra of Nile Red added into H 2 O and particle dispersions . ................................ ................................ .......................... 100 5 1 Room temperature FT IR spectra of PB, g Gd PB and a GdFe PBA particle sample. ................................ ................................ ................................ ............. 113 5 2 The TGA curve of a g Gd PB particle sample. ................................ ................. 113 5 3 Scheme of the gradient K 0.3 Gd 0.2 Fe[Fe(CN) 6 ] 4.9H 2 O, g Gd PB, nanoparticle and the cubic unit cell of Prussian blue. ................................ ........................... 114 5 4 A TEM image of g Gd PB particles, scale bar 100 nm and particle size histograms of g Gd PB , showing an average size of 58 ± 9 nm. ..................... 115 5 5 Scheme showing that EDS spot scan analyses at three different regions of a g Gd PB particle. ................................ ................................ ............................. 116 5 6 TEM image and EDS spot scan analyses determined Gd:Fe ratios for spectra taken at 3 points from the core to the edge of the particle. .................. 117 5 7 Room temperature powder X ray diff raction patterns of GdFe PBA, g Gd PB , and KFe[Fe(CN) 6 ] 4.8H 2 O . ................................ ................................ ............... 118

PAGE 14

14 5 8 Room temperature far IR spectra of PB, g Gd PB , and GdFe PBA particle sample. ................................ ................................ ................................ ............. 119 5 9 g Gd PB suspension (Gd 3+ 8 × 10 5 M) dialyzed against nanopure water and different competing ions, including Zn 2+ , Cu 2+ , Ca 2+ , K + , and Na + . ................... 119 5 10 TEM image of attem pted core shell, GdFe PBA , and PB. ................................ 120 5 11 EDS map of attempted core shell , Fe map , and Gd map only. ........................ 120 5 12 Plot of the proton relaxation rate (1/T 1,2 ) of water suspensions of g Gd PB nanoparticles and the corresponding relaxivities (r 1,2 ) (1.41 T) . ........................ 121 5 13 T 1 weighted MR images for a g Gd PB particle sample at differ ent gadolinium concentrations ranging up to 0.23 mM (4.7 T). ............................... 121 5 14 UV Vis spectrum of g Gd PB showing a broad absorption band around 700 nm. ................................ ................................ ................................ ................... 122 5 15 Temperature profile of g Gd PB under irradiation with an 808 nm laser and IR image. ................................ ................................ ................................ .......... 123 5 16 Confocal microscopic images of CEM cells stained with both FDA and PI followi ng different treatments . ................................ ................................ ........... 124 5 17 C ell viability of g Gd PB nanoparticles at different concentrations without, and with laser irradiation. ................................ ................................ .................. 125 A 1 Schematic representation of three different Gd CAs: Magnevist, gadofullerenes, and gadonanotubes entrapped within the porous SiMPs. ....... 140 A 2 Schematic representation of the gluc ose responsive MSN based delivery system for controlled release of bioactive G Ins and cAMP. ............................ 141 A 3 Scheme of engineered core corona porous iron carboxylates for drug delivery and imaging. ................................ ................................ ........................ 142 A 4 Schematic illustration of the procedure for the synthesis of uniform and water dispersible iron oxide na nocapsules and their TEM images . .................. 143 A 5 Illustration of the synthetic procedure of porous HPGd nanoparticles. ............. 143 A 6 TEM images of g Gd PB template and g Gd PB after NaOH treatment with different concentrations. ................................ ................................ ................... 144 A 7 XRD of g Gd PB nanoparticles after NaOH treatment with different concentrations. ................................ ................................ ................................ . 145

PAGE 15

15 A 8 Images of g Gd PB color change af ter NaOH treatment with different concentrations. ................................ ................................ ................................ . 146 A 9 FT IR analysis results of g Gd PB precursors after base treatment with different concentrations. ................................ ................................ ................... 147 A 10 S amples from left to right are original g Gd PB , HPGd without PEO PAA surface modification, and HPGd with surface modification . .............................. 148 A 11 TEM image of HPGd with out PEO PAA surface modification, and HPGd with surface modification. ................................ ................................ ......................... 148 A 12 FT IR analysis results of HPGd nanoparticles after surface modification with PEO PAA . ................................ ................................ ................................ ......... 149 A 13 Plot of the proton relaxation rate (1/T 1,2 ) of water suspensions of HPGd nanoparticles and the corresponding relaxivities (r 1,2 ) (1.41 T) . ........................ 149 A 14 T 1 weighted MR images (4.7 T) of HPGd at various Gd 3+ concentrations. ....... 150 A 15 Fluorimetric characterization of DOX loaded HPGd nanoparticles. .................. 150 A 16 Confocal imaging of drug loaded HPGd particle and HPGd only . .................... 150 A 17 Confocal images of drug loaded HPGd particle uptake by Hela cells: Hoechst nuclear stain observed in b lue color. Fluorescent Dox are observed in red. .... 151 A 18 Cell viability of HPGd nanoparticles at different concentrations with and with out anticancer drug DOX loading. ................................ .............................. 152

PAGE 16

16 LIST OF ABBREVIATIONS CA Contrast agent CCD Charge coupled device CHN Carbon, hydrogen, nitrogen CLSM Confocal laser scanning mircroscopy CN Cyanide DCC N , N ' dicyclohexylcarbodiimide DCU 1,3 dicyclohexylurea d Distance between diffra cting plane s DLS Dynamic light scattering DOX Doxorubicin DTPA Diethylene triamine pentaacetic acid EDTA Ethylenediaminetetraacetic acid EDX Energy dispersive X ray spectroscopy Em Emission EPR Enhanced permeability and retention Ex Excitation FDA Food and drug administration FDA Fluorescein d iacetate FID Free induction decay FOV Field of view FT IR Fourier transform infrared spectroscopy HER2 Human epidermal growth factor receptor 2 HRTEM High resolution transmission electron microscopy hMS C Human m esenchymal stem cells

PAGE 17

17 HUVEC Human umbilical vein endothelial cells HyPAM Hyperbranched polyamidoamine ICP AES Inductively coupled plasma atomic emission spectroscopy K Kelvin K sp Solubility product constant M Transition metal, Molar MOF Met al organic frameworks MR Magnetic resonance MRI Magnetic resonance imaging MTS 3 (4,5 dimethylthiazol 2 yl) 5 (3 carboxymethoxyphenyl) 2 (4 sulfophenyl) 2H tetrazolium Nile Red 9 diethylamino 5 benzo phenoxazinone NIR Near infrared NMR Nuclear magn etic resonance PA Photoacoustic PAA Poly(acrylic acid) PAMAM Polyamidoamine PB Prussian blue PBA Prussian blue analogue PBS Phosphate buffer saline PEG Poly(ethyleneglycol) PEO Poly(ethylene oxide) PEO PAA Poly(ethylene oxide b acrylic acid). PET Positron emission tomography PI Propidium iodide

PAGE 18

18 PMIDA N phosphonomethyl iminodiacetic acid PTT Photothermal therapy PTSA p toluenesulfonic acid PXRD Powder X ray diffraction QD Quantum dot r 1 Longitudinal relaxivity r 2 Transversal relaxivity R1 Longitudinal relaxation rate R2 Transversal relaxation rate RF Coils Radiofrequency coils SAED Selected area electron diffraction SEM Scanning electron microscopy SPECT Single photon emission computed tomography SPIO Superparamagnetic iron oxide STE M Scanning transmission electron microscopy t Time T Tesla or Temperature T 1 Longitudinal relaxation time T 2 Transversal relaxation time TEM Transmission electron microscopy TGA Thermogravimetric analysis TR Repetition time TE Echo time UV Ultravi olet UV Vis Ultraviolet visible

PAGE 19

19 V Volt XRD X ray diffraction Diffraction angle Wavelength Dielectric constant

PAGE 20

20 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of th e Requirements for the Degree of Doctor of Philosophy LANTHANIDE BASED NANOPARTICLES AS MULTIFUNCTIONAL AGENTS FOR MAGNETIC RESONANCE IMAGING AND IN VITRO CANCER THERAPY By Yichen Li May 2015 Chair: Daniel R. Talham Major: Chemistry Magnetic resonance imaging has been widely used in modern biomedicine with the ability to provide anatomical details of internal structures and to detect lesions based on the relaxation time difference s between tissues . When there are only small differences between the norma l tissues and lesions, MRI contrast agents are used to improve the imaging accuracy and sensitivity. A great deal of efforts has been put into the development of Gd 3+ containing nanoparticles as MRI contrast agent s . Nanop articl e based MRI contrast agents s how a wide range of relaxivity behavior . To begin to understand some of the factors contributing to the relaxivity, different sizes of GdPO 4 nanoparticles have been synthesized to investigate the size influence on relaxivity . The results show an increasing relaxivity with decrease of particle size. A correlation between the surface to volume ratio and relaxivity was demonstrated. Further control over particle size and MRI response was achieved using PAMAM and its derivatives. With PAMAM and HyPAM polymers a s surface coating molecules , u ltrasmall GdPO 4 nanoparticles were obtained. A size dependent rela xivity study gives rise to an optimum particles size, which shows the highest relaxivity for 23 ± 11 nm particles , with lower value s observed for both smaller a nd larger particles. This

PAGE 21

21 behavior is due to the contributing factors that have opposite particle size correlation. One of the factors is surface to volume ratio , which accounts for the most often observed relaxivity increase for smaller particles . On ther other hand, for particles in a certain range of size s , relaxivity increases for larger particles as tumbling time s get long as the hydrodynamic volume of the particle increase s . These two opposite contributing factors are expected to give rise to this opt imum particle size. Additionally, Eu 3+ ions have been doped into the gadolinium phosphate nanoparticles, enabling the nanoparticles to be used for d ual imaging , including MRI and fluorescent imaging. Using a c oordination polymer as a host for Gd 3+ ions is also reported . Gradient gadolinium iron hexacyanoferrate nanoparticles are synthesize d , in which gadolinium ions are introduced and gradiently distributed , presenting less in the particle center and more toward the surface. This gradient strategy can be use d for t he improvement of particle based MRI contrast agent s . In addition, the Prussian blue host is demonstrated to be an efficient photothermal therapy agent for in vitro cancer cells treatment.

PAGE 22

22 CHAPTER 1 INTRODUCTION Biomedical Applications of Nanoparti cles Nanoparticles are composed of chemically diverse and heterogeneous compounds (Figure 1 1) . 1,2 They can be prepared from inorganic compounds such as iron oxide and quantum dots , polymer compounds such as dendrimer and liposomes , and carbon based materi als such as graphene oxide (Table 1 1) . 3 15 Compared with traditional approaches, n anoparticles present some key advantages towards biomedical applications, including enhanced bioavai la bility and easy manipulation to realize multifunction al purposes . 1 Nano particles have been used for a variety of biomedical applications in fields including imaging, specific targeting, diagnostics, and cancer treatment. 3,5,7,15 In the field of biomedical imaging, nanopar ticles have brought an important breakthrough in recent years . For example, fluores cent quantum dots which show long term stability have become an alternative to the conventional organic dyes and proteins for labeling and imaging biological molecules (Figure 1 2 and 1 3 ) . 1 6,17 This opens the possibility for ma ny biologica performed before because of the short fluorescence lifetime and relatively poor photostability of organic dyes . 18 On the other hand , lanthanide doped nanoparticles have drawn a lot of attention recently due to the ir a bility to convert infrared or near infrared light to higher energy (Figure 1 4) . 1 9 The use of infrared light is promising in medical application s because it allows deeper tissue penetrati on and minimizes tissue damage (Figure 1 5) . 20 N anoparticle s have als o been used as Magnetic Resonance Imaging ( MRI ) contrast agent s to improve the MRI contrast , leading to higher sensitivity and better image resolution . For example, the

PAGE 23

23 tumor site of a mouse becomes brighter after introducing manganese oxide nanoparticles, a recently developed MRI contrast agent (Figure 1 6) . 21 Another category of imaging in which nanoparticles have had significant impact is X ray computed tomography (CT) . Due to strong X ray absorption , g old nanoparticles were found to be an efficient CT i maging agent . 22 Last but not least , the use of dense nanoparticles that contain the high atomic number element , ytterbium , as a contrast agent for X ray CT imaging was shown in Figure 1 7 , 2 3 providing new biological applications of lanthanide elements. Not limited to the imaging techniques mentioned above, other biomedical imaging applications of nanoparticles include single photon emission tomography (SPECT), positron emission tomography (PET), ultrasound imaging , and photoacoustic (PA) imaging . 24 27 Besid es imaging, another important field of biomedical applications using nanoparticles relies on drug delivery. The foundation of disease treatment is beginning to be changed by the development of a wide spectrum of nanoparticles . 28 Compared with traditional d rug s , nanoparticles can potentially improve therapeutic effectiveness , overcome drug solubility issues, and minimize side effect s . 28 Therapeutic and diagnostic agents can be encapsulated, cova lently attached, or adsorbed on to such nanoparticles. 3,29,30 For example, these molecules can be physical ly loaded into the pores of the nanoparticles. Porous hollow magnetite nanoparticles, reported by Chen et al., 3 were loaded with the anticancer drug doxorubicin (DOX), which greatly facilitate the in vitro cancer th erapy . In addition, Kang et al . 29 used GdVO 4 :Dy 3+ nano spheres with large cavities as hosts for therapeutic drugs which show a great potential in drug delivery application s . The other routine method for drug loading is by covalent

PAGE 24

24 attachment. That is, t he t herapeutic agents are covalently bonded to the functional groups for which the attachment is able to be cleaved under specific conditions , allowing for the release of drug. Recently , Chen et al . 30 reported a drug nanocarrier, with DOX successfully gra fted to the surface of the core shell nanoparticles via an amide bond, which can be cleaved under acid conditions in the presence of protease . In general , nanoscale based delivery strategies are beginning to make a significant impact on disease treatment . 28,31 N a noparticle s can also be engineered to incorporate targeting capabilities either by a passive or active strategy . R esearchers have developed a wide range of technologies in the nanoscale regime , which can be employed for targeting different cells and extr acellular elements in the body , in order to deliver the therapeutic agents to specific locations. For passive targeting, nanoparticles with small size can accumulate in tumors due to the pathophysiologic characteristics of tumor blood vessels. 32 When tumor cells multiply, cluster together and reach a size of 2 3 mm , new blood vessel formation is required to supply the oxyg en demands of the growing tumor . 33 The incomplete tumor vasculature results in leaky vessels with enlarged gap junctions with sizes of 10 s . 34 This provide s macromolecules , plasma components , and nanoparticles easy access to the tumor site . Moreover, the slow venous return in tumor tissue and the poor lymphatic clearance allow macromolecules to be ret ained in the tumor tissue , 35,36 whereas nanoparticles continue entering into tumor interstitium. This is called the E nhanced P ermeability and Retention (EPR) effect , as depicted in Fig ure 1 8 . 37 For example, Abraxane, an albumin bound paclitaxel nanopartic le for the treatment of breast cancer, has a size of

PAGE 25

25 130 nm and accumulates in tumor si t e s in part through the EPR effect. 38 Clinical studies have shown that Abraxane almost double d the therapeutic response rate and increase d overall survival in patients w ith breast cancer. These effects were achieved without resulting in toxicity to normal tissues (the drug dose was 1.5 times higher than the maximum tolerated dose of traditional paclitaxel) . 38 Generally speaking , t hese agents circulate in the body with a l onger half life and lower toxicity . On the other hand, the active targeting has also brought extensive research inter e st because of it s flexibi li ty and greater targeting elements allowed. Targeting allows therapeutics to be delivered to specific locations , which was not possible before. A common strategy is to attach macromolecules, such as antibodies or aptamer s, which can specifically target the sites of interest (Figure 1 9 ) . 39,40 Other than macro molecules, small molecules such as folic acid can also be used for targeting due to the fact that some cancer cells express more folic acid receptor than normal cells . 41,42 Indeed, research into the targeting delivery of pharmaceutical, therapeutic, and diagnostic agents with nanosized particles is the frontier i n nanomedicine. 28,43 Finally , photothermal therapy (PTT) strategies , have grown with the development of nanoscale technologies. PTT agents convert absorbed light to heat, leading to t hermal ablation of cancer cells. B ecause it is minimally invasive, PTT is recognized as a promising alternative or complement to conventional chemotherapy, surgery and radiotherapy cancer treatments (Figure 1 1 0 ). 44 4 8 There are s everal nanoparticle based systems which are under active investigations for PTT. The most common ex amples include gold, copper sulfide, and reduced graphene oxide. 44 49 For example, Tian et al . 44

PAGE 26

26 reported photothermal ablation of cancer cells with Cu 2 x S, proving these nanoparticles can contribute to localized killing of cancer cells. MRI Contrast Agent Magnetic resonance imaging, with the ability to provide anatomical details of internal structures and detect lesions, has become a routine diagnostic tool for modern biomedicine (Figure 1 1 1 ). 50, 51 Even beyond anatomical imaging for medical care, there is a vast array of potential applications of MRI that includes imaging of cellular and molecular processes to visualize changes in chemical events associated with pathology or controlled external stimulus . 52 55 For anatomical imaging, MRI contrast agents are employed to improve the imaging accuracy and sensitivity when there are only small differences in proton relaxation time between normal tissues and lesions and abnormalities . 56 57 For molecular or cellular imaging, the agents are designed to affect relaxi vity in response to changes in environment or in response to selected stimuli . 52 55 As a result of the expanding use of MRI, the design of effective and innovative MRI contrast agents remains an important topic for biomedic al researchers. One of the classe s of contrast agents is superparamagnetic iron oxide (SPIO) nanoparticles, which has received a lot of attention since their development as MRI contrast agents for the liver. 56 The SPIOs have a different contrast mechanism and are known as T 2 or negative c ontrast agents. 56 The other main class of MRI contrast agent is Gd 3+ complexes with seven unpaired electrons , which are positive contrast agents , also called T 1 contrast agent s , in clinical use (Figure 1 1 2 ). 57,58 The capability of Gd 3+ based T 1 contrast a gents t o introduce signal enhancement is directly proportional to its ability to induce proton relaxation of neigh boring water molecules . 59 This effect is generally divided into two components, includ ing both inner -

PAGE 27

27 sphere (from water molecule s directly coo rdinated to the paramagnetic Gd 3+ ions ) and outer sphere contributions (from water molecules in the second coordination sphere and beyond ). 58,59 The latter effect is usually relatively small compared with effect from inner sphere and is often neglected. 59 For Gd 3+ chelate contrast agents , the inner sphere relaxivity is given by the following equation: 57 60 where is the hydration number of Gd 3+ ion, is the mole fraction of water molecules coordinated to Gd 3+ , is the average length of time a single water molecule resides within the inner sphere coordin ation sphere , and is the relaxation enhancement of the inner sphere water molecules. Based on this equation, f or Gd chelate complexes, i ncreasing can potentially improve relaxivity . However, increasing inner sphere bound water molecules may res ult in unstable complexes , giving release of Gd 3+ in the body. Additionally , while opening up more coordination sites for water ligation, it also allows coordination with other endogenous ligands, such as bicar bonate or phosphate, which in turn decrease s t he relaxivity. 57 As a result , and should be short in order to obtain better relaxation effect . For first generation of Gd 3+ chelate contrast agents, where is much larger than , the relaxation rate enhancement exper ienced by the bulk solvent mainly depend s on . 57,58 In other words, limits the relaxivity. The determination of is through the Solomon Bloembergen Morgan equation outlined below : 57 62

PAGE 28

28 w here is the proton gyromagnetic ratio, is the electron g factor, is the Bohr magneton, S is the number of unpaired electron s in the Gd 3+ ion, is the distance between the water protons and the unpaired electron s of paramagnetic metal ion , and are proton Larmor frequency and electron Larmor frequency , respectively, and is correlation time. This equation shows that modulation of the correlation ti me , , becomes critical if the high relaxivities are to be obtained . 60 The calculation is shown as follows : 57,58,60 The correlation time ( ) has three components, (t he electronic relaxation time of the unpaired electrons), (the water residency lifetime), and (the rotational correlation lifetime). The high field strength s used in MRI simplify th is equation, since is long enough to reasonably i gnore the contributions. 63 is ro tational correlation time and related to physical tumbling time of the MR contrast agents in solution. O ptimizing has been demonstrated to improve the relaxivity of the Gd 3+ complex by slowing down its molecu lar tumbling. 59 , 64 For example, Anderson et al. 64 developed a MRI contrast agent by conjugation of gadolinium chelate, Magnevist, onto a viral capsid with a size of ar o und 27 nm. D ue to the slow tumbling rate of capsids, the contrast agent shows enhanced r 1 relaxivity of 16.9 mM 1 s 1 . 64 Nanoparticles of solid state gadolinium compounds can show impressive relaxivity, r 1 , leading to positive contrast. The predominant mechanism is the interaction of water molecules with Gd 3+ sites at the particle surface. Imp ortantly, i ncreased rotational correlation times because of slower particle tumbling can result in i mpressive values of relaxivity. 61 As an example, nanoparticles of NaGdF 4 , reported by Johnson et

PAGE 29

29 al . 65 show r 1 relaxivity of 7.2 mM 1 s 1 , which compares fav orably to the commercial agent Gd DTPA with r 1 of 4.3 mM 1 s 1 . 66 In addition, Fortin et al . 67.68 reported PEG coating Gd 2 O 3 nanoparticles as a potential MRI contrast agent with r 1 of 9.4 mM 1 s 1 . Prussian blue (PB) particles have also been studied, for whi ch relaxivity as high as 38.5 mM 1 s 1 was reported for K 0.53 Gd 0.89 Fe 4 III [Fe II (CN) 6 ] 3.8 1.2H 2 O nanoparticles . 69 Additionally , Perrier et al . 70 prepared Gd 3+ /[Fe(CN) 6 ] 3 nanoparticles stabilized by polyethylene glycols with an average size of 2.1 nm, and mea sured a relaxivity of 12.6 mM 1 s 1 . Highly porous inorganic nanoparticles are also under active investigations, due to the i ncreased porosity resulting in more Gd 3+ active sites. 71 For example , hollow Gd 2 O 3 nanopa rticles were synthesized, allow ing more wat er reaching the i nner surface of the nanospheres which as a result increas e the interaction between water and Gd 3+ ions. 71 In addition , Ananta et al. 72 loaded Magnevist, gadofullerenes and gadonanotubes individually into highly porous silicon particles. Sp ecifically, gadofullerenes after loading into the particles, shows a boost in r 1 relaxivi ty with a value of 200 mM 1 s 1 . 72 Other than inorganic nanoparticles, polymer based nanoparticles were also studied for the relaxivity enhancement. 73 75 A well know ex ample is dendrimer nanoclusters, which were fabricated by conjugation Gd DTPA to polyamidoamine, allowing for tumor targeted magnetic resonance imaging . 73 Besides dendrimer , a variety of liposome systems were studied with the capability of response to sele cted stimuli . 74,75 For example, Gianolio et al. 74 prepared a liposome system, in which the Gd chelate experiences different intraliposomial distribution depending on the pH condi tion, which shows the potential to provide measurement of the tissue pH in viv o by MRI. In addition,

PAGE 30

30 a temperature sensitive liposome reported by de Smet et al . , 75 which presents st r uctural change upon temperature increase , a llow s for drug delivery under MRI guidance . For negative contrast agent s , other than SPIO, researchers have r ecently developed new types of nano materials, which show the potential to be employed as T 2 MRI contrast agent s. 56 For example, Yang et al. 76 synthesized superparamagnetic MnFe 2 O 4 with tetraethylene glycol as a coordination and stabilization agent. R elaxiv ity measurement s together with additional in vivo MRI studies demonstrate the application of MnFe 2 O 4 nanoparticles as a negative contrast agent. Furthermore , Chou et al . 77 used FePt nanoparticles as T 2 contrast agents which present excellent biocompatibili ty and hemocompatibility. After conjugation with anti Her2 antibody, the FePt nanoparticle is able to selectively enhance the contrast of Her2/neu overexpression cancer lesions. Study Overview In this work, MR contrast enhancement of dif ferent systems has been studied , including inorganic nanoparticle s and coordination polym ers . Different factors affect ing MRI contrast efficiency using nanoparticle based MRI contrast agent are discussed in order to improve MR contrast enhance ment and diagnosis sensitivity, as well as providing route s towards building nanoparticle based contrast age nt s with greater performance . Chapter 2 gives an introduction of the instrumentation and experiment al methods used in this work, including analytical characterization tools , biolog ical assay s , and MRI measurement techniques . Chapter s 3 and 4 focus on discussion s of GdPO 4 nanoparticles, which have proved to be an efficient MRI contrast agent. These two chapters cover the synthetic methods for preparing GdPO 4 nanoparticles of differen t sizes for size dependent

PAGE 31

31 relaxivity studies . Particularly , PAMAM, HyPAM , HyPAM PEG, and HyPAM C 18 PEG polymer s were used to mediate the synthesis of GdPO 4 nanoparticles and realize the size control of the particles . T he relaxivity of different size GdPO 4 nanoparticles were systematically studied. Work presented in Chapter 3 was done in collaboration with Dr. Weihong Tan and Dr. Tao Chen in Department of Chemistry at the University of Florida , while work presented in Chapter 4 was a collaborative initiativ e between the IMRCP laboratory at the University of Toulouse, including members: Dr. Chri stophe Mingotaud, Dr. Jean Daniel Marty, Camille Frangville, M aylis Gallois, Hanh Hong Nguyen , and Dr. Nancy Lauth de Viguerie, and our group at the University of Flor ida . In C hapter 5 , a coordination polymer system , Prussian blue, is introduced. Within the novel trimetallic Prussian blue , gradient G d 3+ distributions were obtained in our g Gd PB particles , with more Gd 3+ ions d istributed close to the surface and less in the center . These particles were prepared by a previous member from our group , Carissa Li. The gradient particles were proved not only as a M RI contrast agent, but also were demonstrated to perform as a photothermal therapy agent , by taking advantage of t he photosensitive PB host . Moreover, PB , whic h has been approved by the US Food and Drug Administration (FDA) in the treatment of cesium and thallium poisoning , offers good biosafety . Appendix A discusses the biological performance of highly porous metal h ydroxide nanoparticles , which were prepared using the g Gd PB nanoparticles synthesized from Chapter 5 with some post synthetic modifications . With more vacancies created in the frameworks , t he MRI contrast enhancement of these particles was further improv ed , giving an even higher relaxivity value. Additionally, the porous

PAGE 32

32 nanostructure provides a platform for drug loading, which can then be used for drug delivery . Finally, Chapter 6 is the summary and conclusion of the overall work.

