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1 ENGINEERED NANOPARTICLE S AS CONTRAST A GEN T S: UPTAKE AND CLEARANCE STUDIES By DEBAMITRA DUTTA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009
2 2009 Debamitra Dutta
3 To my loving husband, my best friend and guarding angel and my affectionate parents and siblings. Without their encourag ement and support I would have never be en able to pursue my dreams
4 ACKNOWLEDGMENTS There we re so many p eople who ha ve been with me on this long journey toward s my doctoral degree that it actually ma k e s me nostalgic while writing this section as I r etrace my footsteps back to the early days of Graduate School. Firstly, I would like to express my sincere gratitude to my advisor and mentor Dr Brij Moudgil, who gave me the opportunity to work in some great research environment s, at PERC UF and at PNNL WA to deal with some of the most challenging scientific problems of our times. Apart from my PhD research work, I have had the opportunity to participate in industrial advisory board meetings conferences and leadership councils that have shaped me as a c onsummate research professional I am grateful to Dr. Swadeshmukul Santra for mentoring me during the early days in PERC and initiating me into the field of bioimaging. His mentorship essentially set the tone for my PhD research and his inputs have been in valuable to my dissertation. I have also had an opportunity to publish a number of journal papers and two book chapters due to his constant encouragement. I am also greatly thankful to Dr. Justin Teeguarden for his invaluable mentor ship during the second p hase of my PhD research work at PNNL. I benefited immensely from th e s e research interactions and thank him for his inputs in my work It helped the transition to partico kinetic studies of this research work much easier, which would have been difficult, if not impossible without his mentorship. I would like to sincerely thank the members of my PhD Committee, Drs. Hassan El -Shall, Christopher Batich, Henry Hess and Steve Roberts for their valuable inputs during the various stages of my research work. It is a matter of great pride for me to have such accomplished researchers and experts evaluate my candidature for the PhD degree. A t every point along th is program they were far less just committee members, and much more my valued mentors who helped to show me directions to get things done in a more effective manner.
5 I gratefully acknowledge all n anotoxicology team members at UF especially Drs Scott Brown, David Barber, Nancy D enslow, David Moraga, Scott Wasdo Kevin Powers, and Maria Palazuelos who helped lay the foundation for this interdisciplinary research work. Further, I also acknowledge the members of EBI at PNNL Drs. Joel Pounds, S.K. Sundaram, T om W eber and Brian Riley for their invaluable help for my research at Richland My heartfelt appreciation i s extended to all past & present Moudgil and El Shall group members who have helped me during this research work : Drs. Madhavan Esayanur, Ayman El -Midany, Suresh Yeruva, Georgios Pyrgiotakis, Parvesh Sharma, Vijay Krishna, Marie Kissinger, KyoungHo Bu, St ephen Tedeschi and Rhye Hamey S pecial thanks to Amit Singh, Ajoy Saha and Paul Carpinone for their help Special appreciations for all PERC and PNNL staff members without whose invaluable help, it would be very difficul t to complete this work in time: Gar y Scheiffele, Gill Brubaker, Greg Norton, Jo -Anne Standridge, Donna Jackson, Sophie Leone, Jamie Anders and James Newman at UF and Vichaya Sukwarotwat, Jeff Creim Jolen Soelberg, Teresa Luders, Terry Curry, Angela Woodstock, Andrea Busby and Barbara Goodw in at PNNL. I am also grateful to National Science Foundation (NSF) Grant (EEC 94 02989), NIRT (EEC 0506560), Particle Engineering Research Center (PERC) and its industrial collaborators for financial support. I acknowledge my friends in Gainesville and Richland. It would not have b een possible for me to come to Gainesville for the short trips from Richland without Sanchii, Bhuvan, Nima, Shrut i and their hospitality. I also acknowledge my extended family TCPC members and all other close friends wh o helped me to remain motivated Finally, as I do not believe in acknowledging my very own thats why this dissertation is dedicated to the people who walked with me every step of this long and difficult journey, my husband, parents and siblings. We made it togeth er so far and theres so much more to travel to pursue all my dreams.