PAGE 33

33 Figure 1 1 . Nanoparticles synthesized with different structures (A) spherical, (B) cubic, and (C) cylindrical topologies. TEM images are accompanied by drawings that represent the morphology of each nanostructure. T he figure was reprinted from Ref 1 with permission from The American Association for the Advancement of Science . Figure 1 2 . Typical sized CQDs optical images illuminated under white (left; daylight lamp) and UV light (right; 365 nm) . The figure was reprinted from Ref 16 with permission .

PAGE 34

34 Figure 1 3 . QD resistance to photobleaching and multicolour labelling. Top row: Nuclear antigens were labelled with QD 630 streptavidin (red), and microtubules were labelled with Alexa 488 conjugated to anti mouse IgG (green) simultaneously in a 3T3 ce ll. Bottom row: Microtubules were labelled with QD 630 streptavidin (red), and nuclear antigens were stained green with Alexa 488 conjugated to anti human IgG . Continuous exposure times in seconds are indicated with light from a 100 W mercury lamp. The fig ure was r eprinted from Ref 17 with permission from the Nature Publishing Group.

PAGE 35

35 Figure 1 4 . Room temperature upconversion emission spectra of ( A ) NaYF 4 :Yb/Er (18/2 mol %), ( B ) NaYF 4 :Yb/Tm (20/0.2 mol %), ( C ) NaYF 4 and ( D ) NaYF 4 :Yb/ (10 mM). The spectra in ( C ) and ( D ) were normalized to Er 3+ 650 nm and Tm 3+ 480 nm emissions, respectively. Compiled luminescent photos showing corresponding colloidal solutions of ( E ) NaYF 4 :Yb/Tm (20/0.2 mol %), ( F J ) NaYF 4 K N ) NaYF 4 mol %). The samples were excited at 980 nm with a 600 mW diode laser. The figure was reprinted from Ref 19 with permission from American Chemical Society .

PAGE 36

36 Figure 1 5 . Whole animal imaging of a BALB/c mouse injected via tail vein with the HA (NaYbF 4 :0.5% Tm 3+ )/CaF 2 core/shell nanoparticles. ( A , D ) UC PL images; ( B , E ) bright field images; and ( C , D ) merged bright field and UC PL images. Mouse was imaged in the belly ( A , B , C ) and the back positions. Inset in ( F ) shows the spectra of the NIR UC PL and background taken from the circled area. The figure was reprinted from Ref 20 with permission from American Chemical Society . Figure 1 6. Breast cancer cells were selectively enhanced in T 1 weighted MRI by the Herceptin functionalized MnO nanoparticles. The figure was reprinted from Ref 21 with permissi on .

PAGE 37

37 Figure 1 7 . In vivo CT coronal view images of a rat after intravenous injection of 1 mL PEG UCNPs (70 m 1 ) solution at timed intervals. (A ) Heart and liver. (B ) Spleen and kidney. ) The corresponding 3D renderings of in vivo CT images. The figure was reprinted from Ref 23 with permission. Figure 1 8 . Nanoparticles accumulate in tumor tissue d ue to the EPR effect .

PAGE 38

38 Figure 1 9 . Proposed m echanism for targeted cancer chemotherapy. With aptamer attachment , nanoparticles specifically enter target cancer cells through receptor mediated endocytosis . I n acidic lysosomes , DOX is released to induce can cer cells apoptosis. Figure 1 1 0 . After injection , these nano particles accumulate into targeted tumor . B y absorbing NIR radiation incident on the nanoparticles and converting it into heat, nanoparticles cause photothermal ablation of tumor .

PAGE 39

39 Figure 1 1 1 . MRI images showing 3 orientations and the 3D view of a brain tumor . The figure was adapted from previously published work. 51 Figure 1 1 2 . Structure of gadolinium chelate based T 1 MRI contrast agent s . The figure was reprinted from Ref 58 with permissi on from American Chemical Society .

PAGE 40

40 Table 1 1. Some nanoparticle c omposition s and b iomedical a pplications . Nanoparticles Composition Biomedical Application SMNs 3 Iron oxide MRI and targeting cancer chemotherapy Ferucarbotran 4 Iron oxide MRI Quantum dots 5 CdS/CdSe/CdTe/PbSe Live cell imaging, in vivo imaging, and diagnostics QD probe 6 Core shell CdSe ZnS In vivo cancer targeting and imaging Gold nanorod 7 Gold nanorod Cancer cell imaging and photothermal therapy Gold nanocages 8 Gold nanocages Phototherma l treatment of cancer Calcium phosphate nanoparticles 9 Calcium phosphate Targeted gene delivery to the liver NGO PEG DOX 10 Graphene oxide Synergistic effect of chemo photothermal therapy GO nS 11 Graphene oxide Gene delivery and fluorescent probe SWNTs 1 2 Carbon nanotube Drug loading and delivery Doxorubicin HPMA copolymer conjugate 13 Liposome Treat subcutaneous murine B16F10 melanoma SL DOX 14 Liposome Cancer treatment Folate PAMAM dendrimer conjugate 15 Dendrimer Targeting of anti arthritic drug to inf lammatory tissues

PAGE 41

41 CHAPTER 2 EXPERIMENTAL METHODS AND TECHNIQUES T he different particle characterization techniques we have used in this work are des cribed in this C hapter. With these techniques , we are able to know the chemical composition s , structure, size, shape, and surface propert ies of the prepared nanoparticles, which are all important parameters affecting their performance in the following MRI a nd biomedical studies. Transmission Electron Microscopy Transmission electron microscopy (TEM) is used to obtain images by applying a beam of electrons onto the specimen. In the TEM column, e lectrons are first emitted from the electron gun . The e lectromagnetic lenses then focus the electrons into a very thin beam before pass ing through and interact ing with the samples . Depending on the density and size of the material present, some of the electrons are scattered. Aft er diffraction by the specimen, the electrons are refocused by the electromagnetic lenses and detected , forming a magnified image on a fluoresce nt screen which is recorded by a CCD camera (Figure 2 1). Due to the small de Broglie wavelength of electrons , t ransmission electron microscopy is able to acquire images with very high resolution , obtaining objects images to the order of a few angstrom s (1 0 10 m). For specimen preparation, samples ( ~ 5 mg) were suspended in water. T he well suspended solution containing nanoparticles is then added dropwise onto the TEM grid (ultrathin carbon film on holey carbon support film, 400 mesh, copper) , wh ich was purchased from Ted Pella, Inc. After the specimen is completely dried, it is processed for imaging. The instrument we have used in our experiment s is JEOL 2010F HRTEM with an operating voltage of 200 kV .

PAGE 42

42 Inductively Coupled Plasma Atomic Emission S pectroscopy Inductively coupled plasma atomic emission spectroscopy (ICP AES) is used to measure the concentration of elements , based on the excited atoms and ions emit ting characteristic radiation at specific wavelengths for each element . Th e emission int ensity is indicative of the concentration of the element within the sample. To introduce samples , a pump is used to send the solution into the nebu lizer, in which it wi ll turn into mist and be introduce d into plasma. The sample break s up into their respect ive atoms and plasma excites the atoms. The radiation from the plasma is then collected and analyzed. A fter the radiation is separated into different wavelengths , the intensity of light is measured. B ased on the created calibration line , the concentration is computed and recorded (Figure 2 2). To measure the concentration s of the target element s , three different standard solution s of the desired elements are prepared (1ppm, 10 ppm, and 100 ppm). Using nanopure water as a blank , the standard solutions are in troduced into the instrument separately i n order to create the calibration line s. Then , the sample solution containing the element s is pumped into the instrument for measurement. The instrument we used is a Perkin Elmer Optima 3200RL inductively couples pl asma atomic emission spectroscopy . U ltraviolet Visible Spectroscopy U ltraviolet visible spectroscopy gives absorption spectroscopy in the range of 200~800 nm. T he sample beam passes through a transparent cuvette containing a solution of the compound of int erest . The intensities of these light beams are then measured by electronic detectors as shown in Figure 2 3. The UV Vis absorption spectra presented in this work were acquired with a Shimadzu UV1701.

PAGE 43

43 Fourier Transform Infrared Spectroscopy Fourier transfo rm infrared spectroscopy , with the advantages of wide scan range, high resolution, short scan time, and high accuracy, is widely used to obtain structural information of molecules. During the measurement, part of the applied infrared radiation passes throu gh the sample while part of it is absorbed by the sample, causing change s of dipole moment of the sample molecules. The number and intensity of the ab sorption peaks is related to the types of chemical bonds of the molecule (Figure 2 4) . No two different mo lecular structures will show the same FT IR spectrum. Therefore, based on the spectra obtained, a lot of structure information can be obtained . I n our study , FT IR was used to obtain info rmation of the molecular structures of the surface coatings , na mely , materials used for surface functionalization and modification discussed in Chapter 3 and Appendix A . In C hapter 5 , FT IR plays an important role for characterizing PB and its analogues . These materials contain characteristic cyanide ligands between two met al centers ( M' C N M ), which give unique absorption peak s between 1900 cm 1 and 2300 cm 1 . The instrument used in our work is Nicolet 6700 Thermo Scientific Fourier transform infrar ed s pectrometer. Powd er samples w ere mixed with KBr and pressed into a pell et using 3000 psi (20 MPa). Typically 16 scans were taken for the measurement. Before measuring the sample , a scan of pure KBr is taken as a background reference. Combustion Analysis Combustion analysis, which is also called CHN analysis, is used to determ ine the elemental composition of a sample, by combusting the sample in a large excess of oxygen gas. The resulting combustion products, including carbon di oxide, water, and

PAGE 44

44 nitric oxide, will be trapped separately from each other. The mass for each product will be weighted and quantitatively analyzed (Figure 2 5 ) . Finally the analytical determination of the amount of combustion products will give the empirical formula. This technique has most often been applied for characterizing organic compounds. For CHN analysis, only few milligrams of sample are required and the sample is fully burned at very high temperature. The analysis is provided by the University of Florida Spectroscopic Services Laboratory. Thermogravimetric Analysis Thermogravimetric a nalysis (TG A) is a technique measuring changes of mass of the sample as a function of temperature . Mass change of the sample is measure d by a highly sensitive balance as illustrated in Fi gure 2 6 . A wide range of materials can be measured by TGA analysis, including p olymer s , nanoparticles, and biological samples. TGA was used to characterize the gadolinium doped Prussian blue nanoparticles synthesized in Chapter 5 . Based on TGA analysis, the water content in the PB particles can be determined. In our work , TGA was per formed on a Perkin Elmer TGA 7. The sample rests on a small platinum pan, which hangs from a small hook conn ected to the balance. The temperature range for TGA measurements was set from 20 °C to 45 0°C at a scan rate of 10°C/min. Dynamic Light Scattering Dyn amic light scattering (DLS) is a well known technique which is used to determine the size and its distribution of various materials, including polymer, nanoparticles, protein, and liposome, based on their Brownian motion in solution . In a typical DLS exper iment, the cuvette containing sample solution, in which the sample has been well dispersed, is irradiated with monochromatic light from a laser

PAGE 45

45 which is shot through a polarizer. T he dispersion should be either centrifuged or filter ed during the sample pre paration process in order to get rid of the dust, which could also cont ribute to the light scattering and cause interference. When light goes through the sample, particles in the soluti on are being hit and the light is diffracted in all directions (Figure 2 7 ) . The diffracted light can either interfere constructively or destructively with the sample , which is then collected by a photomultiplier tube . T he resulting image is projected onto a screen, producing a speckle pattern. The collected data are analyzed to determine size and size distribution of the particles. DLS experiments are performed with Malvern Zetasizer Nano ZS instrument (Malvern Instruments, Ltd . , UK). For each measurement, nanoparticle samples were well dispersed in water and then added into a cuvette. The cuvette was then introduced into the instrument for DLS analysis. Energy Dispersive X ray Spectroscopy Energy dispersive X ray spectroscopy has been extensively used in this work for determining both the localized and average concentrations of elements of the prepared particle samples . It is a quantitative technique which has been widely used to obtain details of the chemical composition of the target material . EDX is usually coupled with scanning electron microscopy (SEM) or transmission ele ctron microscopy due to the use of a strong, focused electron beam to excite the material . For the EDX analysis in this work, they were all performed in conjunctio n with HRTEM. After introduction into the instrument, the specimen is bombarded b y the high e nergy electron beam, causing electron excitations and creating an electron hole in the inner shell of an atom . Another electron from a higher energy level of the same atom will fill in this hole, releasing

PAGE 46

46 energy in the form of X r ay radiation, as illustra ted in Figure 2 8 . The energy of the X ray is the energy difference between the two electron energy states. Since each atom has its unique atomic structure, it will also give unique characteristic X r ay radiation, which is used to determine elemental compo sition s of materials . Besides measuring the average compositions, ED X spot scan s and 2D maps can also be acquired to determine localized elemental percentages of a particle sample . Characteristic X r ay intensity is measured on a specific spot or in an area containing the sample material . The difference in X ray intensity at the characteristic energy value indicates the relative concentration for the applicable element across the specific area of sample. One or more maps on different elements can be recorded at the same time, which provide more details about the elemental distribution of the sample of interest . ED X 2D maps were taken for samples discussed in Chapter 3. Two different elements, ga dolinium and europium, are analyzed to view their d istribution in the particle sample . ED X spot scan s were performed for sample s discussed in Chapter 5 , in which gradient distribution s of gadolinium in the PB nanoparticles were achieved . X ray Powder Diffraction X ray powder diffraction is a technique using X ray diffra ction on powder samples for phase identification of a crystalline material , providing structural information on unit cell dimensions. Powder X ray diffraction, compared with single crystal X ray diffraction, is much more convenient in terms of sample selec tion as it uses powder samples and When X rays hit the surface of the sample , part of them are scattered by the atoms composed of the material , as shown in Figure 2 9. Diffraction patterns are created for crystalline sa mples, that is, samples show a periodic array with long range

PAGE 47

47 order . Amorphous materials like glass will not produce a diffraction pattern. To give a well resolved diffraction pattern , the spacing between atom layers must be close to the radiation waveleng th. If X ray beams are diffracted by two different layers in phase, a peak will show up in the diffraction pattern. However if the two layers are out of phase, destructive interference occurs and there is no peak. Diffraction peaks will occur only when Bra =2 d sin where n is an integer, is the wavelength, is the angle of incident X ray beam with respect to the atomic plane of the sample , and d is the spacing between the atom ic layers. In general, a powder X ray d iffractometer has three parts, the X ra y sour ce, a sample holder, and a detector which is on the opposite side of the X ray source. When X ray s hit the sample from angle , the detector will detect the signal intensity at angle 2 from the path of X ray source, as depicted in F igure 2 10. The d iffractometer used in our work is an sample preparation, powder s amples are mounted with double sided tape on a glass slide and then fixed onto the sample holder. The measurement was taken in steps of 0.008 ° in the range of 2 from 10 ° to 80 ° . After each measure ment, the diffraction pattern will be plotted with intensity as a function of 2 , which is further used to obtain crystallographic information including symmetry and the unit cell parameter s of t he sample. Selected Area Electron Diffraction Selected area electron diffraction (SAED) is a useful scientific tool to identify lattice parameters of the desired sample with showing either spots or rings. In a SAED pattern, each spot corresponds to a satis fied diffraction condition of the sample's crystal

PAGE 48

48 structure . Unlike powder X ray diffraction which requires several milligrams of sample for a measurement, only a small amount of s ample is used for SAED analysis . Typically, SAED is performed inside a tran smission electron microscope. An amorphous sample shows diffuse rings, while samples with high crystallinity gives a series of bright spots. SAED was used in Chapter 4 to confirm the composition of a gadolinium phosphate particle sample . Due to the small a mount of sample that can be prepared each time, powder X ray diffraction is not favorable in this case . Instead, SAED analysis , which only requires few milligrams of sample for each measurement , is performed. Fluorescence Spectroscopy In a fluorescence spe ctrometer, a beam of light strikes the sample and part of the incident light is absorbed, causing excitation of the sample molecules which then emit photons. Generally , there are two different modes to record a fluorescence spectrum . The first one is calle d an emission spectrum, for which the excitation wavelength is fixed while the range of emission detection varies. On the other hand, f or an excitation spectrum , the emission wavelength is fixed while the range of excitation energies varies. For spectra co llected in this work, we mainly focused on the fixed excitation mode . A typical fluorescence spectrometer has three parts: the light source, a sample holder, and a detector. The excitation wavelengths are made selectable with the use of an excitation monoc hromator or a filter (Figure 2 11) . For detecting photons , photomultiplier tubes are used . In our study, fluorescence spectra were collected by a Photon Technology International (PTI) photon counting fluorescence spectrophotometer. Before each measurement, samples were dispersed in water and then transferred into a cuvette.

PAGE 49

49 Based on the type of cuvettes, sample volume varies from ~100 µ L to ~1 mL. The sample analyzed with fluorescence spectro scopy is europium doped gadolinium phosphate nanoparticles, which show extraordinary fluorescent properties including large Stokes shifts, long lifetimes, and narrow emission lines , due to the presence of fluorescent europium ions . C onfocal Laser Scanning Microscopy Confocal laser scanning microscopy has become a useful tool in the fields of biological and biomedical sciences due to the capability of viewing optical sections in both living and fixed specimens. The key advantage of conf ocal laser scanning microscopy relies on the fact that it can be employed to obtain opti cal images in selec ted depths of the sample, allowing interior structure s to be imaged. As a comparison, a conve ntional microscope can only show image s as far into the specimen as the light can penetrate. In a confocal laser scanning microscope, light f rom laser excitation source pass es through a light source pinhole aperture and is reflected by a dichromatic mirror. T he laser is then refocused by the objective lens before hitting the specimen . T he fluorescenc e emitted from desired plan pass es through the d ichromatic mirror. Finally, t he light passing the detector pinhole aperture is detected with a photomultiplier tube (Figure 2 12) . There are two different models of confocal las er scanning microscopes that we used in this work : Olympus FV 500 IX81 and Leic a TCS SP5, which is equipped with violet, indigo, blue, blue green, green, and red lasers. Three fluorescence channels are configurable for any b r and. In addition, t here is an environ mental control, a 37 °C chamber with 5% CO 2 , coupled with Leica TCS SP5 .

PAGE 50

50 I n our study, we used co nfocal laser scanning microscopy to monitor the endocytosis of the nanoparticle samples by cancer cells. I n Chapter 3, europium doped gadolinium phosphate nanoparticles were used as dual imaging agent s : MRI and fluorescent imaging. F or fluorescent imaging, nanoparticles were introduced to both cancer cell and C. elegans . The specimens were imaged separate ly. I n Chapter 6 , it was employed to monitor the anticancer drug nanocarrier, with t he red fluorescence given by doxorubicin. Cell C ulture Cell culture refers to incubation of cells from an animal or plant outside their natural environment. Cell culture is a major technique in cellular and molecular biology, and is t he model system for drug screen ing and many other biomedical applicati ons. In this work, the cells we incubated were all cancer cells. The artificial environments in which the cells are cultured are important, including the temperature, gas mixture, and cell growth medium. The first step for common cell culture is to view th e culture medium color and clarity and then check the cultures under a microscope to assess the degree of confluence and see if the culture is polluted with bacterial or fungal contaminations. The next step is t o remove spent medium, followed by washing th e cells with PBS buffer. If the cells are attached to the bottom of the petri dish, t rypsin is added to the washe d cell layer and incubate d for 3 min or until the cells become detached. Once this is done , cells are resuspended in a small volume of culture medium and then transferred to a new petri dish. For suspended cells, no trypsin treatment is required.

PAGE 51

51 Cytot oxic ity Assay Cytotoxicity assay is widely used in biological scie nce s , biomedical field s , and the pharmaceu tical industry to determine cytotoxicit y of the target sample to specific cells. The cyto to x icity assay we use d is MTS assay. Unlike the conventional MTT assay which requires dissolving MTT reduction products , MTS formazan product is soluble in the culture medium. During the assay, MTS is added into the culture medium which will be bioreduced by cells, forming colored formazan . T he mixture is then introduced in to a 96 well plate reader for measuring absorbance at 490 nm, which is the absorbance of the product material . The absorbance intensity a t this wavelength is directly proportional to the number of living cells. The chemical used in our work is Pro CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). In general, the MTS assay requires four consecutive step s. First, thaw the reagent either at room temperature or at 37 °C and add 20 µ L of it to each well that contain s cells . Then , incubate the mixture (37 °C , humidified 5% CO 2 atmosphere) for 1~4 h. After that, the plate is introduced to a 96 well plate reader for absorbance measurement . Finally, the recorded data are used to analyze the cytotoxicity of the sample. Relaxivity Measurement In MRI, the protons will spin align with an external magnetic field , B 0 , creating a net magnetic moment, M, parallel to B 0 . T he spinning protons wobble at a frequency, called the Larmor frequency , which is shown below: B 0

PAGE 52

52 where and B 0 i s the strength of the applied magnetic field. From th is equation , we can see that t he resonance frequency , , of a spin is proportional to the magnetic field, B 0 . Now if an electromagnetic radio frequency (R F) pulse is applied perpendicular to B 0 at the resonance frequency, the protons can absorb that energy and jump in to a higher energy state. When the RF transmitter is turned off, M will return from the higher energy state back to its equilibrium state, whi ch is parallel to the external magnetic field , B 0 . This process is called relaxation. During this proc ess, energy is emitted and is detectable as an RF signal called the F ID response signal , which is collected and forms MR images . The T 1 relaxation time (a lso known as the spin lattice relaxation time) indicates how quickly the net magnetic moment, M, recovers to thermodynamic equilibrium state, which is parallel to the direction of external magnetic field, B 0 . The return of excited nuclei from a high energy state to low energy or ground state is associated with loss of energy to the surrounding nuclei. R elaxation time, T 2 , also called spin spin relaxation time, describes progressive dephasing of spinning dipoles following the radio frequency pulse. In genera l, T 2 is shorter than T 1 and different tissues usually have different relaxation times. The key parameter to determine whether the material is an effective MRI contrast agent or no t depends on its longitudinal relaxivity (r 1 ) and transverse relaxivity (r 2 ) . Relaxivity indicates how efficient the MRI contrast agent will induce the relaxation time change of the protons. To obtain relaxivity of a specif ic sample, a series of particle dispersion s in water with different concentrations are prepared, followed by measuring

PAGE 53

53 both T 1 and T 2 relaxation times for all the samples. Base d on the recorded data, a line of relaxation rates as a functi on of ion concentrations can be plotted. Relaxivity is calculated as the slop e of this line. C oncentration of the ion is acquir ed with the application of ICP AES as described previously. The instrument we use d for relaxation time measurement is M inispec Contrast Agent Analyzer MQ 60 (Bruker Optics, Billerica, MA, USA) at a constant temperature of 37 °C with magnetic field of 1.41 T . MRI System A typical MRI scanner includes the following key parts: magnet, gradient coil, and RF coil , as shown in Figure 2 13. The m agnetic field has uniform f ield density and strength. There are two types of mag net s : permanent magnet and superconductin g magnet. A p th low magnetic field strength. H igh magnetic field strength can be achieved with the superconducting magnet which is composed of sup erconducting wires. The superconductor has approximately zero resistance when the temperature is close to 0 K. A g radient coil is used to produce a gradient in B 0 in different directions. There are three different types of RF coil: transmit and receive coil, transmit only coil, and r eceive only coil. The t ransm it coil is used to produce the magnetic field, while the receive coil is used to detect the RF signal emitted from the object.

PAGE 54

54 Figure 2 1 . Illustration of a transmission electron microscope.

PAGE 55

55 Figure 2 2. Scheme representing the essential comp on ents of a n inductively coupled plasma atomic emission spectrometer. Figure 2 3. Schematic illustration of the ultraviolet visible spectrometer.

PAGE 56

56 Figure 2 4 . Scheme r epresentation of the principal parts of a Fourier transform infrared spectrometer. Figure 2 5 . Block diagram illustrating the essential components of a CHN elemental analyzer.

PAGE 57

57 Figure 2 6 . Schematic diagram of t hermogravimetric analysis technique. Figure 2 7 . Block diagram illustrating the DLS instrument.

PAGE 58

58 Figure 2 8 . Schematic diagram of ene rgy d isper sive X ray spectroscopy. In EDX, the incoming electron beam ejects an inner shell electron, leaving a vacancy which is filled by an outer shell electron, releasing energy in the form of X ray radiation. Figure 2 9. Schematic illustration of th e X ray diffraction with the sample .

PAGE 59

59 Figure 2 10. Scheme illustrating X ray powder d iffractometer. It consists of X ray source, sample holder, and a det ector to detect the diffracted X rays. Figure 2 11. Block diagram illustrating the essential compon ents of a fluorescence spectrometer.

PAGE 60

60 Figure 2 12. Schematic drawing of the main parts of a co nfocal laser scanning microscope and the light path.

PAGE 61

61 Figure 2 13. Schematic diagram of a MRI scanner.

PAGE 62

62 CHAPTER 3 SIZE DEPENDENT MRI RELAXIVITY AND DUAL IMAGING WITH E u 0.2 G d 0.8 PO 4 ·H 2 O NANOPARTICLES Preface Three different sizes of Eu 0.2 Gd 0.8 PO 4 ·H 2 O nanoparticles have been prepared to investigate the particle size influence on water proton relaxivity. Longitudinal relaxivity (r 1 ) values increase for smaller partic les, reach as high as r 1 = 6.13 mM 1 s 1 for a sample of 40 ± 4 nm particles, which, with a ratio of transverse/longitudinal relaxivity, r 2 /r 1 = 1.27, are shown to be effective positive contrast agents. The correlation between relaxivity and the surface to volume ratio implies that access to surface Gd 3+ sites is the principal factor affecting relaxivity. On the other hand, although ionic molar relaxivity decreases for larger particles, the relaxivity per particle can be significantly greater. Gadolinium ba sed nanoparticles doped with fluorescent lanthanide elements have attracted attention for their dual imaging abilities, combining m agnetic resonance imaging and fluorescence imaging agents. In both in vitro experiments with HeLa cells and in vivo experimen ts with C. elegans , strong red fluorescence is observed from Eu 0.2 Gd 0.8 PO 4 ·H 2 O with high resolution, demonstrating the parallel use of the particles as fluorescence imaging agents. Relevant sections in this C hapter were published in Langmuir , and are copyr ight of American Chemical Society. The online abstract can be found at http://dx.doi.org/10.1021/la500602x. 1 Introduction Multifunctional nanoparticles elicit considerable attention due to their ability to serve simultaneously as agents for important tasks such as MRI, fluorescence imaging, Reprinted with permission fro m Li, Y.; Chen, T.; Tan, W.; Talham, D. R. Langmuir 2014 , 30 , 5873 5879

PAGE 63

63 controlled drug release, and specific targeting. 42, 78,79 A variety of material platforms have been used to develop multiple functions, including gold, silica, polymer, and magnetic oxide nanoparticles. 80 83 Gadolinium io n based nanoparticles such as Gd 2 O 3 , KGdF 4 , and GdPO 4 are a promising category due to their intrinsic ability to affect water proton relaxation and serve as potential T 1 or T 2 contrast agents. 84 86 Combining fluorescent bioimaging with MRI capabilities is attractive because the greater sensitivity and resolution of fluorescence imaging can be used to complement MRI capabilities. 79 , 84,85 , 87 Several nanoparticle based systems have been developed for this purpose, such as superparamagnetic Fe 3 O 4 nanoparticles conjugated with fluorescent quantum dots and organic fluorophores immobilized on Gd 2 O 3 nanoparticles. 84 , 88 The mixing of different lanthanide ions in a single phase system provides an effective way to achieve particles that combine fluorescence imaging and MRI easily while avoiding potential complication associated with more complex architectures. 89 93 Circumventing the need for quantum dots or organic fluorophores, lanthanide ions such as Eu 3+ and Tb 3+ show extraordinary fluorescent properties, including l arge Stokes shifts, long lifetimes, and narrow emission lines. 89 , 93,94 With seven unpaired electrons, Gd 3+ forms the basis of the most widely used T 1 contrast agents. 95 As a result of these advantages, gadolinium based nanoparticles have become a suitable platform for doping with luminescent lanthanide ions to achieve dual mode imaging agents. Previously, Shi et al. 92 used Eu 3+ doped Gd 2 O 3 hybrid nanoparticles to label human mesenchymal stem cells (hMSCs), which was confirmed by confocal laser scanning micr oscopy. In addition, europium doped gadolinium sulfide (GdS:Eu 3+ ) was successfully applied as a fluorescent imaging agent for breast cancer

PAGE 64

64 cells (SK BR 3). 91 In a third example, Ren et al. 89 used Eu 3+ doped GdPO 4 nanorods to observe red luminescence from labeled HeLa cells. Following our earlier report 86 exploring MRI contrast generation with gadolinium phosphate particles stabilized with phosphate terminated oligonucleotides, we became interested in the Eu x Gd 1 x PO 4 ·H 2 O system. The present report describes the synthesis of Eu 0.2 Gd 0.8 PO 4 ·H 2 O, and we show that suspensions can be stabilized by the phosphate containing modifier, PMIDA . The PMIDA modified particles are shown to be compatible with HeLa and A549 cells in viability studies and can be used to genera te fluorescent images of the cells. The particles also enhance water proton relaxivity and are demonstrated to be MRI contrast agents. Our study contributes to a range of relaxivity values reported for the GdPO 4 ·H 2 O and Eu x Gd 1 x PO 4 ·H 2 O systems. 86,89,96,97 Differing particle size is likely one of the parameters contributing to the range of reported values, 65 , 95 , 98 101 so to understand the particle size dependence better, three different samples, ranging from 40 to 140 nm, are investigated. Materials and Me thods Synthesis of Eu 0.2 Gd 0.8 PO 4 ·H 2 O Nanoparticles Sample 1 The synthesis of nanoparticles is based on procedures reported by Dumont et al. 86 with a few modifications. The nanoparticles are obtained by combining two precursor surfactant mixtures, one cont aining the metal ions and the other the phosphate ions. The metal ions, 400 mg of Gd(NO 3 ) 3 ·6H 2 O and 100 mg of Eu(NO 3 ) 3 ·6H 2 O, in 5 mL of water are added to a solution of IGEPAL CO 520 (20 mL) dissolved in 100 mL of cyclohexane under vigorous stirring. In a separate suspension, NaH 2 PO 4 ·H 2 O (700 mg) in 5 mL of water is combined with a solution of IGEPAL CO -

PAGE 65

65 520 (20 mL) dissolved in 100 mL of cyclohexane. The two suspensions were stirred separately for 1 h at room temperature, following which they were combined by the dropwise addition of the phosphate suspension to the metal ion suspension within a time period of 30 min. Once the addition was complete, the mixture was stirred vigorously for 3 h before the microemulsion was broken with 200 mL of acetone. The nano particles were collected by centrifugation and washed with water and acetone. The surface modification of the nanoparticles was performed by dispersing 20 mg of the nanoparticles in 1 mL of water followed by the addition of 30 mg of Gd(NO 3 ) 3 ·6H 2 O and sonic ating for 20 min. To this suspension, 3 mL (pH 7) of 60 mg of PMIDA was added. The mixture was left to sonicate for another 1 h, after which the nanoparticles were collected by centrifugation, washed with water, and redispersed in water for later use. Samp le 1 : 40 ± 4 nm, white color; ICP (Gd/Eu in mg/L) 32.54/7.58; XRD, the XRD pattern can be equally well indexed to the hexagonal phase of either GdPO 4 ·H 2 O (JCPDS no. 39 232) or EuPO 4 ·H 2 O (JCPDS no. 20 1044); IR, <1250 cm 1 characteristic of (PO 4 ) 3 vibratio ns, 2920 and 2851 cm 1 as ) and s 2 ) groups of PMIDA. Sample 2 Hydrothermal methods were used, combining a mixture of Eu 3+ and Gd 3+ ions with (NH 4 ) 2 HPO 4 . 102 A solution of 400 mg of Gd(NO 3 ) 3 ·6H 2 O and 100 mg of Eu(NO 3 ) 3 ·6H 2 O in 15 mL of water was combined with a solution of 0.5734 g of (NH 4 ) 2 HPO 4 in 15 mL of water and stirred for 20 min before transferring the entire contents into a 130 mL Teflon lined autoclave. The autoclave w as sealed and mainta ined at 130 °C for 14 h. After cooling to room temperature, the precipitate was separated by centrifugation and washed with water. The same surface modification

PAGE 66

66 described for sample 1 was used. Sample 2 : 71 ± 14 nm, white color; ICP (Gd/Eu in mg/L) 41.20/10 .05; XRD, all of the peaks can be indexed to the hexagonal phase of GdPO 4 ·H 2 O (JCPDS no. 39 232) or EuPO 4 ·H 2 O (JCPDS no. 20 1044); IR, <1250 cm 1 characteristic of (PO 4 ) 3 vibrations, 2920 and 2851 cm 1 associated with the asymmetric as s 2 ) groups of PMIDA. Sample 3 The same process described for sample 2 was used, changing the precursor solutions to 400 mg of Gd(NO 3 ) 3 ·6H 2 O plus 100 mg of Eu(NO 3 ) 3 ·6H 2 O in 10 mL of water and 700 mg of NaH 2 PO 4 ·H 2 O in 10 mL of water. The same surface modification was used. Sample 3 : 145 ± 36 nm, white color; ICP (Gd/Eu in mg/L) 45.34/11.41; XRD, all of the peaks can be indexed to the hexagonal phase of GdPO 4 ·H 2 O (JCPDS no. 39 232) or EuPO 4 ·H 2 O (JCPDS no. 20 1044); IR, <1250 cm 1 characteristic of (PO 4 ) 3 vibrations, 2920 and 2851 cm 1 ass as s ) 2 ) groups of PMIDA. In Vitro and in Vivo Fluorescent Imaging A confocal laser scanning microscope (Olympus FV 500 IX81) was used to record cellular images. HeLa cel ls were plated in a 35 mm confocal dish (glass bottom dish) and grown to around 60% confluency for 24 h before the experiment. Cells were washed three times with DMEM, supplied with 1 mL DMEM, and then incubated with modified Eu 0.2 Gd 0.8 PO 4 · H 2 O nanoparticles. After 24 h of incubation, cells were washed three times with DMEM, supplied with 1 mL of DMEM, and then subjected to confocal fluorescence imaging. An argon laser (excitation wavelength of 488 nm) was used with a 60× oil dispersion objec tive with a long pass filter at 560IF.

PAGE 67

67 The same confocal laser scanning microscope was used to record in vivo fluorescent images of a small animal, C. elegans , with excitation at 488 nm and a 560IF or 32 mM in Gd 3+ E. coli ) in S basal medium. Two hundred microliters of these solution s were pipetted out onto the assay plate and left to dry, which served as food for the worms. For the control group, no nanoparticles were added. The worms were starved for 48 h before they were fed for 4 h, followed by transferring into a 2 mL tube with w ater and holding at 65 °C for 5 min. Once prepared, the worms were imaged with a 10× objective. Results and Discussion Particle Characterization TEM images (Figure 3 1 ) of each preparation of Eu 0.2 Gd 0.8 PO 4 ·H 2 O show uniform rodlike particles. The size distr ibutions, shown in the histograms of Figure 3 1 , give average particle sizes of 40 ± 4, 71 ± 14, and 145 ± 36 nm for samples 1 3 , respectively. The structure and composition of the particles were determined using powder XRD in conjunction with ICP AES and EDS. The XRD patterns (Figure 3 2 ) from each sample can be indexed to the isostructural hexagonal phase s of either GdPO 4 ·H 2 O (JCPDS no. 39 232) or EuPO 4 ·H 2 O (JCPDS no. 20 1044), and no impurity phase was detected. 103 The resolution of the diffraction patte rns does not discern between a homogeneous phase and a mixture of phases. However, EDS maps (Figure 3 2 ) clearly show the Eu and Gd ions are uniformly distributed within crystallites. Particle surface modification provides colloidal stability and can be us ed to impart other functions such as biocompatibility or mechanisms for vectoring. The most common strategies include polymer modification and silica shell coating. 83 , 104 In the

PAGE 68

68 current study, we use the small molecule PMIDA for postsynthesis surface modif ication following reports of its successful use for other metal phosphate and metal oxide particles. 105,106 The PMIDA molecule is expected to bind the particle surface through the divalent phosphate, 86 , 107 consistent with how it binds to Ca 3 (PO 4 ) 2 and iron oxide surfaces. 105,106 After surface modification, the phosphate groups strongly bind with metal ions on the surface, leaving free carboxyl groups at the periphery, producing a well dispersed suspension through electrostatic repulsions. P revious study of GdPO 4 ·H 2 O particles modified with phosphate terminated oligonucleotides showed that postsynthesis modification with excess Gd 3+ to ensure a surface rich in Gd 3+ led to increased binding of the oligonucleotide in addition to providing more water accessible sites to influence the relaxivity. 86 This result was also observed here for PMIDA modification (Figure 3 3) . FT IR spectra confirm successful surface modification with PMIDA. Figure 3 4 compares FT IR spectra of Eu 0.2 Gd 0.8 PO 4 ·H 2 O before and after surface m odification. The spectra are dominated by vibrations from the lattice PO 4 3 groups and water. 108 However, new bands at 2920 and 2851 cm 1 as ) and s ) stretching vibrations of methyl 2 ) groups of PMIDA. 106 , 109 Surface modification with PMIDA provides an easy and direct procedure for obtaining dispersible and biocompatible particles. Furthermore, the terminal carboxyl groups of the PMIDA can be easily used for later functionali zation with other biologically active molecules. 105,106,110 Size Dependent MRI Relaxivity To begin to understand some of the factors that contribute to the range of relaxivity behavior reported for GdPO 4 ·H 2 O and Eu x Gd 1 x PO 4 ·H 2 O particles, samples of

PAGE 69

69 three different average particle sizes were prepared. Magnetic resonance relaxivity measurements of different concentrations of Eu 0.2 Gd 0.8 PO 4 ·H 2 O particles dispersed in water were performed at 1.4 1 T to determine T 1 and T 2 values (Figure 3 5 and Table 3 1 ). The specific relaxivities were calculated according to where T i app is the apparent T 1 or T 2 , r i represents relaxivities r 1 or r 2 , and [CA] is the concentration of contrast agent. 111,112 The longitudinal relaxivity , r 1 , ranges from 2.34 mM 1 s 1 for the largest particles to 6.13 mM 1 s 1 for the smallest. The data in Figure 3 5 and Table 3 1 clearly show that the smaller particles give rise to higher molar relaxivity. Although the exact knowledge of how the gadolini um phosphate nanoparticles induce water proton relaxation is unknown, we would speculate that Gd 3+ at the outer surface should contribute more to the relaxivity than ions in the core of the particle as water molecules have direct access to complexes at the surface. The correlation between the S/V ratio and relaxivity of Eu 0.2 Gd 0.8 PO 4 ·H 2 O nanoparticles is plotted in Figure 3 6 . As expected, the smaller particle of sample 1 with the largest surface to volume ratio of 0.49 nm 1 shows the highest relaxivity, wh ich then decreases nearly linearly as the S/V ratio decreases, indicating that the availability of surface sites is primarily responsible for inducing relaxation. The correlation with S/V ratio is in line with other studies of nanoparticle based contrast a gents. Relaxivity studies of Gd 2 O 3 report that particles smaller than 10 nm have much higher r 1 than particles on the order of 30 nm diameter. 68,84,95 Similarly, r 1 values were found to be higher for smaller MnO particles, leading to speculation that the

PAGE 70

70 p aramagnetic Mn 2+ ions on the surface of the nanoparticles are responsible for the shortening of the T 1 relaxation times. 98 Both Rieter et al. 99 and Nishiyabu et al. 101 report on relaxivity studies with Gd 3+ MOF systems. In both cases, an inverse dependence on nanoparticle size was observed, with the smaller nanoparticles possessing a larger r 1 relaxivity, suggesting that the Gd 3+ ions at or near the surface are primarily responsible for the observed relaxivities. Whether the availability of surface ions is the only important factor is unclear from these observations, but certainly surface sites are the principal agents affecting the relaxivity with these paramagnetic metal oxide and metal phosphate systems. Although the molar relaxivity decreases for larger particles, the relaxivity per particle can be significantly greater than for the small particles. Such a characteristic could allow for enhanced local contrast while using particle based contrast agents, which would be highly beneficial for targeted imagin g. 65 Recasting the relaxivity in units of relaxivity per particle, 99 (mM particle) 1 s 1 , shows the enhancement (Figure 3 6 ). The 1 s 1 for the 40 nm particle to 1 s 1 for the 145 nm particles (Figure 3 6 ). Even for the larger particles, the per ion relaxivity is of the same order as for known gadolinium chelates, 89,96,113 but with so many more ions in a particle, they provide a mechanism to concentrate and localize the induced relaxation effectively. With r 1 = 6.13 mM 1 s 1 and r 2 = 7.78 mM 1 s 1 , the low value of r 2 / r 1 = 1.27 suggests usefulness as a positive contrast agent. To demonstrate this, sample 1 , with the highest longitudinal relaxivity, was used to obtain T 1 weighted MR images. Figure 3 -

PAGE 71

71 7 shows the T 1 weighted images obtained at 4.7 T at concentrations ranging up to 8 mM. Enhanced MRI brightness is observed with the increase in particle concentration. It is useful to compare the relaxivity data for the materials descr ibed in this study to those of other gadolinium phosphate and Eu 3+ containing gadolinium phosphate systems. Eu 0.2 Gd 0.8 PO 4 ·H 2 O samples 1 3 are compared to other reported particle systems in Table 3 2 . Using particles containing 2% Eu 3+ , Rodriguez Liviano et al. 97 reported relaxivity values of r 1 = 0.19 mM 1 s 1 and r 2 = 17.33 mM 1 s 1 for Eu 0.02 Gd 0.98 PO 4 ·H 2 O, and by taking advantage of a large r 2 / r 1 ratio, they used the particles to effectively generate negative contrast in MRI phantom images at 9.4 T. Inter estingly, studies of GdPO 4 ·H 2 O also report a wide range of relaxivities and r 2 / r 1 ratios, with some studies emphasizing utility as positive contrast agents with others focusing on a large r 2 value and using the particles to demonstrate negative contrast. P article size explains some of the differences, but it is clear that there are also other factors that influence the relaxivity behavior of the gadolinium phosphate system. In addition to the different particle sizes represented in Table 3 2 , each study use s a different surface modifier. Details of the significance of these factors remain to be resolved. Fluorescence Imaging Suspensions of the particles exhibit strong visible fluorescence in the red region under UV irradiation, a result of the Eu 3+ in the p articles ( Figure 3 8 ). 89 , 97 Such fluorescence properties have attracted considerable attention and show great potential for bioimaging applications. For example, Ren et al. 89 used Eu 3+ doped GdPO 4 nanorods modified with PVP to observe red luminescence from labeled HeLa cells. In addition, Patra et al. 94 , 114 successfully used pure EuPO 4 nanorods without a surface

PAGE 72

72 modifier to label both 786 O cells and HUVEC cells. To demonstrate the potential of the Eu 0.2 Gd 0.8 PO 4 ·H 2 O system as a dual imaging agent, the HeLa cells were imaged with confocal laser scanning microscopy following incubation with the PMIDA modified particles. After treatment, strong fluorescence was associated with nanoparticle uptake by the cells (Figure 3 9 ). Further evidence of the fluorescence i maging potential of the particles is presented in Figure 3 10 showing confocal laser scanning microscopy images of C. elegans following inclusion of the Eu 0.2 Gd 0.8 PO 4 ·H 2 O particles with different concentrations in the culture medium of the worms. Bright re d fluorescence was observed due to the presence of the Eu 3+ doped nanoparticles in C. elegans , where a stronger intensity can be seen when higher particle concentrations were used. No fluorescence was observed in the red region in the control. Together wit h the cell imaging and MRI contrast capability, these experiments demonstrate the potential of the PMIDA modified Eu 0.2 Gd 0.8 PO 4 ·H 2 O particle system for dual imaging. Cytotoxicity Measurements When evaluating the clinical potential of nanoparticles, their t oxicity is an important factor that should be taken into consideration. Therefore, we have studied the cytotoxicity of the as prepared nanoparticles. Cytotoxicity testing was performed on PMIDA modified Eu 0.2 Gd 0.8 PO 4 ·H 2 O sample 1 using HeLa cells and A549 cells in solution up to 1 mM Gd 3+ . As shown in Figure 3 11 , the average cell viability is 93 % and 90% for HeLa cells and A549 cells, respectively. Therefore, PMIDA coated Eu 0.2 Gd 0.8 PO 4 ·H 2 O nanoparticles are nontoxic up to 1 mM Gd 3+ .

PAGE 73

73 Conclusions The phospha te containing PMIDA molecule stabilizes colloidal suspensions of particles of the mixed lanthanide Eu 0.2 Gd 0.8 PO 4 ·H 2 O, which with fluorescent Eu 3+ ions and paramagnetic Gd 3+ ions can be used for both fluorescence imaging and to generate MRI contrast. Relaxi vity values vary with particle size, increasing with available surface area, implying that access to Gd 3+ complexes at the surface is the principal factor influencing molar relaxivity. On the other hand, as the particles become larger, the relaxivity per p article increases, suggesting that larger particles might be useful for concentrating relaxivity effects in applications such as cellular or molecular imaging.

PAGE 74

74 Figure 3 1 . TEM images of PMIDA modified Eu 0.2 Gd 0.8 PO 4 ·H 2 O, samples 1 ( A ), 2 ( B ), and 3 ( C ); particle length distributions obtained from the TEM images ( D ) sample 1 : 40 ± 4 nm; ( E ) sample 2 : 71 ± 14 nm; and ( F ) sample 3 : 145 ± 36 nm.

PAGE 75

75 Figure 3 2. ( A ) XRD patterns of Eu 0.2 Gd 0.8 PO 4 ·H 2 O samples 1 3 and a JCPDS file of hexagonal GdPO 4 ·H 2 O (JCPDS no. 39 232) and EDS maps across a Eu 0.2 Gd 0.8 PO 4 ·H 2 O nanoparticle; ( B ) overlapped Gd and Eu detection; ( C ) gadolinium map only; and ( D ) europium map only.

PAGE 76

76 Figure 3 3. From left to right: Eu 0.2 Gd 0.8 PO 4 ·H 2 O nanoparticles; Eu 0.2 Gd 0.8 PO 4 ·H 2 O nanoparticles after modification with PMIDA only; Eu 0.2 Gd 0.8 PO 4 ·H 2 O nanoparticles after modification with both Gd 3+ and PMIDA. Before taking image, all the samples are left standing for 3 days. Nanoparticles after both Gd 3+ and PMIDA modification remain well suspended, wherea s the other two samples show sedimentation . Figure 3 4. FT IR spectra of Eu 0.2 Gd 0.8 PO 4 ·H 2 O nanoparticles before (red) and after (blue) modification with PMIDA.

PAGE 77

77 Figure 3 5. Plot of the proton relaxation rate (1/T 1 ) of water suspensions of Eu 0.2 Gd 0.8 PO 4 ·H 2 O nanoparticles at various Gd 3+ concentrations and the corresponding relaxivites for samples 1 (40 nm), 2 (71 nm), and 3 (145 nm). The measurement was performed at 1.41 T.

PAGE 78

78 Figure 3 6. ( A ) Molar relaxivity, r 1 , as a function of the surface to volume ra tio, S/V, for samples 1 3 . ( B ) Relaxation enhancement per particle as a function of nanoparticle size. Figure 3 7. T 1 weighted MR images (4.7 T) of Eu 0.2 Gd 0.8 PO 4 ·H 2 O particles from sample 1 at various Gd 3+ concentrations.

PAGE 79

79 Figure 3 8. Emission spectra of Eu 0.2 Gd 0.8 PO 4 ·H 2 O nanoparticles (excitation wavelength 250 nm); The inset shows an aqueous sample solution before and after irradiation with a UV lamp.

PAGE 80

80 Figure 3 9. Confocal laser scanning microscopy i mages of Hela cells incubated (A) with and (B ) with out sample 1 nanoparticles. Figure 3 10. Confocal laser scanning microscopy images of C. elegans fed (A ) with the higher co ncentration of nanoparticles, (B ) with the lower concen tration of nanoparticles, and (C ) without nanoparticles.

PAGE 81

81 Figure 3 11. In vitro cell v iability of (A) Hela cells (B ) A549 cells incubated with Eu 0.2 Gd 0.8 PO 4 ·H 2 O sample 1 at different concentrations (0, 0.25, 0.5, 0.75 and 1mM Gd 3+ ). Table 3 1. Size dependent relaxivity data for s amples 1 3 . Sample Length (nm) Width (nm) Molar Relaxivity r 1 /[Gd 3+ ] (mM 1 s 1 ) Relaxation Enhancement Per Nanoparticle r 1 /NP (mM particle) 1 s 1 Surface to Volume Ratio (nm 1 ) 1 40±4 9±2 6.13±0.1 0.49±0.13 2 71±14 13±4 2.56±0.1 0.34±0.14 3 145±36 14±4 2.34±0.1 0.30±0.13

PAGE 82

82 Table 3 2. Chemical and physical properties of GdPO 4 based contrast agents . Name Core material Surface Morphology Length×Width (nm×nm) r 1 /[Gd 3+ ] (mM 1 s 1 ) r 2 /[Gd 3+ ] (mM 1 s 1 ) B0 (T) PGP/dextran K01 85 GdPO 4 Dextran Nanorod (20 30)×(6 15) 13.93 14.96 0.47 Sample 1 Eu 0.2 Gd 0.8 PO 4 PMIDA Nanorod 40×9 6.13 7.78 1.41 Sample 2 Eu 0.2 Gd 0.8 PO 4 PMIDA Nanorod 71×13 2.56 4.54 1.41 Sample 3 Eu 0.2 Gd 0.8 PO 4 PMIDA Nanorod 145×14 2.34 4.43 1.41 GdPO 4 78 GdPO 4 PVP Nanorod 100×10 2.08 4.7 GdPO 4 75 GdPO 4 DNA Nano rod 50×10 0.2 12.8 14.1 Eu 0.02 Gd 0.98 PO 4 86 Eu 0.02 Gd 0.98 PO 4 No Nanocube 75×75 0.19 17.33 0.47

PAGE 83

83 CHAPTER 4 POLYAMIDOAMINE AN D ITS DERIVATIVES MEDIATED SIZE CONTROLLED SYNTHESIS OF GADOLINIUM PHOSPHATE NANOPARTICLES: COLLOIDAL PROPERTIES AND MRI RELAXIVITY ST UDY Preface Polyamidoamine (PAMAM) and its derivatives, including hyperbranched polyamidoamine (HyPAM) , HyPAM PEG, and also HyPAM C 18 PEG, were used to synthesize gadolinium phosphate nanowires under mild conditi ons. The resulting particles were coated by the polymer, and the control of particle size was achieved by adjusting polymer concentration . R elaxivity measurements of HyPAM coated GdPO 4 nanowires reveal an optimum particle size, showing relaxivity values as high as 55 ± 9 mM 1 s 1 for r 1 and 67 ± 11 mM 1 s 1 for r 2 . The colloidal stability of these hybrid systems were then optimized through the use of functionalized core shell polymers containing PEG segments and C 18 PEG segments, offer ing the possibility of imparting additional function s into the pol ymer particle hybrids. This work is expected to be submitted to scientific journal for publication in the near future. 2 Introduction As discussed in Chapter 3, gadolinium phosphate nanoparticles with sizes ranging from ~ 40 to ~ 140 nm have been successfully synthesized and employed as a dual imaging contrast agent after doping with europium. Nev ertheless, the MRI properties of GdPO 4 nanoparticles with sizes down to few nanometers are still unrevealed , due to the difficulty in synthesizing ultrasmall particle s under regular synthetic conditions. Frangville, C.; Gallois, M.; Li, Y.; Nguyen, H. H.; Viguerie, N. L.; Talham, R. T.; Mingotaud, C.; Marty, J. Manuscript in preparation.

PAGE 84

84 To achieve this objective, PAMAM was initially introduced as the surface coating molecules to mediate the synthesis of GdPO 4 nanoparticles. Due to the structural similarity b etween PAMAM and HyPAM as well as the lower cost and easy preparation of HyPAM , HyPAM instead of PAMAM is selected as the surface coater for further relaxivity study . Similar to gold, silver or platinum nanoparticles for which precise particle size control has been achieved, 115 118 GdPO 4 nanoparticl es of different sizes were synthesized by complexing with HyPAM, that is, b y mixing Gd 3+ with the polymer and replacing chemical reduction with a precipitation step. In addition, p olymers with HyPAM core functionalized with different shells using PEG were also synthesized to add stability, biocompatibility , 119 and drug lo ading capacity to the hybrid nanoparticles . The ability of such structures to both stabilize and control particle growth was discussed in this C hapter . Materials and Methods Materials Gadol inium (III) nitrate hexahydrate, sodium phosphate dibasic, and Nile Red (9 diethylamino 5 benzo phenoxazinone) were purchased from Sigma Aldrich Co. Ltd. at generation 4 was pu rchased from Polymer Source Inc . Water was purified through a filter and ion exchange resin using a Purite device (resistivity 18.2 Polymer Synthesis Preparation of h yperbranched p olyamidoamine c ore The synthesis of the H y PAM cores were carried out following previously published work . 115,117,120 T he HyPAM was obtained by stirrin g the mixture of tris(2 aminoethyl)amine (17.1 mmol) with tris(2 di(methylacrylate)aminoethyl)amine (1.7

PAGE 85

85 mmol) under argon atmosphere at 75°C for two days . HyPAM : 1 H NMR (DMS O d 6 , 500 MHz): 2.24 (m, N CH 2 CH 2 N); 2.45 (m, CO NH CH 2 CH 2 N); 2.51 (DMSO and m, CH 2 NH 2 ); 2.63 (m, N CH 2 CH 2 CO); 2.7 (m, N CH 2 CH 2 CO); 3.1 (m, CH 2 NH CO ); 3.45 (NH); 13 C NMR (DMSO d6, 500 MHz): 33.79 (N CH 2 CH 2 N); 37.34 ( CH 2 NH CO ); 41.62 (N CH 2 CH 2 CO); 46.15 ( CH 2 NH 2 ); 47.43 (N CH 2 CH 2 CO); 56.61 (CO NH CH 2 CH 2 N); 172.0 ( CO ); IR (HyPAM): = 3345, 3283, 3078, 2943, 2858, 2824, 1645, 1557, 1460, 1354, 1291, 1096, 1062, 946, 915 cm 1 . Double and s ingle s hell s ynthesis T he synthesis of the b uilding block PEG 750 C 17 COOH was performed with a method that has been previously described , 121 while t he single shell building block mPEG 750 COO H was prepared by oxidation with Jones reagent. 1 H NMR (400 MHz, CDCl 3 ) : 4.01 (s, O CH 2 COOH); 3.80 3.44 (m large, PEG backbone); 3.35 (s, O CH 3 ); 3.04 (q, Et 3 N traces: N( CH 2 CH 3 ) 3 ); 1.25 (t, Et 3 N traces: N(CH 2 CH 3 ) 3 ). 13 C NMR (100 MHz, CDCl 3 ) : 174.19 ( O CH 2 COOH ); 71.86 ( CH 2 O CH 3 ); 70.49 (br PEG ); 70.00 ( O CH 2 COOH ); 58.95 ( O CH 3 ) ppm. Grafting of PEG 750 C 17 COOH or PEG 750 COOH onto the H y PAM c ore To couple the shell to HyPAM , the t erminal carboxyl groups, after activation by coupling with N hydroxysuccinimide (SuOH) using N, N' dicyclohexylcarbodiimide (DCC) , was added to the hyperbranched core HyPAM in methanol and stirred for 24 h . H y PAM PEG: 1 H NMR (400MHz, D 2 O) : 3.71 (br, O CH 2 CH 2 O from mPEG); 3.34 (s, O CH 3 from mPEG); (3.39 (br, CO NH CH 2 ); 2.79 (br, N CH 2 CH 2 CO); 2.79 (br, NH 2 CH 2 ); 2.67 (br, N CH 2 CH 2 NH CO); 2.67 (br, N CH 2 CH 2 N ); 2.45 (br, CH 2 CO ) ppm. 13 C NMR (100 MHz, D 2 O) : 174.5 ( NH CO CH 2 from H y PAM core and core shell link); 71.02 ( CH 2 O CH 3 ); 69.7 ( O CH 2 C H 2 O); 60.4 ( O CH 3 ); 58.1 (NH CO -

PAGE 86

86 CH 2 C H 2 O); 51.4 53.6 (N CH 2 CH 2 N and CONH CH 2 CH 2 N); 52.4, 49.2 50.3 (N CH 2 CH 2 CO); 37.1 (CO NH CH 2 CH 2 O) ppm. IR (KBr): (cm 1 ) = 1107, 1539, 1644, 1733, 3428. HYPAM C 18 PEG: 1 H NMR (400MHz, CDCl 3 ) : 4.18 (t, COO CH 2 ); 3.82 3.40 (br , O CH 2 CH 2 O from mPEG ); 3.35 (s, O CH 3 ); 3.21 (br, CO NH CH 2 ); 2.69 (br, CO NH CH 2 CH 2 N from NHCO CH 2 CH 2 N from HYPAM); 2.52 (br, N CH 2 CH 2 N and NH CH 2 CH 2 NH from HYPAM); 2.30 (t, CH 2 COO ); 2.19 (br, NHCO CH 2 ); 1.58 (br, NHCO CH 2 CH 2 (CH 2 ) 12 CH 2 CH 2 COO); 1.21 (m, NHCO CH 2 CH 2 ( CH 2 ) 12 CH 2 CH 2 COO ) ppm. 13 C NMR (100 MHz, CDCl 3 NH CO CH 2 from HYPAM core and core shell link); 173. 7 ( CH 2 COO ); 71.9 ( CH 2 O CH 3 ); 70.5 ( O CH 2 CH 2 O); 69.1 ( COO CH 2 CH 2 ); 63.3 ( COO CH 2 ); 58.9 ( O CH 3 ); 53.4 55.1 (N CH 2 CH 2 N); 51.1 52.4 (CONH CH 2 CH 2 N); 49.4 50.6 (N CH 2 CH 2 CO); 37.6 ( CO NH CH 2 CH 2 N ); 36.4 (CO NH CH 2 from the core shell li nk); 34.1 (NH CO CH 2 ); 28.8 30.9 (NHCO CH 2 CH 2 ( CH 2 ) 12 CH 2 CH 2 COO ); 25.9 ( NH CO CH 2 CH 2 ); 24.8 ( CH 2 CH 2 COO ) ppm. IR (KBr): (cm 1 ) = 1032, 1530, 1644, 3280. Gd 3+ /HyPAM I nteractions Stock solutions of HyPAM (2 × 10 4 M) and Gd(NO 3 ) 3 (4 × 1 0 4 M) were prepared in D 2 O. Aliquots of 1 mL were prepared by adding 500 µL of HyPAM, 0 to 500 µL of Gd(NO 3 ) 3 and completed at 1 mL with D 2 O. The pH of each aliquot was then carefully adjusted between 8 and 9 with NaOD (1M). 1 H NMR spectra were recorded on a BrukerARX500 equipped with a cryogenic probe (400MHz for 1 H). Calibration was performed using the chloroform peak at 7.26 ppm for 1 H. GdPO 4 / Pol ymer Nanowire Synthesis and A nalysis Two precursors were prepared for the synthesis of HyPAM based nanowire s . Typically, precursor 1 was prepared by adding 250 3 ) 3 (1 × 10 3 M) stock

PAGE 87

87 solution to 1 mL o f water containing various polymer concentrations (from 0 to 8 µM) . Precursor 2 was prepared by dissolving 2 PO 4 (1 × 10 3 M) in 1.25mL of water . The pH of precursor 1 was adjusted to pH 8 9 with NaOH (0.01 M) or HCl (0.01 M). Then, precursor 2 was added dropwise to precursor 1 under stirring , using a s yringe pump with flowrate = 1. 6 mL/h, (Neolus needle , Ø 0.8 mm) . A dro p of each aliquot was then droppe d on a carbon coated copper TEM grid (Ted Pel la Inc.) and left to dry under air. The samples were analyzed with a MET Hitachi HT7700 transmission electron microscope operating at 80 kV. Size distribution histograms were determined by using magnified TEM images and by measuring a minimum of 200 partic les of each sample, using ImageJ software . NaCl Stability S tudy 100 µL of NaCl (0.02 M, 0.2 M and 2 M) stock solutions were added to 100 µL of GdPO 4 nanowires aliquots synthesized with HyPAM, HyPAM PEG, or HyPAM C 18 PEG (5 µM). These solutions were left 15 days to equilibrate and then analyzed though DLS using a Zetasizer Nano ZS (Malvern Instruments, Ltd, UK) with integrated 4 mW He Ne method (NNLS) to obtain the distribution of diffusion coefficients of the solutes. The apparent equivalent hydrodynamic diameter was then determined using the Stokes Einstein equation. Z average was obtained from five different runs of the number plot. Standard deviations were evaluated from the diameter distribution. Drug Loading C apacity 1 µL of Nile Red (9 diethylamino 5 benzo phenoxazinone) stock solution (1 × 10 3 M in THF) were added on aliquots of GdPO 4 nano wires synthesized with HyPAM, HyPAM PEG, or HyPAM C 18 PEG (5 µM) (200 µL completed at 1 mL with

PAGE 88

88 water). Fluorescence measurements were then performed on a Photon Technology International photon counting fluorescence spectrophotometer with a xenon lamp EIMAC of 175W. The spectrofluorimeter was set with excitation and emission slits of 4 nm. Excitat ion and emission spectra were record ed separately em =660 nm and ex =590 nm. Results and Discussion Particle Characterization The TEM image and electron diffraction pattern of GdPO 4 / HyPAM nanoparticles (Figure 4 1) suggests formation of amorphous GdPO 4 nanowires (Figure 4 2) , although the exact exten t of hydration was not determined (Ta ble 4 1 and Table 4 2) . By using PAMAM as the surfactant, ultrasmall GdPO 4 nanowire s were successfully obtained . It was also observed that the particle size decreased from ~15 nm to ~8 nm by adding more polymer during t he synthetic process (Figure 4 3) , providing an efficient method for precise size control of the material. In addition to PAMAM, HyPAM shows similar results in terms of size control. As shown in Figure 4 4 and Figure 4 5 , the average length of the nanowire s can be adjusted by chan ging the polymer concentration. The average length of the nanowires decreases from ~ 120 nm to ~ 6 nm, with nearly isotropic nanoparticles forming at the higher polymer concentration (above 3.5 µmol L 1 ). The ability of these polymer s to control particle sizes during the synthesis process can be explained by the interaction between tertiary amines on HyPAM and Gd 3+ prior to the addition of sodium phosphate, confirmed by the shift and widening of the 1 H NMR vicinal proton resonance of the tertiary amine upon adding Gd 3+ to the HyPAM polymers ( Figure 4 6 ). Also , the presence of HyPAM stabilizes the nanowires , as suggested by their higher colloi dal stability after adding the polymer . Both factors contribute to control

PAGE 89

89 over the nucleation and growth processes upon the addition of phosphate. This polymer mediated synthesis is the first report for controlling sizes of GdPO 4 nanoparticles down to 5 nm , fabricated under aqueous, room temperature conditions. The precise size control allows syste matic analyses of size effect s of GdPO 4 nanoparticles/nanowires as MRI contrast agents . Size D ependent Relaxivity Study Transversal and longitudinal relaxation times T 1 and T 2 were recorded to evaluate the size effect of the prepared GdPO 4 nanowires . Dialy sis and Gd ion titration were performed on the samples before relaxivity measurements. There is a clear dependence of the length of the nanowires on r 1 and r 2 values . As shown in Figure 4 7, the re is an optimum particle size of 23 nm which shows the highes t relaxivity value , similar to the trends reported by Park et al. 95 for Gd 2 O 3 nanoparticles. For the hybrid GdP O 4 /HyPAM nanowires, the optimal length was found to be 23 ± 11 nm with exceptionally high relaxivity values of r 1 = 55 ± 9 mM 1 s 1 and r 2 = 67 ± 11 mM 1 s 1 . These values are higher than the conventional molecule based T 1 contrast a gents and most gadolinium based particles explored to date. For example, Hifumi et al. 96 previously reported gadolinium based hybrid nanoparticles (PGP/dextran K01) wit h r 1 of 13.9 mM 1 s 1 . Johnson et al. 65 has studied relaxivity dependence of NaGdF 4 nanoparticles of different sizes, with the smallest size of 2.5 nm showing r 1 of 7.2 mM 1 s 1 . Another example is ultrasmall gadolinium hydrated carbonate nanoparticles, st udied by Liang et al. , 122 showing high r 1 of 34.8 mM 1 s 1 . The particle size dependences of r 1 and r 2 show a maximum for the 23 nm particle sample, with lower values of relaxivity for bot h larger and smaller particles. Similar observations of an optimal p article size have been observed for other particle

PAGE 90

90 systems, 95 , 123 including NaGdF 4 and Gd 2 O 3 , and is attributed to contributing factors influencing relaxivity that have opposite particle size corelatio ns. One of the possible factors that influences the rel axivity value is the availability of Gd 3+ ions at the surface, as direct chemical exchange of water molecules is the largest co ntributor to proton relaxation. As discussed in Chapter 3, t he surface to volume ratio increases as particle size gets smaller, a ccounting for the most often observed increase in relaxivi ty as particles become smaller. On the other hand , t umbling times get longer as the hydrodynamic v olume of the particle increases. 57, 65 ,1 24 ,1 25 S o , for particles in a certain range of sizes, relaxiv ity inc rease s as particles get bigger. These opposing contributions are then expected to give rise to an optimum particle size for any set of conditions, as observed in Figure 4 7 . I t is important to realiz e that correlation times will be affected by other other parameters, also, such as medium viscocity and applied magnetic field strength, so any particle size will depend on the application . Since the tumbing time depends on the hydrodynamic volume of the composite polymer particle object, the po lymer mediated preparation affords the potential for tuning the response through synthetic variation of both the size of the inorganic nano particle s and the thickness of the polymer coating. Core S hell HyPAM Polymer The hyperbranched polymer mediated synt hesis of GdPO 4 was extended to the functionalized core shell HyPAM analogues, HyPAM PEG and HyPAM C 18 PEG, in order to confer additional properties such as biocompatibility, additional colloidal stability , and drug loading capacity. Similar size contr ol wa s achieved with these two hyperbranched polymers (Figure 4 8) .

PAGE 91

91 The stability of colloidal suspensions of the polymer stabilized gadolinium phosphate hybrids was analyzed with dynamic light scattering, measuring the average hydrodynamic radius under differe nt conditions of added salt ( Figure 4 9 ) . Whereas GdPO 4 nanowires coated by HyPAM aggregate upon addition of NaCl, the PEG functionalized core shell polymers ensure high stability of the polymer particle hybrids against ionic strength, up to 1 M NaCl. The suc cessful stabilization of the GdPO 4 nanowires by the hyperbranched polymers is in line with studies of other hybrid organic/inorganic system s. 116,117,126,127 Furthermore, other examples have shown that pegylated hybrid systems can be dried and easily red ispersed in water or organic solvents compatible with the PEG moieties, 118,120 a property that could be exploited for storage or transport of these potential contrast agents. The polymer particle hybrids can impart additional functions beyond solub ility an d colloidal stability. For example, the hydrophobic C 18 layer of core shell shell HyPAM C 18 PEG colloids has also been exploited for it s drug loading capacity. To highlight the possibility of this added function, the well known polarity probe Nile Red 128 1 30 was added as a surrogate for a hydrophobic drug and the resulting fluorescence intensities were recorded to assess the potential for molecule uptake ( Figure 4 10 ). As expected, the GdP O 4 /HyPAM and GdPO 4 / HyPAM PEG polymer particle hybrids do not exhibit any shift of their maximum emission wavelength compared to water or GdP O 4 controls as would be indicative of a change in environment of the Nile red, and thus do not possess any potential as drug carriers. On the other hand, the GdP O 4 /HyPAM C 18 PEG systems possess an apolar layer in which the Nile Red becomes localized, resulting in a shifted emission wavelength corresponding to a dielectric constant of

PAGE 92

92 combined with imagi ng probe. Lastly, r 1 and r 2 relaxivity measurements were performed for the GdP O 4 particles stabilized with the PEG con taining hyperbranched polymers. Comparisons of the GdP O 4 relaxivities for the different particles are gathered in Table 4 3. The GdP O 4 na nowires stabilized by HyPAM and HyPAM PEG have hydrophilic coatings and therefore exhibit similar r 1 and r 2 values, due to the similarly hydrated environment around the inorganic particle t hat these two coatings provide. In contrast, the HyPAM C 18 PEG stab ilized GdP O 4 nanoparticles exhibit lower r 1 and r 2 values, most likely as a result of the hydrophobic C 18 layer of the hyperbranched structure, which reduces the diffusion of water molecules between the particle surface and the bulk solvent. Nevertheless, the relaxivities of the HyPAM C 18 PEG stabilized particles are still quite rea s onable. Conclusions Hyperbranched polymers and functionalized core shell polymers were used to govern the synthesis of size controlled GdP O 4 nanowires in aqueous solutions at ro om temperature. The GdP O 4 nanowires were obtained in a range of 120 ± 39 nm down to 6 ± 2 nm with micromo lar concentrations of the HyPAM . Relaxivity measurements of the H y PAM coated particles over the range of GdP O 4 particle sizes revealed an optimal lengt h at ~ 23 nm for which promising r 1 and r 2 values were obtained (r 1 = 55 ± 9 mM 1 s 1 ; r 2 = 67 ± 11 mM 1 s 1 ). The functionalized core shell polymers HyPAM PEG and HyPAM C 18 PEG were then used to form similar polymer particle hybrids in order to improve stabi lity, biocompatibility , and potentially permit payload loading.

PAGE 93

93 Figure 4 1 . ( A ) TEM pictures of GdPO 4 /HyPAM nanoaprticles ; ( B ) Selected area electron diffraction pattern of the GdPO 4 /HyPAM nanoaprticles.

PAGE 94

94 Figure 4 2. ( A ) JCPDS file of GdPO 4 ·H 2 O (JCPDS n o. 39 232), ( B ) . JCPDS file of GdPO 4 ·1.5H 2 O (JCPDS no. 21 337)

PAGE 95

95 Figure 4 3. TEM images of GdP O 4 nanoparticles synthesized with increasing amounts of PAMAM ([ PAM AM]=1.7 85 µmol.L 1 ; pH adjusted = 8 9) , scale bars = 100nm. Figure 4 4. TEM images of GdPO 4 nanoparticles synthesized with increasing amounts of HyPAM ([HyPAM]=0 8 µmol.L 1 ; pH adjusted = 8 9) , before dialysis, scale bars = 200nm.

PAGE 96

96 Figure 4 5. Average lengths of GdP O 4 nanoparticles, determined from TEM analysis, versus increase concentration of HyPAM, HyPAM PEG, and HyPAM C 18 PEG.

PAGE 97

97 Figure 4 6. 1 H NMR study for [HyPAM]=1 × 10 4 M and increasing Gd 3+ concentration in D 2 O; ( A ) HyPAM proton classification , ( B ) . 1 H NMR spectra, ( C ) . 1 H NMR peak shifts .

PAGE 98

98 Figure 4 7. GdP O 4 /HyPAM size dependence on r 1 and r 2 relaxivities (1.4 1 T). Particle lengths were determined before dialysis.

PAGE 99

99 Figure 4 8. TEM pictures of GdPO 4 nanowires synthesized with increasing amount of ( A ) HyPAM PEG, ( B ) HyPAM C 18 PEG, scale bars = 200nm.

PAGE 100

100 Figure 4 9 . DLS measurements of t he GdP O 4 /hyperbranched polymers (5 µmol L 1 ) upon NaCl addition (0, 0.01, 0.1, 1 M). . Figure 4 10 . Normalized emission fluorescence spectra of 0.5 µM Nile Red added in to H 2 O (black), GdPO 4 dispersion (grey), GdPO 4 /HyPAM dispersion (yellow), GdPO 4 /HyPAM PEG dispersion (blue) and GdPO 4 /HyPAM C 18 PEG dispersion (red) . ex =590nm .

PAGE 101

101 Table 4 1. Comparison between the experimentally obtained diffraction spacing and the values reported in JCPDS no. 39 232 . (hkl) Intensity Reported d spacing (Å) Measured d spacing (Å) Ring 1 29.85 31.99 (2 0 0) (1 0 2) 100 98 2.991 2.795 2.861±0.013 Ring 2 42.44 42.84 (2 1 1) (0 03) 54 21 2.128 2.109 2.118±0.023 Ring 3 47.81 49.53 (3 0 1) (2 1 2) 21 64 1.901 1.839 1.865±0.018 Table 4 2. Comparison between the experimentally obtained diffraction spac ing and the values reported in JCPDS no. 21 337 . (hkl) Intensity Reported d spacing (Å) Measured d spacing (Å) Ring 1 29.84 32.10 (2 0 0) (1 0 2) 100 100 2.992 2.786 2.861±0.013 Ring 2 42.70 (0 03) 60 2.116 2.118±0.023 Ring 3 47.86 49.58 (3 0 1) (2 1 2) 40 70 1.899 1.837 1.865±0.018 Table 4 3. Relaxivity comparison of GdP O 4 coated with H y PAM and functionalized H y PAM polymers (field 1.4 1 T, [Polymer]=5 µmol L 1 ) . HyPAM HyPAM PEG HyPAM C 18 PEG r 1 (mM 1 .s 1 ) 26.2 0.3 23.8 0.8 13.9 0.1 r 2 (mM 1 .s 1 ) 33.5 0.3 36.8 1.2 17.2 0.2 r 2 /r 1 1.3 1.5 1.2 L ength (nm) 15 7 15 5 18 10

PAGE 102

102 CHAPTER 5 ONE STEP SYNTHESIS OF GRADIENT GADOLINIUM IRONHEXACYANOFERRATE NANOPARTICLES: A NEW PARTICLE DESIGN EASILY COMBINING MRI CONTRAST AND PHOTOTHERMAL THERAPY Preface A one step synthesis of Prussian blue nanoparticles possessing a concentration gradient of Gd 3+ counterions, g Gd PB , has been developed, and the potential for the particles to perform as both MRI positive contrast agents and photothermal therapy agents is demon strated. The synthesis of potassium /gadolinium ironhexacyanoferrate is performed under increasing concentration of Gd 3+ ions forming particles with a higher concentration of gadol inium toward the outer layers. The proton relaxivity (r 1 ) measured for the pa rticles is 12.3 mM 1 s 1 . T 1 weighted images of phantoms containing the particles show their po tential as MRI contrast agents. In addition, the Prussian blue host can rapidly and efficiently convert energy from near IR (NIR) light into thermal energy, allow ing g Gd PB to be used as a ph otothermal therapy agent. The photothermal properties are demonstrated by measuring temperature changes of particle suspensions under irradiation and by photothermal ablation of CCRF CEM cancer cells. Relevant sections in this C hapter were published in Nanoscale , and are copyright of Royal Society of Chemistry. The online abstract can be found at http://dx.doi.org/10.1039/C4NR06481J . 3 Introduction Nanoparticle platforms provide opportunity to realize multiple functions and this is also the case for particle based MRI contrast agents. 56, 131,132 In C hapter 3, by doping europium ions into gadolinium phosphate, the resulting particle can be used for both Reprinted with permission from Li, Y.; Li, C. H. ; Talham, R. T. Nanoscale 2015 , DOI: 10.1039/C4NR06481J.

PAGE 103

103 MRI and fluorescent imaging . Recently, Kang et al. 1 33 reported poly(acrylic aci d) modified lanthanide doped GdVO 4 hollow spheres as a carrier of the anticancer drug doxorubicin hydrochloride, combining MRI i maging and chemotherapy agents. Other biomedical tools, including fluorescent imagin g and photothermal therapy , have also been i ncorporated into multifunctional nanoparticles. 84,134 The combination of PTT and MRI allows simultaneous imaging and near IR induced local cancer ablation. 44 PTT converts absorbed light to heat, leading to thermal ablation of cancer cells, and because it i s minimally invasive, is recognized as a promising alternative or complement to conventional chemotherapy, surgery and radiotherapy cancer treatments. 44 48 Importantly, the ability of MRI to detect the size and location of tumor sites and also the potentia l to localize appropriately modified particles at affected sites can highly increase the efficacy and minimize side effects. 44 Several systems are under active investigations for synergistic MRI and PTT. The most common systems include gold, copper sulfide and reduced graphene oxide. 44 49 However, these materials have confronted li mitations to wide application. The thermal stability of gold nanoparticles is poor due to morphological changes after long periods of laser irradiation, 1 35 leading to a decre ase o f photothermal efficiency. Biosafet y is also an important concern. For example, copper sulfide and carbon based nanomaterials are found to be poorly metabolized. 136 At the same time, most of these examples are complex systems that combine different materia ls into single particles, thereby requiring multistep preparations, which can be time consuming and ultimately costly . 137 In this C hapter , a concentration gradient gadolinium ion containing Prussian blue nanoparticle , g Gd PB , is described that combines th e ability to enhance proton

PAGE 104

104 relaxivity for MRI with PTT capabilities. With Gd 3+ ions located closer to the outer surface of the particles, water molecules still have access to the paramagnetic metal center, yet less gadolinium is required than for a compar ably sized pure phase. Moreover, the Prussian blue host has recently been shown to be an efficient PTT agent, 138 and the new gradient particles are shown to be effective agents for the phototh ermal ablation of cancer cells. A similar result might be expect ed from a simple core shell strategy, but core shell structures are not always easy to achieve, as is the case for the current system where we were unable to prepare analogously sized potassium Prussian blue particles with a gadolinium Prussian blue shell. Furthermore, the gradient strategy is new for these coordination polymer solids and allows us to manipulate the ion distribution in the nanostructure in a single pot one step preparation, unlike most core shell preparations that require additional is olati on and redispersion steps. Finally, it is worth noting that PB has previously been approved by the FDA as an effective therapy agent in the treatment of cesium and thallium poisoning, 138 140 attesting to its biosafety. Materials and Methods Materials Gadol inium(III) nitrate hexahydrate (99.90%), c itric acid monohydrate, and a nhydrous f erric c hloride were purchased from Fisher Scientific (Waltham, MA, USA). Potassium ferrocyanide (Baker Analyzed, ACS reagent) was purchased from J.T. Baker (Phillipsburg, NJ, USA). MTS ( c ellTiter 96 ® aq ueous o ne s olution c ell p roliferation a ssay) was purchased from Promega Corporation (Madison, WI, USA). All commercial chemicals were used without further purification.

PAGE 105

105 Concentration G radient K 0.3 Gd 0. 2 Fe[Fe(CN) 6 ] 4.9 H 2 O, g Gd PB , Synthe s is The synthesis was performed at room temperature. During the reaction, a 150 mL aqueous solution of Gd(NO 3 ) 3 ·6H 2 O (0.271 g, 0.60 mmol) and citric acid (3.152 g, 15 mmol) was added dropwise to a continuously stirred Fe 3+ aqueous solution, initiall y containing FeCl 3 (0.097 g, 0.60 mmol) and citric acid (3.152 g, 15 mmol) in 150 mL of water, thereby gradually changing the Gd 3+ concentration. The mixed Fe 3+ Gd 3+ solution, with gradually increasing Gd 3+ concentration, and a 300 mL aqueous solution of K 4 Fe(CN) 6 ·3H 2 O (0.507 g, 1.20 mmol) were simultaneously pumped into 300 mL of nanopure water at a rate of 150 mL/h. The mixture was stirred for 18 h after complete addition. For collection and analysis, the particles were centrifuged at 1 2 00 0 rpm for 15 min , washed with water , and redispersed in ~ 100 mL water . The chemical composition was determined by i nductively c oupled p l asma a tomic e mission s pectro scopy and t hermogravimetric analysi s . K 0.3 Gd 0.2 Fe[Fe(CN) 6 ] 4.9H 2 O (citrate) 0.15 . 58 ± 9 nm. Dark blue powder . Yield: 190 mg (80%). IR (KBr): 2082 cm 1 (s, CN , Fe III NC Fe II ) (Figure 5 1 ) ; IR (ATR) 496 cm 1 ( Fe C ); ICP AES (K/Gd/Fe in mg/L) 3.08: 6.63: 27.42. TGA analysis (Figure 5 2) gives 22% H 2 O per unit formula. CHN analysis calcd. for C 6.9 H 10.6 N 6.0 O 6.0 Fe 2. 0 Gd 0.2 K 0.3 : C, 19.32; H, 2.47; N, 19.6. Found: C, 19.40; H, 1.92; N, 18.65. Measurement of P hotothermal P erformance To measure the photothermal conversion performance of g Gd PB nanoparticles, a 808 nm NIR laser was used to irradiate an aqueous dispersion ( 5 of 0.46 mM g Gd PB nanoparticle suspension). The NIR light output power is 2.5 W/cm 2 with an area of 0.2 cm 2 . The temperature and IR images were recorded with a

PAGE 106

106 FLIR A325 infrared camera (Wilsonville, OR, USA). The temperature profile was plotted b y measuring the temperature change within a time range of 3 min (2 min for the heating process and 1 min for the cooling process) . Photothermal Ablation of Cancer C ells For photothermal ablation experiments, CCRF CEM (human T cell acute lymphoblastic leuke mia cell line) cancer cells were used and treated with g Gd PB particles and laser light. A rapid double staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) is applied to label viable and dead cells. 141 Five groups of experiments and controls included treating the CEM cells: 1. w ithout g Gd PB or laser irradiation; 2. w ith laser irradiation only; 3. w ith g Gd PB (0.46 mM) only; 4. w ith both g Gd PB (lower concentration of 0.046 mM) and laser irradiation; 5. w ith both g Gd PB (high er concentration of 0.46 mM) and laser irradiation. For each group, 100 µL of CEM cells with concentration of 10 6 /mL were transferred to a tube. The g Gd PB particles were then added into groups 3 5 . After adding the nanoparticles, the mixture was treated with 808 nm laser irradiation for groups 2 , 4 and 5 for 10 min with a power of 2.5 W/cm 2 . The mixtures were then cent rifuged at 1300 rpm for 3 min and washed 2 times with DMEM. Before confocal scanning microscope imaging, cells were double stained with FDA and PI. FDA powder was previously dissolved in acetone (1 mg/mL) while PI was dissolved in water (1 mg/mL) and 3 µL of each were added into the mixture and shaken for 6 min . Finally, the mixture was centrifuged at 13 00 rpm for 3 min and washed 2 more time s. An Olympus FV 500 IX81 confocal microscope was used to record cellular images with a 20× objective.

PAGE 107

107 Cell Viability Assay with and without Laser T reatment Cell viability analysis was performed on cells exposed to g Gd PB nanoparticles both with and witho ut laser irradiation. CEM cells, 100 µL of 10 5 /mL concentration, were transferred to a tube with g Gd PB add ed at different concentrations. The mixture was treated with a NI R laser at 808 nm for 10 min with a power of 2.5 W/cm 2 followed by centri fugation a t 1300 rpm for 3 min. Cells were then transferred to 96 well plates and incubated at 37°C for 36 h in a humidified, 5% CO 2 atmosphere. After incubation, cells were centrifuged at 1300 rpm for 3 min and washed with DMEM one more time. T he cell viability was determined with an MTS assay (cellTiter 96 ® aqueous one solution cell proliferation assay), adding 20 µL MTS assay reagent into each well. After incubation for another 1.5 h, the absorbance at 490 nm was recorded using a 96 well plate reade r. To measure t he effects of g Gd PB nanoparticles alone, no laser irradiatio n was applied. Gd 3+ R elease from g Gd PB N anoparticles A dialysis experiment was performed to determine whether Gd 3+ ions could be released from the g Gd PB nanoparticles. The dialysis bag (3,00 0 Da cutoff size) was filled with 500 µL of a suspension of g Gd PB nanoparticles (Gd 3+ 0.8 × 10 4 M ), then dialyzed for 48 hours against 4 mL of nanopure water and different competing ions, including Zn 2+ (5 × 10 4 M) , Cu 2+ (5 × 10 4 M) , Ca 2+ (5 × 10 4 M) , K + (1 M ) , and Na + (1 M) . The Gd 3+ concentration in the dialysis solution was measured by ICP AES. Results and Discussion Particle S ynthesis and C haracterization The one step synthesis of g Gd PB nanoparticles was achieved by slowly adding the Gd 3+ solution, during the co precipitation reaction of Fe 3+ and [Fe(CN) 6 ] 4 , into the

PAGE 108

108 Fe 3+ precursor solution. As a result, more Gd 3+ locates closer to the outer surface as illustrated in Figure 5 3 . Figure 5 4 shows a TEM image of the g Gd PB particle sample, showing uniform cubic particles with an average size of 58 ± 9 nm. In order to better understand the ion distribution in the gradient particles, three spots were picked from the center to the edge of a particle for EDS analysis (Figure 5 5 and Figure 5 6 ). Spot 1 samples primarily the edge of a particle while for spot 3, the electron beam samples both the edges and the center of the particle. The results indicate that from the center to the edge (spectrum 3 to 1) the Gd:Fe ratio increases from 0.05 to 0.14 (Figure 5 5) , me aning that the concentration of Gd 3+ at the edge is clearly higher than at the center, demonstrating the gradient ion distribution. The present method provides a novel route to synthesize concentration gradient coordination polymers with nanometer scale di mensions. The Gd 3+ ions in the solid are determined to be counter cations residing in the cubic PB structure ( Figure 5 3 ) . All peaks in the X ray diffraction pattern of the g Gd PB can be indexed to the cubic phase, confirming formation of the Prussian blu e structure (Figure 5 7 ) . A Prussian blue analogue, KGd[Fe(CN) 6 ] 3.5H 2 O (GdFe PBA), with Gd 3+ linking the hexacyanoferrate ions as part of the framework, is known and the X ray diffraction pattern of a sample is included in Figure 5 7 . 1 42 None of the pure phase KGd[Fe(CN) 6 ] 3.5H 2 O d iffraction peaks were observed. Also, far IR spectroscopy shows the Fe C mode characteristic of the Prussian blue framework at 496 cm 1 , whereas the analogous peak for the GdFe PBA is absent ( Figure 5 8). The ICP results further c onfirm this assignment by showing a lower potassium content relative to Prussian blue, KFe[Fe(CN) 6 ] 4.8H 2 O, with three moles of K + being replaced by each mole of

PAGE 109

109 incorporated Gd 3+ as expected on the basis of charge balance (Table 5 1 ) . On the other hand, t he gradient particles have a larger ratio of K:Gd than the homogeneous phase mixing K + and Gd 3+ as counterions, K 0.53 Gd 0.89 Fe 4 III [Fe II (CN) 6 ] 3.8 1.2H 2 O, reported by Dumont et al. 6 9 Similar to the previously reported homogeneous phase, the gradient particles do not leach Gd 3+ i o n when dialyzed against common metal ions ( Figure 5 9). An attractive feature of the gradient synthesis is that it can be achieved in one step. Furthermore, it could be imagined that a similar distribution of Gd 3+ ions near the particl e surface could be achieved with a core shell heterostructure using PB as the core and gadolinium hexacyanoferrate as the shell. Despite success with other Prussian blue analogue heterostructures, 143 14 6 we were unable to successive ly form this core shell system. Instead of forming heterostructures, the system favors separate homogeneous precipitation of PB and gadolinium hexacyanoferrate particles, giving a mixture that quickly aggregates ( Figure 5 10 and 5 11 ). MRI Relaxivity S tudy The gradient particles were used to measure water proton NMR relaxation times (T 1 , 2 ) at different gadolinium concentrations ( Figure 5 12 ) to obtain relaxivity values. The relaxivity, r 1 , obtained from the slope of this plot was 12.3 mM 1 s 1 for g Gd PB . This value is higher tha n found for commonly used molecular agents, such as Gd DTPA, for which r 1 is 4.3 mM 1 s 1 , 6 6 and is in the range of several other Gd containing particle systems recently repo rted. For example, Johnson et al. 65 reported different sizes of NaGdF 4 nanoparticle s as MRI contrast agents, with the smallest particles (average size = 2.5 nm) showing a relaxivity of 7.2 mM 1 s 1 . In addition, both Bridot et al. 84 and Park et al. 95 have reported on Gd 2 O 3 particles, which show a relaxivity value of 9.9 mM 1 s 1 for a

PAGE 110

110 part i cle size of approximately 1 nm. Recently, Dumont et al. 69 reported K 0.53 Gd 0.89 Fe 4 III [Fe II (CN) 6 ] 3.8 1.2H 2 O nanoparticles with an average size of 33 nm, exhibiting a relaxivity of 38.5 mM 1 s 1 . Direct comparisons of one system to another are complicated by the fact that factors including particle size and surface coating significantly impact molar relaxivity because of changing surface to volume ratios and particle tumbling rates, 65,95,122,123 but the results show that the gradient particles exhibi t reasonab le relaxivity values. The ability of the particles to induce contrast is demonstrated in T 1 weighted MR images ( Figure 5 13 ) , which shows dose dependent contrast . Photothermal T herapy The UV Vis spectrum of a suspension of g Gd PB nanoparticles shows a bro ad absorption band ranging from 500 to1000 nm with max = 720 (Figure 5 1 4 ), 69 which is due to the intervalence charge transfer transition between Fe(II) and Fe(III). 138 The photothermal properties of the g Gd PB nanoparticles were studied by recording the temperature change of the particle suspension ( 0.4 6 mM ) after irradiation with a 808 nm laser for 2 min with a power of 2.5 W/cm 2 . A rapid temperature increase was observed in the temperature profile of the nanoparticle solution ( F igure 5 1 5 a). The temperature increase is also revealed in IR imaging (Figu re 5 1 5 b) and can be directly attributed to the efficient absorpti on of NIR light by the PB host. These results indicate that g Gd PB nanoparticles can rapidly and efficiently convert the energy from the NIR light into thermal energy and act as a PTT agent . To further illustrate the photothermal therapy potential of the gradient particles they were utilized for the phototherma l ablation of CEM cancer cells. To distinguish live

PAGE 111

111 and dead cells, double sta ining with FDA and PI was used. FDA labels live cells, emitting bright green, whereas PI labels necrotic or apoptotic cell nuclei with membrane damage by emitting bright red. 141 For the control group, CEM cells only show green fluorescence after staining with FDA and PI. In addition, CEM cells in the presence of the g Gd PB nanoparticles without irradiation, or irradiated in the absence of particles, only exhibit green emission, demonstrating the particles by themselves or the laser alone will not induce photothermal ablation of the cancer cells (Figur e 5 1 6 ) . Cells treated with both g Gd PB nanoparticles and laser ligh t were irradiated for 10 min . Significant red fluorescence represe nting non viable cells results. The higher concentration g Gd PB (0.46 mM), results in additional red emission (Figure 5 1 6 ), conf irming the potential of the g Gd PB nanoparticles for photothermal therapy. The PTT capability of the g Gd PB nanoparticles was fur ther studied with a MTS assay. For nanoparticles without laser irradiation, cell viability remains 100% at various Gd concent rations from 0 0.46 mM, demonstrating that the g Gd PB particle sample itself is no n toxic under these conditions. This observation is consistent with the fact that Prussian blue capsules (Radiogardase) ha ve been approved by the FDA as an effective therapy agent in the treatment of cesium and thallium poisoning. 138 140 In the presence of particles and after laser treatment, cell viability decreases dramatically as the concentration of t he applied particles increases. At 0.051 mM, cell viability had decreased to approximately 50%. At 0.46 mM, cell viability was less than 10% (Figure 5 1 7 ) . These results show that g Gd PB nanoparticles can be used as an effective PTT agent for cancer cells.

PAGE 112

112 Conclusion s In conclusion, a new synthesis method for coordination polymer particles with the ability to manipulate ion distributions in a nanostructur e was described in this report. Based on this method, w e have synthesized g G d PB nanoparticles, in which more Gd 3+ is located closer to the surface than in the core of the particle. The gradient particles are shown to facilitate proton relaxivity and MRI contrast, and this new synthetic strategy could help to improve the efficienc y of particle based MRI contrast agents by reducing the overall number of ac tive ions in a particle sample. In addition, in the present example which uses Prussian blue as the host, the gradient particle rapidly and efficiently converts energy from a near IR source into thermal energy. Therefore, the unique particle structure of g Gd PB has potentia l as both an MRI and PTT agent. Compared with conventional multimodal materials, g Gd PB can be easily prepared in a one step synthesis, has good photothermal st ability and, based on other clinical applications of Prussian blue, has a high likelihood of acceptable biosafety.

PAGE 113

11 3 Figure 5 1. Room temperature FT IR spectra of PB , g Gd PB and a GdFe PBA particle sample . Figure 5 2 . The TGA curve of a g Gd PB particl e sample .

PAGE 114

114 Figure 5 3 . ( A ) Scheme of the gradient K 0.3 Gd 0.2 Fe[Fe(CN) 6 ] 4.9H 2 O, g Gd PB, nanoparticle. The Gd rich outer layer provides for MR imaging and the PB host can be used for photothermal therapy; ( B ) the cu bic unit cell of Prussian blue. The cyano metallate vacancies and coordinated and zeolitic water molecules are omitted for clarity.

PAGE 115

115 Figure 5 4 . ( A ) A TEM image of g Gd PB particles, scale bar 100 nm; ( B ) particle size histograms of g Gd PB , showing an average size of 58 ± 9 nm.

PAGE 116

116 Figure 5 5 . ( A ) Scheme showing that EDS spot scan analyses at three different regions of a g Gd PB particle evaluate the core and edge of the particle to different extents; ( B ) the corresponding TEM image and EDS determined Gd:Fe ratios for spectra taken at points 1 3.

PAGE 117

117 Figure 5 6 . TEM image and EDS spot scan analys es determined Gd:Fe ratio s for spectra taken at 3 points from the core to the edge of the particle .

PAGE 118

118 Figure 5 7 . Room temperature powder X ray diffraction patterns of GdFe PBA, g Gd PB , and KFe[Fe(CN) 6 ] 4.8H 2 O from

PAGE 119

119 Figure 5 8. Room temperature far IR spectra of PB , g Gd PB , and GdFe PBA particle sample. Figure 5 9. g Gd PB suspension (Gd 3+ 8 × 10 5 M) dialyzed against nanopure water and different competing ions, including Zn 2+ (5 × 10 4 M), Cu 2+ (5 × 10 4 M), Ca 2+ (5 × 10 4 M), K + (1 M), Na + (1 M) for 48 hours. Then Gd 3+ concentration in dialysis solution was measured by ICP AES.

PAGE 120

120 Figure 5 10 . ( A ) TEM image of attempted core shell ( instead of forming heterostructures, the system favors separate homo geneous precipitation of PB and gadolinium hexacyanoferrate particles ), ( B ) GdFe PBA and ( C ) PB. Figure 5 11 . ( A ) EDS map of attempted core shell (instead of forming heterostructures, the system favors separate homogeneous precipitation of PB and gadoli nium hexacyanoferrate particles) , ( B ) Fe map and ( C ) Gd map only.

PAGE 121

121 Figure 5 12 . Plot of the proton relaxation rate (1/T 1,2 ) of water suspensions of g Gd PB nanoparticles at various Gd 3+ concentrations and the corresponding relaxivities (r 1,2 ) (1.41 T) . Figure 5 13 . T 1 weighted MR images for a g Gd PB particle sample at different gadolinium concentrations ranging up to 0.23 mM (4.7 T) .

PAGE 122

122 Figure 5 1 4 . UV Vis spectrum of g Gd PB showing a broad absorption band around 700 nm.

PAGE 123

123 Figure 5 1 5 . ( A ) Temperature profile of g Gd PB under irradiation with an 808 nm laser for 2 min followed by 1 min cooling; ( B ) IR image after i rradiation for 2 min. Scale bar = 3 mm.

PAGE 124

124 Figure 5 1 6 . Confocal microscopic images of CEM cells stained with both FDA and PI following differe nt treatments. Five different experiments involved CEM cells treated ( A , B ) without g Gd PB or laser ; ( C , D ) with laser irradiation only; ( E , F ) with g Gd PB only; ( G , H ) with both g Gd PB (0.046 mM) and laser light; ( I , J ) with both g Gd PB (0.46 mM) and laser light. The scale bar is 50 µm.

PAGE 125

125 Figure 5 1 7 . C ell viability of g Gd PB nanoparticles at different concentrations ( A ) without, and ( B ) with laser irradiation. Table 5 1. ICP results of the g Gd PB and pure PB. g Gd PB PB K/Fe 0.16/1 0.42/1 Gd/Fe 0.09/1 0/1

PAGE 126

126 CHAPTER 6 C ONCLUSION S Research presented in the dissertation is summarized in this C hapter , focusing on the development of nanoparticle based M RI contrast agents for improved MR contrast. In addition, the nanoparticle platform can be readily manipula ted with some synthetic modifications to incorporate other biological functions, including specific ta rgeting, drug delivery, and photothermal therapy for multifunctional purposes. Chapter 1 introduces the variety of biomedical applications of nanopa rticle s and MRI contrast agents, while Chapter 2 represents an introduction of the instrumentation and experim ental methods used in this work. This includes the analytical characterization tools, biological assays, and MRI measurement techniques. In Chapter 3, e uropium doped gadolinium phosphate nanoparticles of three different sizes have been success fully synthesized and investigated for a size dependent relaxivity study. Particle s ize is the first factor we studied that clearly impact s the efficiency of MRI con trast . The results sh ow an increasing relaxivity with decrease of particle size. A correlation between the surface to volume ratio and relaxivity was demonstrated . As expected, smaller particle s with a larger surface to volume ratio deliver the highest rel axivity , indicating that Gd 3+ sites at the surface should contribute more to the relaxivity than ions in the core of the particle as water molecules have direct access to Gd 3+ complexes at the surface. Importantly, d ue to the dopi ng of fluorescent europium ions in the particle sample, the as pr epared nanoparticles can serve as both MRI contrast agent and fluorescent imaging agent. In Chapter 4, we achieved fine control of particle s ize over a wide r range , especially for dimensions less than 40 nm . This allo w s more precise studies of the size

PAGE 127

127 dependence on relaxivity, for small particles with sizes down to few nanometer s . To control particle size s , different polymer s , PAMAM and its derivatives HyPAM, have been used as the surface coating molecules. The averag e length of the nanoparticle can be adjusted by changing polymer concentration, with more polymer s leading to a decrease of the average nanoparticle length. Similar to the results discussed in Chapter 3 , larger particles lead to smaller relaxivity values. However, we also observed an optimum relaxivity value for 23 nm particles, from which relaxivity decreases as particle becomes smaller. The surface to volume ratio increases as particle size gets smaller, accounting for the most often observed increase in relaxivity as particles become smaller. On the other hand, tumbling times and therefore correlation times are another important factor aff ecting relaxivity . Tumbling times get longer as the hydrodynamic volu me of the particle increases. As a result, for a certain range of particle size, relaxiv ity should increase as particle get s bigger. These opposing contributions are then expected to give rise to an optimum particle size for any set of conditions. In additon to PAMAM and HyPAM, t he functionalized core sh ell polymers, HyPAM PEG and HyPAM C 18 PEG, were used to form similar polymer particle hybrids in order to improve stability, biocompatibility , and potentially permit payload loading. The PEG functionalized core shell polymers ensure high stability of the p olymer particle hybrids against high ionic strength. It is noticed that HyPAM C 18 PEG stabilized gadolinium phosphate nanoparticles exhibit lower r 1 and r 2 values than HyPAM PEG/GdPO 4 and HyPAM/GdPO 4 , most likely as a result of the hydrophobic C 18 layer of the hyperbranched structure, which reduces the diffusion of water molecules between the particle surface and the bulk solvent.

PAGE 128

128 In Chapter 5, instead of using metal phosphate particles, a novel Gd containing coordination polymer particle, g Gd PB , was succ essfully synthesized and used as an efficient MRI contrast agent. The facile, one step synthesis of Prussian blue nanoparticles possessing a concentration gradient of Gd 3+ counterions provides a new synthetic method to improve performance while realiz ing m ultiple functions . In this particle, more gadolinium ions were distributed closer to the outer surface while less in the center. The gradient particles were shown to facilitate proton relaxivity and MRI contrast, and this new synthetic strategy could help to improve the efficiency of particle based MRI contrast agents by reducing the overall number of Gd 3+ ions in a particle sample. As the Prussian blue host can rapidly and efficiently convert energy from near IR light into thermal energy, the gradient nano particles can also function as a photothermal therapy agent. Compared with conventional multimodal materials, g Gd PB can be easily prepared in a one step synthesis, has good photothermal stability and, based on other clinical applications of Prussian blue approved by FDA, has a high likelihood of acceptable biosafety. In addition, we further expand the applications of this gradient nanoparticle by changing the nanostructure with base treatment. In the Appendix A , gadolinium doped iron hydroxide nanoparticl es are prepared by adding different concentrations of NaOH solutions to g Gd PB nanoparticle samples , creating large cavities through the cubic nanostructure. The resulting highly porous structure is able to further increase the availability of Gd 3+ sites to water molecules. This has been demonstrated by both the high relaxivity values and T 1 weighted MR i mages. The unique porous structure also enables the nanoparticles to be used as a drug nanocarrier. The a nticancer drug, DOX, was used as a model drug to demonstrate this

PAGE 129

129 capa bility. The se h ighly porous nanoparticles show the potential to be employed as both a MR I contrast agent and an anticancer drug nanocarrier. In general, this dissertation provides insights into the factors that will impact the efficie ncy of MRI contrast agents, which can be used to improve other nanoparticle based MRI contrast agent system s . It also opens up biomedical applications of the coordination polymer nanostructures, including MRI, PTT, and drug delivery after further synthetic modification. Finally, all the work would not have been finished without the guidance and support from Dr. Talham. I also sincerely appreciate all the efforts from our collaborators on the projects.

PAGE 130

130 APPENDIX A HIGHLY POROUS NANOPARTICLES FOR MRI CONTRAST ENHANCEMENT AND ANTICANCER DRUG DELIVERY Preface In this C hapter, highly porous gadolinium doped iron hydroxide nanoparticles , HP Gd , were synthesized with g Gd PB as precursor s , as described in C hapter 5. These highly porous nanoparticle s , with lots of voi ds and a larger surface area, allow more Gd 3+ sites get ting access to water molecules to induce MR contrast . R elaxivity measurement shows high r 1 and r 2 of 42.1 mM 1 s 1 and 79.0 mM 1 s 1 , respectively , which is almost 10 times greater than the value obtaine d for the comme rcially used MRI contrast agent, Gd DTPA, with r 1 of 4.3 mM 1 s 1 . 66 The use of HPGd nanoparticles as MRI contrast agent was further demonstrated with T 1 weighted MR images, from which we observed the contrast becomes stronger as the nanopart icle concentration increases . The HPGd with the unique highly porous nano structure, holds the promise to incorporate drug delivery capability into the platform. Doxorubicin was used as a model drug to demonstrate the application of the prepared nanoparticl es as a drug nanocarrier . Fluorescent spectra , together with confocal images prove the successful loading of anticancer drug , by showing emission in the red region or red light, resulting from the loaded DOX . Additionally, t he in vitro cancer treatment was evaluated by both confocal laser scanning microscopy and cytotoxicity assay . Introduction D ifferent factors , including particle size, surface coating, and Gd 3+ ion distribution, which affect the MRI contrast efficiency have been studied. W ith the decrease of size, the Gd containing particles have a larger surface to volume ratio, leading to increase of relaxivity. Impo rtantly, as the particle size becomes very small as

PAGE 131

131 discussed in Chapter 4 , the increase trend will be reversed because the smaller particl e possesses shorter tumbling time, which is responsible for the decrease of relaxivity. It is also noticed that relaxivity is tunable as the surface coating material is changed . 147 I n Chapter 5, we manipulated Gd 3+ distribution s in the coordination polymer particles as a novel strategy for the improvement o f MRI contrast agents. In this C hapter, to further explore the functionalities and improve MRI contrast efficiency of the gradient particles , g Gd PB , a base treatment method was applied to achieve a highl y porous nano structure based on the as prepared g Gd PB particles . 148 The resulting highly porous structure is proved to be able to further enhance the availability of Gd 3+ sites as direct chemical exchange of water molecules. Previou sly, Ananta et al. 72, 1 49 reported highly porous silicon microparticles individually load ed with Magnevist, gadofulleren s, and gadonanotubes, from which a boost in longitudinal proton relaxivity r 1 was observed (Figure A 1) . As it is well known, s ome anticancer drugs suffer from problems such as low solubility and short circulation time. Alternatively, using nanoparticles as a carrier for drug delivery confront these limitations , showing longer circulation time, better solubility, control led release, targeting , and minimum side e ffect . 28,31 Among various nanocarrier systems, porous nanoparticle is a hot topic under active investigations . For example, Zhao et al. 150 reported porous silica nanoparticles functionalized with boronic acid as drug delivery system , with controlled releas e under glucose stimuli, which is promising to be used as self regulated insulin releasing devices (Figure A 2 ) . In addition, Horcajada et al . 151 show ed non toxic porous iron(III) based metal organic frameworks with engineered cores and surfaces, function as superior nanocarriers for efficient

PAGE 132

132 controlled delivery of antitumoural and retroviral drugs against cancer and AIDS (Figure A 3 ) . Recently, n anocarriers that combin e imaging with drug delivery have become significant , because of their capability to pro vide diagnosis and therapy at the same time. 3,56 ,1 52 Imaging is able to tel l where the lesion is, followed by chemo therapy by the loaded drug. Among multiple medical imaging techniques, MRI is more favorable to combine wit h drug delivery, due to its abilit y to provide anatomical details of internal structures and lesions. 50,51 For example, Chen et al. 3 reported hollow iron oxide nanoparticles which combine MRI and drug delivery in a single particle . In addition , the prepared nanoparticles were surface modif ied with aptamer , realizing specific cancer cell targeting. Another example is water dis persible magnetite nanocapsules , which were synthesized by Pia o et al . 56, 152 The nanocapsules were used not only as a drug delivery vehicle, but also as a T 2 magnetic r esonance imaging contrast agent , as shown in Fi gure A 4 . 56,1 52 Compared with iron oxide, which has been widely used as T 2 MRI contrast agent, gadolinium based nanoparticles are more promising due to the fact that it will brighten the lesion area. However, the combination of MRI contrast from gadolinium based nanopoarticles with drug delivery is seldom reported. In this C hapter , we pre pared highly porous HPGd nanoparticles . With more gadolinium sites available to water molecules, MR contrast efficiency was f urther improved relative to g Gd PB . In the meantime , with this unique porous structure, HPGd nanoparticles are demonstrated to be a superior drug nano carrier for in vitro cancer treatment.

PAGE 133

133 Materials and Methods Materials Hoechst 33342 was purchased from S igma (St. Louis, MO, USA). Gadolinium(III) nitrate hexahydrate (99.90%), c itric acid monohydrate, and a nhydrous f erric c hloride were purchased from Fisher Scientific (Waltham, MA, USA). Potassium ferrocyanide (Baker Analyzed, ACS reagent) was purchased fro m J.T. Baker (Phillipsburg, NJ, USA). PAA 3.7k PEO 11k was purchased from Polymer Source Inc. MTS ( c ellTiter 96 ® aq ueous o ne s olution c ell p roliferation a ssay) was purchased from Promega Corporation (Madison, WI, USA). All commercial chemicals were used with out further purification. HP Gd Nanoparticle Synthesis The g Gd PB was prepared following the method present ed in Chapter 5. T o create the hollow structures, 5 mL of the as prepared g Gd PB particle solution was completely mixed with 10 mL of ethanol , f ollo wed by adding 10 mL of NaOH (concentration ranges from 0~0.2 M) . The mixture was kept shaking for 5 min and centri fuged immediately at 12500 rpm for 15 min . The supernatant was decanted . To wash the nanoparticles, 15 mL of water was added and centrifuged , and repeated for three cycles . The surface coating was performed by mixing 1 mL of the nanoparticle solution after base treatment and 400 µL of PAA 3.7k PEO 11k , sonicating for 30 min, and washi ng three times with water . Anticancer Drug Loading 1 mL of HPGd nanoparticle solution was mixed with 500 µ L of doxorubicin with a concentration of 4 mg/mL at room temperature and kept shaking for 24 h. The drug loaded HPGd were precipitated by centrifugation at 12500 rpm for 15 min and washed three times with water .

PAGE 134

134 In Vitro Drug D elivery A confocal laser scanning microscope (Leica TCS SP5) was used to record cellular images. HeLa cells were plated in a 35 mm confocal dish (glass bottom dish) and grown to around 60% confluency for 24 h before the experiment. Cells were washed three times with DMEM, supplied with 1 mL DMEM, and then incubated with DOX loaded p Gd . After 1 h and 5 h , cells were further incubated with 5 g/mL Hoechst 33342 for 10 min in incubator . T he loading solution was removed and Hela cell mon olayers were washed three times with PBS and examined by confocal laser scanning mic roscopy . Cell Viability A ssay with and without D rug L oading HPGd Nanoparticles Cell viability analysis was performed on CEM cells exposed to nanoparticles with and without DOX loading. CEM cells, 100 µL of 10 5 /mL concentration, were transferred to a 96 well plate with adding DOX loading HPGd nanoparticles at different concentrations, and incubated at 37°C for 36 h in a humidified, 5% CO 2 atmosphere. After incubation, cells w ere centrifuged at 1300 rpm for 3 min and washed with DMEM. T he cell viability was determined with a MTS assay (cellTiter 96 ® aqueous one solution cell proliferation assay), adding 20 µL MTS assay reagent into each well. After incubation for another 1.5 h, the absorbance at 490 nm was recorded with a 96 well plate reade r. To measure the effect of HPGd nanoparticles alone, no anticancer drug was loaded . Results and Discussion Particle S ynthesis and C haracterization To synthesize highly porous HPGd nanopartic le s , the cubic g Gd PB particles were used as the template of the HPGd structures . P orous structures were obtained by

PAGE 135

135 precisely controlling the chemical reaction bet ween g Gd PB and NaOH with varying concentrations of base, as reported earlier, 148 followin g the reaction described below: 12OH 1 (aq) + Fe 4 [Fe(CN) 6 ] 3(s) 3Fe(CN) 6 4 (aq) + 4Fe(OH) 3(s) 148 The PB is insoluble in water with a very small solubility product constant (K sp =3.3 × 10 41 ) . In the presence of NaOH, PB with Gd 3+ as counterions undergo es an ion exchange react ion forming porous insoluble metal hydroxide as depicted in F igure A 5 . This ion exchange reaction is driven by the high c oncentration of hydroxide ions. 148 After base tre atment, cavities wer e formed through the particles. The cubic sh ape of g Gd PB wa s still remained as shown in Figure A 6 . At lower base concentrations , only sma ll amount of cavities were created . The porosity could be further improved by increasing the concentration of NaOH to 0. 2 M, from which a highly porous structur e was obtained (Figure A 6 ) . Along with formation of cavities , the composition of particles change s from coordination polymer to metal hydroxide , which was demonstrated by powder X ray diffraction ( Figure A 7 ) . For the initial template g Gd PB , characteris tic peaks corres ponding to PB were observed. As NaOH concentration increases , th e peak intensity becomes smaller and an amorphous structure was indicated by the XRD analysis after treating with 0.2 M NaOH . In addition, the color change of solutions also im plies t he composition change from PB to metal hydroxide. T he characteristic blue color of PB started t o disappear, while the yellow color became more and more intense as the concentration of NaOH increases, due to the formation of Fe(OH) 3 ( Figure A 8 ) . Als o , the stretching frequencies of the cyanide triple bond, , in the region of 1900 cm 1 and 2300 cm 1 features the presence of PB. Based on FT IR analysis , w ith the

PAGE 136

136 increase of NaOH concentration , the stretching signal kept decreasing and finally disappear ed ( Figure A 9 ) . All these results demonstrate the f ormation of metal hydroxide from the original g Gd PB nanoparticles. To investigate the dispersity of the HPGd nanoparticle s , TEM image s ( Figure A 6 ) indicate that the nanoparticles aggregate , especially after high concentration of alkaline treatment. To c ircumvent this drawback , an surface coating experiment was performed before testing the bio functionalities of the particle sample . PAA 3.7k PEO 11k was chosen as the coating molecule because of its good biocompatibility and easy manipulation. As reported pr eviously, Pothayee et al . 153 reported the use of PEO PAA to modify nanoparticle surface, in which PEO PAA wa s found to bind to the surface of magnetic nanoparticle s by ligand adsorption of the PAA block, thereby creating a double corona structure. The noni onic PEO shell serves as a block copolymer segment to improve biocompatibility. Also, Kirby et al . 154 tested the effects of PEO PAA polymers on the stability of BaTiO 3 nano particles under variety conditions, including different pH and different salt specie s. Similarly, after PAA block binding to the nanoparticles, the neutral charged PEO teeth effectively protect the stability of the nanoparticles over a wide range of pH and ionic strength. 154 In our work, we also noticed that dispersity and stability of HP Gd is highly improved after surface modification with PEO PAA polymer. This is demonstrated by images of the n anoparticle suspension in Figure A 10 . A fter surface modification, HPGd nanoparticles were well dispersed in water without aggregation over 2 days , whereas nanoparticles without surface coating aggregated within the initial 3 h, showing separated layers, with clear upp er level and yellow lower level (Figure A 10 ) . The improvement of dispersity was further proved by TEM images

PAGE 137

137 (Figure A 11 ) . For the surface coated sample, single particle can be viewed as opposed to particle clusters. Additionally, s uccessful surface coating with PEO PAA was demonstrated by FT IR , showing distinct C H stretching vibration at 2850 3000 cm 1 , whereas the analogous peak f or the particles without surface modification is absent (Figure A 12 ). Relaxivity M easurements and M R I maging The longitudinal (T 1 ) and transverse (T 2 ) relaxation times of HPGd nanoparticles were measured at various gadolinium concentrations , with r 1 and r 2 determined from the slope of the plot shown in Figure A 13 . The HPGd nanoparticle exhibits superior r 1 relaxivity , 42.1 mM 1 s 1 , among the reported inorganic nanoparticle based T 1 MRI contrast agents . 68 , 89 , 96 98 For example, dextran coated gadolinium ph osphate nanopart icles, reported by Hifumi et al., 96 shows r 1 relaxivity of 13.9 mM 1 s 1 . Park et al. 95 used ultrasmall gadolinium oxide nanoparticles as an in vivo T 1 MRI contrast agent, which gives r 1 of 9.9 mM 1 s 1 . The favorable relaxivity suggest s the particles could generate contrast in MRI. Furthermore , T 1 weighted MR image s were obtained with Agilent 4.7 T system to demonstrate this capability . Figure A 14 shows the T 1 weighted MR image of HPGd suspensions at various concentrations ranging from 0 ~ 0.8 6 mM. Dose dependent contrast was shown, with stronger contrast observed as the increase of nan op article concentration. This result agre es well with the relaxivity measurement , indicating the HPGd particle with large r 1 value and low r 2 /r 1 ratio of 1.88 ca n be employed as an effective positive MRI contrast agent.

PAGE 138

138 In V itro D rug D elivery Doxorubicin was used as a model drug to demonstrate the drug delivery capability of the porous HPGd nanoparticles. DOX loading was performed based on a previously reported me thod , simply by mixing the DOX with nanoparticles , followed by incubation for 24 h and centrifugation to wash off the extra DOX . 155,156 The successful drug loading was proved by fluorescent emission spectra after excitation at 480 nm , which shows a m aximum emission peak around 600 nm , coming from loaded DOX (Figure A 15 ). An Olympus FV 500 IX81 confocal microscope was used to record particle images with a 10× objective, with red light shown for the DOX loaded sample (Figure A 16 ). These results demonstrate the success loading of DOX onto the HPGd nanoparticles. The fluorescent DOX molecule s further facilitate monitor ing DOX intracellular delivery with the HPGd nano carriers by using confocal microscopy analysis . Hela cells, after incubation with nanoparticles for different times, were treated with Hoechst 33342 for nucleus stain. For the control group ( Hela cells without incubation with DOX loaded HPGd particles), no red light was observed, with only blue color coming from the nucleus Hoechst stain (Figure A 1 7 ). For the positive group with incubation time of 1 h , only weak red signal was detected from the Hela cells (Figure A 1 7 ). After incubation for 5 h (Figure A 1 7 ), the intensity of red light increase s, indicating more DOX loaded nano carriers were taken up by the Hela cells . The results demonstrate the efficient intracellular delivery of DOX by the HPGd particles. Cyto to xicity Assay After confirming its cellular uptake and in vitro drug release, the cytotoxicity of DOX loaded HPGd nanoparticles was evaluate d by a MTS assay. This method is based

PAGE 139

139 on the formation of dark red formazan by the metabolically active cells after their exposure to MTS. Cell viability is directly proportional to the amount of formazan produced, monitored by the absorbance at 490 nm. T he control experiment reveals HPGd nanoparticle by itself has no obvious cytotoxicity on CEM cells and more than 90% of ce lls remained alive (Figure A 1 8 ). This indicates the good biocompatibility of the prepared particle sample . DOX loaded HPGd nanopartic les exhibited an increasing inhibition against CEM cells with the increase of concentration s (Figure A 1 8 ). These results demonstrate the potential of HPGd nanoparticles to be used as an anticancer drug nanocarrier . Conclusion s In this study, the PEO PAA c oblock polymer coated highly porous HPGd nanoparticles were synthesized with superior stability. The porous structure further improved the MRI contrast. Both relaxivity measurement s and T 1 wighted MR images demonstrated HPGd can be employed as a highly eff icient MRI contrast agent. In addition, DOX loading onto the nanoparticles enables the system to be used for anticancer drug delivery , as confirmed by both confocal laser scanning microscopy and cytotoxicity assay.

PAGE 140

140 Figure A 1. Schematic representation of three different Gd CAs: (A) Magnevist, (B) gadofullerenes, and (C) gadonanotubes. Scanning electron micrographs of quasi hemispherical (H SiMP: diameter, 1.6 mm; thickness, 0.6 mm) (D) and discoidal (D SiMP: diameter, 1.0 mm; thickness, 0.4 mm) particles (E). (F), Cartoons showing Magnevist, gadofullerenes and gadonanotubes (left to right) entrapped within the porous structure of the SiMPs. The figure was reprinted from Ref 72 with permission from the Nature Publishing Group .

PAGE 141

141 Figure A 2 . ( A ) Schematic re presentation of the glucose responsive MSN based delivery system for controlled release of bioactive G Ins and cAMP. Transmission electron micrographs of ( B ) boronic acid functionalized MSN and ( C ) FITC G Ins capped MSN . The figure was reprinted from Ref 1 50 with permission from American Chemical Society .

PAGE 142

142 Figure A 3 . Scheme of engineered core corona porous iron carboxylates for drug delivery and imaging. The figure was reprinted from Ref 1 51 with permission from Nature Publishing Group .

PAGE 143

143 Figure A 4 . ( A ) Schematic illustration of the procedure for the synthesis of uniform and water dispersible iron oxide nanocapsules and their TEM images. ( B ) T 2 wighted MR images of the magnetite nanocapsules. ( C ) In vitro cytotoxicity of free DOX and DOX loading nanocapsu les against SKBT 3 cells. The figure was reprinted from Ref 56 with permission . Figure A 5 . Illustration of the synthetic procedure of porous HPGd nanoparticles .

PAGE 144

144 Figure A 6 . TEM images of g Gd PB template and g Gd PB after NaOH treatment with differ ent concentrations (ranging from 0.002 M to 0.2 M).

PAGE 145

145 Figure A 7 . XRD of g Gd PB nanoparticles after NaOH treatment with different concentrations ( A ) . 0 M , ( B ) . 0.005 M, ( C ) . 0.0075 M, ( D ). 0.02 M .

PAGE 146

146 Figure A 8 . Images of g Gd PB color change after NaOH treatment with different concentrations ( ranging from 0~ 0.2 M) .

PAGE 147

147 Figure A 9 . FT IR analysis results of g Gd PB precursors after base treatment with different concentrations (ranging from 0~0.2 M) .

PAGE 148

148 Figure A 10 . In both ( A ) and ( B ) , samples from left to r ight are original g Gd PB , HPGd without PEO PAA surface modification, and HPGd with surface modification. ( A ) image was taken at 3 h ( B ) image was taken at 2 days. Figure A 1 1 . TEM image of HPGd ( A ) without PEO PAA surface modification, ( B ) HPGd with su rface modification.

PAGE 149

149 Figure A 1 2 . FT IR analysis results of HPGd nanoparticles after surface modification with P EO P AA . Figure A 1 3 . Plot of the proton relaxation rate (1/T 1,2 ) of water suspensions of HPGd nanoparticles at various Gd 3+ concentrations a nd the corresponding relaxivities (r 1,2 ) (1.41 T) .

PAGE 150

150 Figure A 1 4 . T 1 weighted MR images (4.7 T) of HPGd at various Gd 3+ concentrations (ranging from 0~0.86 mM) . Figure A 1 5 . Fluorimetric characterization of DOX loaded HPGd nanoparticles. (Emission spectr ex = 480 nm) Fig ure A 1 6 . Confocal imaging of (A) drug loaded HPGd particle; (B) HPGd only. Scale bar 200 µm .

PAGE 151

151 Fig ure A 1 7 . Confocal images of drug loaded HPGd particle uptake by Hela cells: Hoechst nuclear stain observed in blue color. Fluorescent Dox are observed in red. Control cells are incubated without nanoparticles. For positive group s , DOX loaded HPGd particles are incubated with Hela cells for 1h and 5 h respectively . Scale bar 50 µm.

PAGE 152

152 Figure A 1 8 . Cell viability of HPGd nanoparticles at di fferent concentrations ( A ) without, and ( B ) with anticancer drug DOX loading.

PAGE 153

153 APPENDIX B COPYRIGHT CLEARANCE FORMS Title: Carving at the Nanoscale: Sequential Galvanic Exchange and Kirkendall Growth at Room Temperature Author: Edgar González, Jordi Arb iol, Víctor F. Puntes Publication: Science Publisher: The American Association for the Advancement of Science Date: Dec 9, 2011 Copyright © 2011, American Association for the Advancement of Science Logged in as: Yichen Li Order C ompleted Thank you very much for your order. This is a License Agreement between Yichen Li ("You") and The American Association for the Advancement of Science ("The American Association for the Advancement of Science"). The license consists of your order details, the terms and conditions provided by The American Association for the Advancement of Science, and the payment terms and conditions. License Number 3557391393655 License date Jan 27, 2015 Licensed content publisher The American Associa tion for the Advancement of Science Licensed content publication Science Licensed content title Carving at the Nanoscale: Sequential Galvanic Exchange and Kirkendall Growth at Room Temperature Licensed content author Edgar González, Jordi Ar biol, Víctor F. Puntes Licensed content date Dec 9, 2011 Volume number 334 Issue number 6061 Type of Use Thesis / Dissertation Requestor type Scientist/individual at a research institution Format Print and electronic Port ion Figure Number of figures/tables 1 Order reference number None Title of your thesis / dissertation LANTHANIDE BASED NANOPARTICLES AS MULTIFUNCTIONAL AGENTS FOR MAGNETIC RESONANCE IMAGING AND CANCER TREATMENT Expected completion date May 2015 Total 0.00 USD

PAGE 154

154 Title: Water Soluble Fluorescent Carbon Quantum Dots and Photocatalyst Design Author: Haitao Li,Xiaodie He,Zhenhui Kang,Hui Huang,Yang Liu,Jinglin Liu,Suoyuan Lian,Chi Him A. Tsang,Xiaobao Yang,Shuit Tong Lee Public ation: Angewandte Chemie International Edition Publisher: John Wiley and Sons Date: May 11, 2010 Copyright © 2010 WILEY VCH Verlag GmbH & Co. KGaA, Weinheim Logged in as: Yichen Li Order Completed Thank you for your order. This Agreement between Yichen Li ("You") and John Wiley and Sons ("John Wiley and Sons") consists of your license details and the terms and conditions provided by John Wiley and Sons and Copyright Clearance Center. Your confirmation email will contain your or der number for future reference. License Number 3557651251629 License date Jan 28, 2015 Licensed Content Publisher John Wiley and Sons Licensed Content Publication Angewandte Chemie International Edition Licensed Content Title Water So luble Fluorescent Carbon Quantum Dots and Photocatalyst Design Licensed Content Author Haitao Li,Xiaodie He,Zhenhui Kang,Hui Huang,Yang Liu,Jinglin Liu,Suoyuan Lian,Chi Him A. Tsang,Xiaobao Yang,Shuit Tong Lee Licensed Content Date May 11, 2010 Licensed Content Pages 5 Type of use Dissertation/Thesis Requestor type University/Academic Format Print and electronic Portion Figure/table Num ber of figures/tables 1 Original Wiley figure/table number(s) Figure 3 Will you be translating? No Title of your thesis / dissertation LANTHANIDE BASED NANOPARTICLES AS MULTIFUNCTIONAL AGENTS FOR MAGNETIC RESONANCE IMAGING AND CANCER TREATME NT Expected completion date May 2015 Total 0.00 USD

PAGE 155

155 Title: Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots Author: Xingyong Wu,Hongjian Liu,Jianquan Liu,Kari N. Haley,Joseph A. Treadway et al. Publication: Nature Biotechnology Publisher: Nature Publishing Group Date: Jan 1, 2003 Copyright © 2003, Rights Managed by Nature Publishing Group Logged in as: Yichen Li Order Completed Thank you very much for your order. This is a License Agreement between Yichen Li ("You") and Nature Publishing Group ("Nature Publishing Group"). The license consists of your order details, the terms and conditions provided by Nature Publishing Group, and the payment terms and conditions. License Number 3557660111127 License date Jan 28, 2015 Licensed content publisher Nature Publishing Group Licensed content publication Nature Biotechnology Licensed content title Immunofluorescent labeling of cancer marke r Her2 and other cellular targets with semiconductor quantum dots Licensed content author Xingyong Wu,Hongjian Liu,Jianquan Liu,Kari N. Haley,Joseph A. Treadway et al. Licensed content date Jan 1, 2003 Type of Use reuse in a dissertation / t hesis Volume number 21 Issue number 1 Requestor type academic/educational Format print and electronic Portion figures/tables/illustrations Number of figures/tables/illustrations 1 High res required no Figures Figure 6 Author of this NPG article no Your reference number None Title of your thesis / dissertation LANTHANIDE BASED NANOPARTICLES AS MULTIFUNCTIONAL AGENTS FOR MAGNETIC RESONANCE IMAGING AND CANCER TREATMENT Expected completion date May 2 015 Total 0.00 USD

PAGE 156

156 Title: Upconversion Multicolor Fine Tuning: Visible to Near Infrared Emission from Lanthanide Doped NaYF4 Nanoparticles Author: Feng Wang, Xiaogang Liu Publication: Journal of the American Chemical Society Publisher: Amer ican Chemical Society Date: Apr 1, 2008 Copyright © 2008, American Chemical Society Logged in as: Yichen Li PERMISSION/LICENSE IS GRANTED FOR YOUR ORDER AT NO CHARGE This type of permission/license, instead of the standard Terms & Conditions, is sent to you because no fee is being charged for your order. Please note the following: Permission is granted for your request in both print and electronic formats, and translations. If figures and/or tables were requested, they may be ad apted or used in part. Please print this page for your records and send a copy of it to your publisher/graduate school. Appropriate credit for the requested material should be given as follows: "Reprinted (adapted) with permission from (COMPLETE REFERENC E CITATION). Copyright (YEAR) American Chemical Society." Insert appropriate information in place of the capitalized words. One time permission is granted only for the use specified in your request. No additional uses are granted (such as derivative works or other editions). For any other uses, please submit a new request. If credit is given to another source for the material you requested, permission must be obtained from that source.

PAGE 157

157 Title: NaYbF4:Tm3+)/CaF2 Core/Shell Nanoparticles with Efficient Near Infrared to Near Infrared Upconversion for High Contrast Deep Tissue Bioimaging Author: Guanying Chen, Jie Shen, Tymish Y. Ohulchanskyy, et al Publication: ACS Nano Publisher: American Ch emical Society Date: Sep 1, 2012 Copyright © 2012, American Chemical Society Logged in as: Yichen Li PERMISSION/LICENSE IS GRANTED FOR YOUR ORDER AT NO CHARGE This type of permission/license, instead of the standard Terms & Cond itions, is sent to you because no fee is being charged for your order. Please note the following: Permission is granted for your request in both print and electronic formats, and translations. If figures and/or tables were requested, they may be adapted o r used in part. Please print this page for your records and send a copy of it to your publisher/graduate school. Appropriate credit for the requested material should be given as follows: "Reprinted (adapted) with permission from (COMPLETE REFERENCE CITAT ION). Copyright (YEAR) American Chemical Society." Insert appropriate information in place of the capitalized words. One time permission is granted only for the use specified in your request. No additional uses are granted (such as derivative works or oth er editions). For any other uses, please submit a new request. If credit is given to another source for the material you requested, permission must be obtained from that source.

PAGE 158

158 Title: Development of a T1 Contrast Agent for Magnetic Resonance Imagin g Using MnO Nanoparticles Author: Hyon Bin Na,Jung Hee Lee,Kwangjin An,Yong Il Park,Mihyun Park,In Su Lee, Do Hyun Nam,Sung Tae Kim,Seung Hoon Kim,Sang Wook Kim,Keun Ho Lim, Ki Soo Kim,Sun Ok Kim,Taeghwan Hyeon Publication: Angewandte Chemie Internationa l Edition Publisher: John Wiley and Sons Date: Mar 13, 2007 Copyright © 2007 WILEY VCH Verlag GmbH & Co. KGaA, Weinheim Logged in as: Yichen Li Order Completed Thank you for your order. This Agreement between Yichen Li ("You") and John Wiley and Sons ("John Wiley and Sons") consists of your license details and the terms and conditions provided by John Wiley and Sons and Copyright Clearance Center. Your confirmation email will contain your order number for future reference. Lic ense Number 3557671279190 License date Jan 28, 2015 Licensed Content Publisher John Wiley and Sons Licensed Content Publication Angewandte Chemie International Edition Licensed Content Title Development of a T1 Contrast Agent for Magnet ic Resonance Imaging Using MnO Nanoparticles Licensed Content Author Hyon Bin Na,Jung Hee Lee,Kwangjin An,Yong Il Park,Mihyun Park,In Su Lee, Do Hyun Nam,Sung Tae Kim,Seung Hoon Kim,Sang Wook Kim,Keun Ho Lim, Ki Soo Kim,Sun Ok Kim,Taeghwan Hyeon Licensed Content Date Mar 13, 2007 Licensed Content Pages 5 Type of use Dissertation/Thesis Requestor type University/Academic Format Print and electronic Portion Figure/table Number of figures/tables 1 Original Wiley fig ure/table number(s) Figure 3 Will you be translating? No Title of your thesis / dissertation LANTHANIDE BASED NANOPARTICLES AS MULTIFUNCTIONAL AGENTS FOR MAGNETIC RESONANCE IMAGING AND CANCER TREATMENT Expected completion date May 2015 Total 0.00 USD

PAGE 159

159 Title: A High Performance Ytterbium Based Nanoparticulate Contrast Agent for In Vivo X Ray Computed Tomography Imaging Author: Yanlan Liu,Kelong Ai,Jianhua Liu,Qinghai Yuan,Yangyang He,Lehui Lu Publication: Angewandte Chemie Inter national Edition Publisher: John Wiley and Sons Date: Dec 30, 2011 Copyright © 2012 WILEY VCH Verlag GmbH %26 Co. KGaA, Weinheim Logged in as: Yichen Li Order Completed Thank you for your order. This Agreement between Yichen Li ("You") and John Wiley and Sons ("John Wiley and Sons") consists of your license details and the terms and conditions provided by John Wiley and Sons and Copyright Clearance Center. Your confirmation email will contain your order number for future refere nce. License Number 3557680112533 License date Jan 28, 2015 Licensed Content Publisher John Wiley and Sons Licensed Content Publication Angewandte Chemie International Edition Licensed Content Title A High Performance Ytterbium Based N anoparticulate Contrast Agent for In Vivo X Ray Computed Tomography Imaging Licensed Content Author Yanlan Liu,Kelong Ai,Jianhua Liu,Qinghai Yuan,Yangyang He,Lehui Lu Licensed Content Date Dec 30, 2011 Licensed Content Pages 6 Type of use Dissertation/Thesis Requesto r type University/Academic Format Print and electronic Portion Figure/table Number of figures/tables 1 Original Wiley figure/table number(s) Figure 4 Will you be translating? No Title of your thesis / dissertation LANTHANIDE B ASED NANOPARTICLES AS MULTIFUNCTIONAL AGENTS FOR MAGNETIC RESONANCE IMAGING AND CANCER TREATMENT Expected completion date May 2015 Total 0.00 USD

PAGE 160

160 Title: Inorganic Nanoparticles for MRI Contrast Agents Author: Hyon Bin Na,In Chan Song,Ta eghwan Hyeon Publication: Advanced Materials Publisher: John Wiley and Sons Date: Mar 4, 2009 Copyright © 2009 WILEY VCH Verlag GmbH %26 Co. KGaA, Weinheim Logged in as: Yichen Li Order Completed Thank you for your order. This A greement between Yichen Li ("You") and John Wiley and Sons ("John Wiley and Sons") consists of your license details and the terms and conditions provided by John Wiley and Sons and Copyright Clearance Center. Your confirmation email will contain your orde r number for future reference. License Number 3557690318256 License date Jan 28, 2015 Licensed Content Publisher John Wiley and Sons Licensed Content Publication Advanced Materials Licensed Content Title Inorganic Nanoparticles for MRI Contrast Agents Licensed Content Author Hyon Bin Na,In Chan Song,Taeghwan Hyeon Licensed Content Date Mar 4, 2009 Licensed Content Pages 16 Type of use Dissertation/Thesis Requestor type University/Academic Format Print and e lectronic Portion Figure/table Number of figures/tables 1 Original Wiley figure/table number(s) Figure 8 Will you be translating? No Title of your thesis / dissertation LANTHANIDE BASED NANOPARTICLES AS MULTIFUNCTIONAL AGENTS FOR M AGNETIC RESONANCE IMAGING AND CANCER TREATMENT Expected completion date May 2015 Total 0.00 USD

PAGE 161

161 Title: Gadolinium(III) Chelates as MRI Contrast Agents: Structure, Dynamics, and Applications Author: Peter Caravan, Jeffrey J. Ellison, Thomas J. McMurry, et al Publication: Chemical Reviews Publisher: American Chemical Society Date: Sep 1, 1999 Copyright © 1999, American Chemical Society Logged in as: Yichen Li PERMISSION/LICENSE IS GRANTED FOR YOUR ORDER AT NO CHARGE This type of permission/license, instead of the standard Terms & Conditions, is sent to you because no fee is being charged for your order. Please note the follow ing: Permission is granted for your request in both print and electronic formats, and translations. If figures and/or tables were requested, they may be adapted or used in part. Please print this page for your records and send a copy of it to your publis her/graduate school. Appropriate credit for the requested material should be given as follows: "Reprinted (adapted) with permission from (COMPLETE REFERENCE CITATION). Copyright (YEAR) American Chemical Society." Insert appropriate information in place of the capitalized words. One time permission is granted only for the use specified in your request. No additional uses are granted (such as derivative works or other editions). For any other uses, please submit a new request. If credit is given to another source for the material you requested, permission must be obtained from that source.

PAGE 162

162 Title: Mesoporous Silica Nanoparticle Based Double Drug Delivery System for Glucose Responsive Controlled Release of Insulin and Cyclic AMP Author: Yannan Zhao, Br ian G. Trewyn, Igor I. Slowing, et al Publication: Journal of the American Chemical Society Publisher: American Chemical Society Date: Jun 1, 2009 Copyright © 2009, American Chemical Society Logged in as: Yichen Li PERMISSION/L ICENSE IS GRANTED FOR YOUR ORDER AT NO CHARGE This type of permission/license, instead of the standard Terms & Conditions, is sent to you because no fee is being charged for your order. Please note the following: Permission is granted for your request in both print and electronic formats, and translations. If figures and/or tables were requested, they may be adapted or used in part. Please print this page for your records and send a copy of it to your publisher/graduate school. Appropriate credit for th e requested material should be given as follows: "Reprinted (adapted) with permission from (COMPLETE REFERENCE CITATION). Copyright (YEAR) American Chemical Society." Insert appropriate information in place of the capitalized words. One time permission is granted only for the use specified in your request. No additional uses are granted (such as derivative works or other editions). For any other uses, please submit a new request.

PAGE 163

163 Title: Porous metal organic framework nanoscale carriers as a potentia l platform for drug delivery and imaging Author: Patricia Horcajada, Tamim Chalati, Christian Serre, Brigitte Gillet, Catherine Sebrie, Tarek Baati Publication: Nature Materials Publisher: Nature Publishing Group Date: Dec 13, 2009 Copyright © 2009, R ights Managed by Nature Publishing Group Logged in as: Yichen Li Order Completed Thank you very much for your order. This is a License Agreement between Yichen Li ("You") and Nature Publishing Group ("Nature Publishing Group"). Th e license consists of your order details, the terms and conditions provided by Nature Publishing Group, and the payment terms and conditions. License Number 3557690077380 License date Jan 28, 2015 Licensed content publisher Nature Publishing Gr oup Licensed content publication Nature Materials Licensed content title Porous metal organic framework nanoscale carriers as a potential platform for drug delivery and imaging Licensed content author Patricia Horcajada, Tamim Chalati, Christian Serre, Brigitte Gillet, Catherine Sebrie, Tarek Baati Licensed content date Dec 13, 2009 Type of Use reuse in a dissertation / thesis Volume number 9 Issue number 2 Requestor type academ ic/educational Format print and electronic Portion figures/tables/illustrations Number of figures/tables/illustrations 1 High res required no Figures Figure 1 Author of this NPG article N o Your reference number None Title of your thesis / dissertation LANTHANIDE BASED NANOPARTICLES AS MULTIFUNCTIONAL AGENTS FOR MAGNETIC RESONANCE IMAGING AND CANCER TREATMENT Expected completion date May 2015 Total 0.00 USD

PAGE 164

164 Title: Size Dependent MRI Relaxivity and D ual Imaging with Eu0.2Gd0.8PO4·H2O Nanoparticles Author: Yichen Li, Tao Chen, Weihong Tan, et al Publication: Langmuir Publisher: American Chemical Society Date: May 1, 2014 Copyright © 2014, American Chemical Society Logged in as: Yichen Li PERMISSION/LICENSE IS GRANTED FOR YOUR ORDER AT NO CHARGE This type of permission/license, instead of the standard Terms & Conditions, is sent to you because no fee is being charged for your order. Please note the following: Permission is granted for your request in both print and electronic formats, and translations. If figures and/or tables were requested, they may be adapted or used in part. Please print this page for your records and send a copy of it to your publisher/graduate school . Appropriate credit for the requested material should be given as follows: "Reprinted (adapted) with permission from (COMPLETE REFERENCE CITATION). Copyright (YEAR) American Chemical Society." Insert appropriate information in place of the capitalized wo rds. One time permission is granted only for the use specified in your request. No additional uses are granted (such as derivative works or other editions). For any other uses, please submit a new request.

PAGE 165

165 Title: Geometrical confinement of gadoliniu m based contrast agents in nanoporous particles enhances T1 contrast Author: Jeyarama S. Ananta, Biana Godin, Richa Sethi, Loick Moriggi, Xuewu Liu, Rita E. Serda Publication: Nature Nanotechnology Publisher: Nature Publishing Group Date: Oct 24, 2010 Copyright © 2010, Rights Managed by Nature Publishing Group Logged in as: Yichen Li Order Completed Thank you very much for your order. This is a License Agreement between Yichen Li ("You") and Nature Publishing Group ("Nature Publishing Group"). The license consists of your order details, the terms and conditions provided by Nature Publishing Group, and the payment terms and conditions. License Number 3591960541832 License date Mar 18, 2015 Licensed content publishe r Nature Publishing Group Licensed content publication Nature Nanotechnology Licensed content title Geometrical confinement of gadolinium based contrast agents in nanoporous particles enhances T1 contrast Licensed content author Jeyarama S. Ananta, Biana Godin, Richa Sethi, Loick Moriggi, Xuewu Liu, Rita E. Serda Licensed content date Oct 24, 2010 Type of Use reuse in a dissertation / thesis Volume number 5 Issue number 11 Requestor type academic/educational Form at print and electronic Portion figures/tables/illustrations Number of figures/tables/illustrations 1 High res required N o Figures Figure 1 Author of this NPG article N o Your reference number None Title of your thesis / d issertation LANTHANIDE BASED NANOPARTICLES AS MULTIFUNCTIONAL AGENTS FOR MAGNETIC RESONANCE IMAGING AND CANCER TREATMENT Expected completion date May 2015 Total 0.00 USD

PAGE 166

166 LIST OF REFERENCES (1) Gonzalez, E.; Arbiol, J.; Puntes, V. F. Science 2011 , 334 , 1377. ( 2 ) Webster, T. J. Safety of Nanoparticles from Manufacturing to Medical Applications. In Nanostructure Science and Technology; Springer: New York, 2009 . ( 3 ) Chen, T.; Shukoor, M. I.; Wang, R.; Zhao, Z.; Yuan, Q.; Bamrungsap, S.; Xiong, X. ; Tan, W. ACS Nano 2011 , 5 , 7866. ( 4 ) Wang, Y. X.; Hussain, S. M.; Krestin, G. P. Eur. J. Radiol . 2001 , 11 , 2319. ( 5 ) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Sci ence 2005 , 307 , 538. (6) Gao, X.; Cui, Y.; Levenson, R. M.; Chung, L. W. K.; Nie, S. Nat. Biotechnol . 2004 , 22 , 969. (7) Huang, X.; El Sayed, I. H.; Qian, W.; El Sayed, M. A. J. Am. Chem. Soc. 2006 , 128 , 2115. (8) Chen, J.; Glaus, C.; Laforest, R.; Zhang, Q.; Yang, M.; Gidding, M.; Welch, M. J.; Xia, Y. Small 2010 , 6 , 811. (9) Roy, I.; Mitra, S.; Maitra, A.; Mozumdar, S. Int. J. Pharm . 2003 , 250 , 25. (10) Zhang, W.; Guo, Z.; Huang, D.; Liu, Z.; Guo, X.; Zhong, H. Biomaterials 2011 , 32 , 8555. (11) Wang, Y.; Li, Z.; Hu, D.; Lin, C. T.; Li, J.; Lin, Y. J. Am. Chem. Soc . 2010 , 132 , 9274. (12) Liu, Z.; Sun, X.; Nakayama Ratchford, N.; Dai, H. ACS Nano 2007 , 1 , 50. (13) Seymour, L. W.; Ulbrich, K.; Steyger, P. S.; Brereton, M.; Subr, V.; Strohalm, J.; Duncan, R. B r. J. Cancer 1994 , 70 , 636. (14) Huang, S. K.; Mayhew, E.; Gilani, S.; Lasic, D. D.; Martin, F. J.; Papahadjopoulos, D. Cancer Res. 1992 , 52 , 6774. (15) Chandrasekar, D.; Sistla, R.; Ahmad, F. J.; Khar, R. K.; Diwan, P. V. Biomaterials 2007 , 28 , 504. (16) L i, H.; He, X.; Kang, Z.; Huang, H.; Liu, Y.; Liu, J.; Lian, S.; Tsang, C. H. A.; Yang, X.; Lee, S. T(S. T.). Angew. Chem., Int. Ed. 2010 , 49 , 4430. (17) Wu, X.; Liu, H.; Liu, J.; Haley, K. N.; Treadway, J. A.; Larson, J. P.; Ge, N.; Peale, F.; Bruchez, M. P. Nat. Biotechnol . 2003 , 21 , 41.

PAGE 167

167 (18) Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Nat. Mater. 2005 , 4 , 435. (19) Wang, F.; Liu, X. J. Am. Chem. Soc. 2008 , 130 , 5642. (20) Chen, G.; Shen, J.; Ohulchanskyy, T. Y.; Patel, N. J.; Kutikov, A.; Li, Z.; Song, J.; Pandey, R. K.; Ã…gren, H.; Prasad, P. N., Han, G. ACS Nano 2012 , 6 , 8280. (21) Na, H. B.; Lee, J. H.; An, K.; Park, Y. I; Park, M.; Lee, I. S.; Nam, D. H.; Kim, S. T.; Kim, S. H.; Kim, S. W.; Lim, K. H.; Kim, K. S.; Kim, S. O.; Hyeon, T. A ngew. Chem., Int. Ed. 2007 , 46 , 5397. (22) Popovtzer, R.; Agrawal, A.; Kotov, N. A.; Popovtzer, A.; Balter, J.; Carey, T. E.; Kopelman, R. Nano Lett. 2008 , 8 , 4593. (23) Liu, Y.; Ai, K.; Liu, J.; Yuan, Q.; He, Y.; Lu, L. Angew. Chem., Int. Ed. 2012 , 51 , 14 37. (24) Adv. Mater. 2011 , 23 , H18. (25) Cheon, J. and Lee, J. H. Acc. Chem. Res. 2008 , 41, 1630. (26) Mallidi, S.; Larson, T.; Tam, J.; Joshi, P. P.; Karpiouk, A.; Sokolov, K.; Emelianov, S. Nano Lett. 2009 , 9 , 2825. (27) De La Zerda, A.; Zavaleta, C.; Keren, S.; Vaithilingam, S.; Bodapati, S.; Liu, Z.; Levi, J.; Smith, B. R.; Ma, T. J.; Oralkan, O.; Cheng, Z.; Chen, X. Y.; Dai, H. J.; Khuri Yakub, B. T.; Gambhir, S. S. Nat. Nan otechnol. 2008 , 3 , 557. (28) Moghimi, S. M.; Hunter, A. C.; Murray, J. C. FASEB J. 2005 , 19 , 311. (29) Kang, X.; Yang, D.; Ma, P.; Dai, Y.; Shang, M.; Geng, D.; Cheng, Z.; Lin, J. Langmuir 2013 , 29 , 1286. (30) Chen, F. H.; Gao, Q.; Ni, J. Z. Nanotechnology 2008 , 19 , 165103. (31) Farokhzad, O. C.; Langer, R. ACS Nano 2009 , 3 , 16. (32) Yu, M. K.; Park, J.; Jon, S. Theranostics 2012 , 2 , 3. (33) Folkman, J. Nat. Med. 1995 , 1 , 27. (34) Reddy, S. M.; Sinha, V. R.; Reddy, D. S. Drugs Today 1999 , 35 , 537. (35) Maed a, H.; Greish, K.; Fang, J. Adv. Polym. Sci. 2006 , 193 , 103. (36) Iyer, A. K.; Khaled, G.; Fang, J.; Maeda, H. Drug Discovery Today 2006 , 11 , 812.

PAGE 168

168 (37) Duncan, R. Nat. Rev. Drug Discov . 2003 , 2 , 347. (38) Miele, E.; Spinelli, G. P.; Miele, E.; Tomao, F.; T omao, S. Int. J. Nanomed. 2009 , 4 , 99. (39) Ulbrich, K.; Hekmatara, T.; Herbert, E.; Kreuter, J. Eur. J. Pharm. Biopharm. 2009 , 71 , 251. (40) Cheng, J.; Teply, B. A.; Sherifi, I.; Sung, J.; Luther, G.; Gu, F. X.; Levy Nissenbaum, E.; Radovic Moreno, A. F.; Langer, R.; Farokhzad, O. C. Biomaterials 2007 , 28 , 869. (41) Stella, B.; Arpicco, S.; Peracchia, M. T.; Desmaële , D.; Hoebeke, J.; Renoir, M.; D'Angelo, J.; Cattel, L.; Couvreur, P. J. Pharm. Sci. 2000 , 89 , 1452. (42) Liong, M.; Lu, J.; Kovochich, M.; X ia, T.; Ruehm, S. G.; Nel, A. E.; Tamanoi, F.; Zink, J. I. ACS Nano 2008 , 2 , 889. (43) Liu, Y.; Miyoshi, H.; Nakamura, M. Int. J. Cancer 2007 , 120 , 2527. (44) Tian, Q.; Hu, J.; Zhu, Y.; Zou, R.; Chen, Z.; Yang, S.; Li, R.; Su, Q.; Han, Y.; Liu, X. J. Am. C hem. Soc. 2013 , 135 , 8571. (45) Dickerson, E. B.; Dreaden, E. C.; Huang, X.; El Sayed, I. H.; Chu, H.; Pushpanketh, S.; McDonald, J. F.; El Sayed, M. A. Cancer Lett. 2008 , 269 , 57. (46) Wang, C. G.; Chen, J.; Talavage, T.; Irudayaraj, J. Angew. Chem., Int. Ed. 2009 , 48 , 2759. (47) Yang, K.; Hu, L.; Ma, X.; Ye, S.; Cheng, L.; Shi, X.; Li, C.; Li, Y.; Liu, Z. Adv. Mater. 2012 , 24 , 1868. (48) Song, X.; Gong, H.; Yin, S.; Cheng, L.; Wang, C.; Li, Z.; Li, Y; Wang, X.; Liu, G.; Liu, Z. Adv. Funct. Mater. 2014 , 24 , 1194. (49) Zhang, S.; Zha, Z.; Yue, X.; Liang, X.; Dai, Z. Chem. Commun. 2013 , 49 , 6776. (50) Degani, H.; Gusis, V.; Weinstein, D.; Feilds, S.; Strano, S. Nat. Med. 1997 , 3 , 780. (51) Yu, C. P.; Ruppert, G. C. S.; Nguyen, D. T. D.; Falcao, A. X.; Liu, Y. Biosignals 2012 , 527. (52) Louie, A. Chem. Rev. 2010 , 110 , 3146. (53) Oghabian, M. A; Farahbakhsh, N. M. J. Biomed. Nanotechnol. 2010 , 6 , 203. (54) Terreno, E.; Castelli, D. D.; Viale, A.; Aime, S. Chem. Rev. 2010 , 110 , 3019.

PAGE 169

169 (55) Viswanathan, S.; Kovacs, Z.; Green, K. N.; Ratnakar, S. J.; Sherry, A. D. Chem. Rev. 2010 , 110 , 2960. (56) Na, H. B.; Song, I. C.; Hyeon, T. Adv. Mater. 2009 , 21 , 2133. (57) Caravan, P. Chem. Soc. Rev. 2006 , 35 , 512. (58) Caravan, P.; Ellison, J. J.; McMurry, T. J.; Lauffer, R. B . Chem. Rev. 1999 , 99 , 2293. (59) Raymond , K. N.; Pierre , V. C. Bioconjugate Chem. 2005 , 16 , 3 . (60) Werner, E. J.; Datta, A.; Jocher, C. J.; Raymond, K. N. Angew. Chem., Int. Ed. 2008 , 47 , 8568. (61) Caravan, P.; Farrar, C. T.; Frullano, L.; Uppal, R. Con trast Media Mol. Imaging 2009 , 4 , 89. (62) Que , E. L. ; Chang , C. J . Chem. Soc. Rev. 2010 , 39 , 51. (63) Quiñónez, C. J. Ph.D. Thesis, California Institute of Technology, 2003 . (64) Anderson, E. A.; Isaacman, S.; Peabody, D. S.; Wang, E. Y.; Canary, J. W.; K irshenbaum, K. Nano Lett . 2006 , 6 , 1160. (65) Johnson, N. J. J.; Oakden, W.; Stanisz, G. J.; Scott Prosser, R.; van Veggel, F. C. J. M. Chem. Mater. 2011 , 23 , 3714. (66) Lauffer, R. B. Chem. Rev . 1987 , 87 , 901. (67) Hu, F. Q.; Zhao, Y. S. Nanoscale 2012 , 4 , 6235. (68) Fortin, M. A.; Petoral, R. M.; Söderlind, F.; Klasson, A.; Engström, M.; Veres, T.; Käll, P. O.; Uvdal, K. Nanotechnology 2007 , 18 , 395501. (69) Dumont, M. F.; Hoffman, H. A.; Yoon, P. R. S.; Conklin, L. S.; Saha, S. R.; Paglione, J. P.; Sze, R. W.; Fernandes, R. Bioconjugate Chem. 2014 , 25 , 129. (70) Perrier, M.; Kenouche, S.; Long, J.; Thangavel, K.; Larionova, J.; Goze Bac, C.; Lascialfari, A.; Mariani, M.; Baril, N.; Guérin , C.; Donnadieu, B.; Trifonov, A.; Guari, Y. Inorg. Chem. 2013 , 52 , 13402. (71) Huang, C. C.; Liu, T. Y.; Su, C. H.; Lo, Y. W.; Chen, J. H.; Yeh, C. S. Chem. Mater. 2008 , 20 , 3840. (72) Ananta, J. S.; Godin, B.; Sethi, R.; Moriggi, L.; Liu, X.; Serda, R. E.; Krishnamurthy, R.; Muthupillai, R.; Bolskar, R. D.; Helm, L.; Fe rrari, M.; Wilson, L. J.; Decuzzi, P. Nat. Nanotechnol. 2010 , 5 , 815.

PAGE 170

170 (73) Cheng , Z.; Thorek, D. L. J.; Tsourkas, A. Angew. Chem., Int. Ed. 2010 , 49 , 346. (74) Gianolio, E.; Porto, S.; Napolitano, R.; Baroni, S.; Giovenzana, G. B.; Aime, S. Inorg. Chem. 20 12 , 51 , 7210. (75) De Smet , M.; La ngereis , S.; van den Bosch , S.; Grüll , H. J. Controlled Release 2010 , 143 , 120 . (76) Yang, H.; Zhang, C.; Shi, X.; Hu, H.; Du, X.; Fang, Y.; Ma, Y.; Wu, H.; Yang, S. Biomaterials 2010 , 31 , 3667. (77) Chou, S. W.; Shau, Y. H.; Wu, P. C.; Yang, Y. S.; Shieh, D. B.; Chen, C. C. J. Am. Chem. Soc. 2010 , 132 , 13270 (78) Drug Delivery 2009 , 6 , 865. (79) Chen, D.; Jiang, M.; Li, N.; Gu, H.; Xu, Q.; Ge, J.; Xia, X.; Lu, J. J. Mater. Chem . 2010 , 20 , 64 22. (80) Kim, D.; Jeong, Y. Y.; Jon, S. ACS Nano 2010 , 4 , 3689. (81) Ow, H.; Larson, D. R.; Srivastava, M.; Baird, B. A.; Webb, W. W.; Wiesner, U. Nano Lett . 2005 , 5 , 113. (82) Li, Y. L.; Zhu, L.; Liu, Z.; Cheng, R.; Meng, F.; Cui, J. H.; Ji, S. J.; Zhong, Z. Angew. Chem., Int. Ed. 2009 , 48 , 9914. (83) Chen, F. H.; Zhang, L. M.; Chen, Q. T.; Zhang, Y.; Zhang, Z. J. Chem. Commun . 2010 , 46 , 8633. (84) Bridot, J. L.; Faure, A. C.; Laurent, S.; Riviere, C.; Billotey, C.; Hiba, B.; Janier, M.; Josserand, V.; Col l, J. L.; Elst, L. V.; Muller, R.; Roux, S.; Perriat, P.; Tillement, O. J. Am. Chem. Soc . 2007 , 129 , 5076. (85) Ryu, J.; Park, H. Y.; Kim, K.; Kim, H.; Yoo, J. H.; Kang, M.; Im, K.; Grailhe, R.; Song, R. J. Phys. Chem. C 2010 , 114 , 21077. (86) Dumont, M. F .; Baligand, C.; Li, Y.; Knowles, E. S.; Meisel, M. W.; Walter, G. A.; Talham, D. R. Bioconjugate Chem. 2012 , 23 , 951. (87) Talanov, V. S.; Regino, C. A.; Kobayashi, H.; Bernardo, M.; Choyke, P. L.; Brechbiel, M. W. Nano Lett . 2006 , 6 , 1459. (88) Insin, N. ; Tracy, J. B.; Lee, H.; Zimmer, J. P.; Westervelt, R. M.; Bawendi, M. G. ACS Nano 2008 , 2 , 197. (89) Ren, W.; Tian, G.; Zhou, L.; Yin, W.; Yan, L.; Jin, S.; Zu, Y.; Li, S.; Gu, Z.; Zhao, Y. Nanoscale 2012 , 4 , 3754.

PAGE 171

171 (90) Rodriguez Liviano, S.; Nuñez, N. O. ; Rivera Fernández, S.; de la Fuente, J. M.; Ocaña, M. Langmuir 2013 , 29 , 3411. (91) Jung, J.; Kim, M. A.; Cho, J. H.; Lee, S. J.; Yang, I.; Cho, J.; Kim, S. K.; Lee, C.; Park, J. K. Biomaterials 2012 , 33 , 5865. (92) Shi, Z.; Neoh, K. G.; Kang, E. T.; Shut er, B.; Wang, S. C. Contrast Media Mol. Imaging 2010 , 5 , 105. (93) Petoral, R. M.; Söderlind, F.; Klasson, A.; Suska, A.; Fortin, M. A.; Abrikossova, N.; Selegård, L.; Käll, P. O.; Engström, M.; Uvdal, K. J. Phys. Chem. C 2009 , 113 , 6913. (94) Patra, C. R. ; Bhattacharya, R.; Patra, S.; Basu, S.; Mukherjee, P.; Mukhopadhyay, D. J. Nanobiotechnol . 2006 , 4 , 1. (95) Park, J. Y.; Baek, M. J.; Choi, E. S.; Woo, S.; Kim, J. H.; Kim, T. J.; Jung, J. C.; Chae, K. S.; Chang, Y.; Lee, G. H. ACS Nano 2009 , 3 , 3663. (96 ) Hifumi, H.; Yamaoka, S.; Tanimoto, A.; Citterio, D.; Suzuki, K. J. Am. Chem. Soc. 2006 , 128 , 15090. (97) Rodriguez Liviano, S.; Becerro, A. I.; Alcántara, D.; Grazú, V.; de la Fuente, J. M.; Ocaña, M. Inorg. Chem . 2013 , 52 , 647. (98) Na, H. B.; Lee, J. H .; An, K.; Park, Y. I.; Park, M.; Lee, I. S.; Nam, D. H.; Kim, S. T.; Kim, S. H.; Kim, S. W.; Lim, K. H.; Kim, K. S.; Kim, S. O.; Hyeon, T. Angew. Chem., Int. Ed. 2007 , 119 , 5493. (99) Rieter, W. J.; Taylor, K. M. L.; An, H.; Lin, W.; Lin, W. J. Am. Chem. Soc . 2006 , 128 , 9024. (100) Della Rocca, J.; Liu, D.; Lin, W. Acc. Chem. Res. 2011 , 44 , 957. (101) Nishiyabu, R.; Hashimoto, N.; Cho, T.; Watanabe, K.; Yasunaga, T.; Endo, A.; Kaneko, K.; Niidome, T.; Murata, M.; Adachi, C.; Katayama, Y.; Hashizume, M.; Ki mizuka, N. J. Am. Chem. Soc . 2009 , 131 , 2151. (102) Lu, S.; Zhang, J.; Zhao, H.; Luo, Y.; Ren, X. Nanotechnology 2010 , 21 , 365709. (103) Sahu, N. K.; Ningthoujam, R. S.; Bahadur, D. J. Appl. Phys . 2012 , 112 , 014306. (104) Gupta, A. K.; Gupta, M. Biomateria ls 2005 , 26 , 3995. (105) Rout, S. R.; Behera, B.; Maiti, T. K.; Mohapatra, S. Dalton Trans . 2012 , 41 , 10777. (106) Das, M.; Mishra, D.; Dhak, P.; Gupta, S.; Maiti, T. K.; Basak, A.; Pramanik, P. Small 2009 , 5 , 2883.

PAGE 172

172 (107) Queffélec, C.; Petit, M.; Janvier, P.; Knight, D. A.; Bujoli, B. Chem. Rev . 2012 , 112 , 3777. (108) Huang, C. C.; Lo, Y. W.; Kuo, W. S.; Hwu, J. R.; Su, W. C.; Shieh, D. B.; Yeh, C. S. Langmuir 2008 , 24 , 8309. (109) Bhattacharya, D.; Baksi, A.; Banerjee, I.; Ananthakrishnan, R.; Maiti, T. K .; Pramanik, P. Talanta 2011 , 86 , 337. (110) Medley, C. D.; Bamrungsap, S.; Tan, W.; Smith, J. E. Anal. Chem . 2011 , 83 , 727. (111) Gillis, P.; Koening, S. H.Transverse Magn. Reson. Med . 1987 , 5 , 323. (112) Arsalani, N.; Fattahi, H.; Nazarpoor, M. Polym. Le tt . 2010 , 4 , 329. (113) Bottrill, M.; Kwok, L.; Long, N. J. Chem. Soc. Rev . 2006 , 35, 557. (114) Patra, C. R.; Bhattacharya, R.; Patra, S.; Basu, S.; Mukherjee, P.; Mukhopadhyay, D. Clin. Chem . 2007 , 53 , 2029. (115) Pérignon, N.; Mingotaud, A.; Marty, J.; Rico lattes, I.; Mingotaud, C. Chem. Mater . 2004 , 16 , 4856. (116) Pérignon, N.; Haag, R.; Marty, J.; Thomann, R.; Lauth de Viguerie, N.; Mingotaud, C. Macromolecules 2005 , 38 , 8308. (117) Pérignon, N.; Marty, J.; Dumont, M.; Rico lattes, I.; Mingotaud, C. Macromolecules 2007 , 10 , 3034. (118) Keilitz, J.; Radowski, M. R.; Marty, J.; Haag, R.; Gauffre, F.; Mingotaud, C. Chem. Mater. 2008 , 20 , 2005. (119) Beija, M.; Salvayre, R.; Lauth de Viguerie, N.; Marty, J. D. Trends Biotechnol . 2012 , 30 , 485. (120) Salib a, S.; Valverde Serrano, C.; Keilitz, J.; Kahn, M. L.; Mingotaud, C.; Haag, R.; Marty, J. D. Chem. Mater . 2010 , 22 , 6301. (121) Radowski, M. R.; Shukla, A.; von Berlepsch, H.; Böttcher, C.; Pickaert, G.; Rehage, H.; Haag, R. Angew. Chem., Int. Ed. 2007 , 46 , 1265. (122) Liang, G.; Cao, L.; Chen, H.; Zhang, Z.; Zhang, S.; Yu, S.; Shen, X.; Kong, J. J. Mater. Chem. B 2013 , 1 , 629. (123) Hou, Y.; Qiao, R.; Fang, F.; Wang, X.; Dong, C.; Liu, K.; Liu, C.; Liu, Z.; Lei, H.; Wang, F.; Gao, M. ACS Nano 2013 , 7 , 330. (124) Villaraza, A. J. L.; Bumb, A.; Brechbiel, M. W. Chem. Rev . 2010 , 110 , 2921.

PAGE 173

173 (125) Wiener, E.; Brechbiel, M. W.; Brothers, H.; Magin, R. L.; Gansow, O. A.; Tomalia, D. A.; Lauterbur, P. C. Magn. Reson. Med . 1994 , 31 , 1. (126) Ye, H.; Scott, R. W. J.; Crooks, R. M. Langmuir 2004 , 20 , 2915. (127) Marty, J. D.; Martinez Aripe, E.; Mingotaud, A. F.; Mingotaud, C. J. Colloid Interface Sci . 2008 , 326 , 51. (128) Greenspan, P.; Fowler, S. D. J. Lipid Res . 1985 , 26 , 781. (129) Sackett, D. L.; Wolff, J . Anal. B iochem . 1987 , 167 , 228. (130) Behnke, T.; Würth, C.; Hoffmann, K.; Hübner, M.; Panne, U.; Resch Genger, U. J. Fluoresc. 2011 , 21 , 937. (131) Reddy,L. H.; Arias, J. L.; Nicolas , J.; Couvreur , P. Chem. Rev . 2012 , 112 , 5818. (132) Sapsford, K. E.; Algar, W. R .; Berti, L.; Gemmill, K. B.; Casey, B. J.; Oh, E.; Stewart , M. H.; Medintz, I. L. Chem. Rev . 2013 , 113 , 1904. (133) Kang, X.; Yang, D.; Dai, Y.; Shang, M.; Cheng, Z.; Zhang, X.; Lian, H.; Ma , P.; Lin, J. Nanoscale 2013 , 5 , 253. (134) Kim, J.; Park, S.; Le e, J. E.; Jin, S. M.; Lee, J. H.; Lee, I. S.; Yang, I.; Kim, J.; Kim, S. K.; Cho, M.; Hyeon, T. Angew. Chem. 2006 , 118 , 7918. (135) Link, S.; Burda, C.; Nikoobakht, B.; El Sayed, M. A. J. Phys. Chem. B 2000 , 104 , 6152. (136) Tian, Q.; Wang, Q.; Yao, K. X.; Teng, B.; Zhang, J.; Yang, S.; Han, Y. Small 201 4 , 10 , 106 3 . (137) Cheng, Z.; Al Zaki, A.; Hui, J. Z.; Muzykantov, V. R.; Tsourkas, A. Science 2012 , 338 , 903. (138) Fu, G.; Liu, W.; Feng, S.; Yue, X. Chem.Commun. 2012 , 48 , 11567. (139) Altagracia Martinez , M.; Kravzov Jinich, J.; Martínez Núñez, J. M.; Ríos Castañeda, C.; López Naranjo, F. Orphan Drugs: Research and Reviews 2012 , 2 , 13 . (140) Shokouhimehr, M.; Soehnlen, E. S.; Hao, J.; Griswold, M.; Flask, C.; Fan, X.; Basilion, J. P.; Basu, S.; Huang , S. D. J . Mater. Chem. 2010 , 20 , 5251. (141) Jones, K. H.; Senft, J. A. J. Histochem. Cytochem. 1985 , 33 , 77. (142) Goubard, F.; Tabuteau, A. J. Solid State Chem. 2002 , 167 , 34 .

PAGE 174

174 (143) Asakura, D.; Li, C. H.; Mizuno, Y.; Okubo, M.; Zhou, H.; Talham, D. R. J. Am . Chem. So c . 2013 , 135 , 2793 . (144) Dumont, M. F.; Knowles, E. S.; Guiet, A.; Pajerowski, D. M.; Gomez, A.; Kycia, S. W.; Meisel , M. W.; Talham, D. R. Inorg. Chem. 2011 , 50 , 4295 . (145) Okubo, M.; Li, C. H.; Talham, D. R. Chem. Commun . 2014 , 50 , 1353. (146 ) Peprah, M. K.; Li, C. H.; Talham, D. R.; Meisel, M. W. Polyhedron 2013 , 66 , 264. (147) Tong, S.; Hou, S.; Zheng, Z.; Zhou, J.; Bao, G. Nano lett. 2010 , 10 , 4607. (148) Zhang, L.; Wu, H. B.; Lou, X. W. J. Am. Chem. Soc . 2013 , 135 , 10664. (149) Santos, H. A.; Bimbo, L. M.; Lehto, V. P.; Airaksinen, A. J.; Salonen, J.; Hirvonen, J. Curr. Drug Discovery Technol. , 2011, 8 , 228. (150) Zhao, Y.; Trewyn, B. G.; Slowing, I. I.; Lin, V. S. Y. J. Am. Chem. Soc . 2009 , 131 , 8398. (151) Horcajada, P.; Chalati, T.; Serr e, C.; Gillet, B.; Sebrie, C.; Baati, T.; Eubank, J. F.; Heurtaux, D.; Clayette, P.; Kreuz, C.; Chang, J. S.; Hwang, Y. K.; Marsaud, V.; Bories, P. N.; Cynober, L.; Gil, S.; Férey, G.; Couvreur, P.; Gref, R. Nat. Mater . 2010 , 9 , 172. (152) Piao, Y.; Kim, J .; Na, B. H.; Kim, D.; Baek, J. S.; Ko, M. K.; Lee, J. H.; Shokouhimehr, M.; Hyeon, T. Nat. Mater . 2008 , 7 , 242. (153) Pothayee, N.; Pothayee, N.; Jain, N.; Hu, N.; Balasubramaniam, S.; Johnson, L. M.; Davis, R. M.; Sriranganathan, N.; Riffle, J. S. Chem. Mater . 2012 , 24 , 2056. (154) Kirby, G. H.; Harris, D. J.; Li, Q.; Lewis, J. A. J. Am. Ceram. Soc . 2004 , 87 , 181. (155) Chen, Y.; Chen, H.; Zeng, D.; Tian, Y.; Chen, F.; Feng, J.; Shi, J. ACS Nano 2010 , 4 , 6001. (156) Yang, J.; Lee, J.; Kang, J.; Lee, K.; S uh, J. S.; Yoon, H. G.; Huh, Y. M.; Haam, S. Langmuir 2008 , 24 , 3417.

PAGE 175

175 BIOGRAPHICAL SKETCH Yichen Li was born in Xuzhou , China . After graduating from high school in 2006, he joined Nanjing University of Science and Technology, which is right beside the pu rple mountain (Zijin Shan) . In 2010, he graduated with a in bioengineering. He then came to University of Florida in the fall of 2010. H e is one of the seven students who joined Professor that year . He st arted his doctoral research since then. He passed his oral qualification exam in the fall of 2012. Yichen defended his research work and graduated in the spring of 2015 from the University of Florida with a Ph.D. in chemistry.