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................................... 4 LIST OF TABLES ................................................................................................................................ 9 LIST OF FIGURES ............................................................................................................................ 10 ABSTRACT ........................................................................................................................................ 12 CHAPTER 1 INTRODUCTION AND SCIENTIFIC BACKGROUND ....................................................... 14 1.1 Historical Perspective ......................................................................................................... 15 1.2 Engineered Nanomaterials ................................................................................................. 17 1.3 Nanobiotechnology ............................................................................................................. 18 1.4 Cancer Nanotechnology ..................................................................................................... 19 1.5 Optical Imaging................................................................................................................... 20 1.6 Optical Imaging Techniques .............................................................................................. 21 1.7 Optical Contrast Agents ..................................................................................................... 22 1.8 Nanoparticle Based Optical Contrast Agents .................................................................. 23 1.8.1 Fluorescent Silica Nanoparticles (FSNPs) ......................................................... 25 1.8.2 Quantum Dot (QD) Nanoparticles ...................................................................... 28 1.8.3 Gold Based Nanoparticles ................................................................................... 31 1.9 Surface Functionalization and Bioconjugation ............................................................... 33 1.10 Biodistribution and Toxicity Studies of Engineered Nanoparticles ............................... 34 1.10.1 Amorphous Silica Nanoparticles ........................................................................ 35 1.10.2 Qua ntum Dots (QD) Nanoparticles .................................................................... 36 1.10.3 Gold Nanoparticles .............................................................................................. 40 1.11 Physicochemical Parameters of Nanoparticles affecting their Bioactivity .................... 42 1.11.1 Effect of Elemental Composition ....................................................................... 43 1.11.2 Effect of Particle Size, Surface Area and Number ............................................ 46 1.11.3 Effect of Shape ..................................................................................................... 48 1.11.4 Effect of Crystal Structure .................................................................................. 49 1.11.5 Effect of Surface Chemistry ................................................................................ 49 1.11.6 Opsonization and Phagocytosis .......................................................................... 50 1.11.7 Pegylation of Nanoparticles ................................................................................ 51 2 SPECIFIC OBJECTIVES AND RESEARCH APPROACH ................................................... 53 2.1 Gap Analysis ....................................................................................................................... 53 2.2 Hypothesis ........................................................................................................................... 54 2.3 Specific Objective ............................................................................................................... 54 2.4 Research Approach ............................................................................................................. 55 2.5 Dissertation Outline ............................................................................................................ 56
7 3 SYNTHESIS AND CHARACTERIZATION OF ENGINEERED NANOPARTICLES ..... 57 3.1 Synthesis and Bioconjuga tion Techniques ....................................................................... 57 3.1.1 Fluorescent Silica Nanoparticles (FSNPs) ........................................................... 59 22.214.171.124 Synthesis strategies: FSNPs ................................................................. 60 126.96.36.199 Surface functionalization and bioconjugation: FSNPs ........................ 63 3.1.2 Colloidal Gold Nanoparticles (NPs) ..................................................................... 65 188.8.131.52 Synthesis strategy: Gold NPs ................................................................ 65 184.108.40.206 Surface functionalization and bioconjugation: Gold NPs ................... 66 3.1.3 Silica Coated Quantum Dot Nanoparticles (QDS NPs) ...................................... 67 220.127.116.11 Synthesis strategy: QDS NPs ................................................................ 67 3.1.4 Gold Speckle d Silica Coated Quantum Dot Nanoparticles (QDSG NPs) ......... 70 3.2 Characterization of Nanoparticles (NPs) .......................................................................... 71 3.2.1 Fluorescent Sili ca Nanoparticles (FSNPs) .......................................................... 71 18.104.22.168 Particle size measurements ................................................................... 71 22.214.171.124 Surface charge measurements .............................................................. 72 126.96.36.199 Absorbance and fluorescence spectroscopy ........................................ 73 3.2.2 Colloidal Gold Nanoparticles (NPs) ..................................................................... 75 188.8.131.52 Particle size measurements ................................................................... 75 184.108.40.206 Surface charge measurements .............................................................. 76 220.127.116.11 Absorbance and fl uorescence spectroscopy ........................................ 76 3.2.3 Silica Coated Quantum Dot Core Nanoparticles (QDS and QDSG NPs) .......... 77 18.104.22.168 Particle size m easurements ................................................................... 77 22.214.171.124 Surface area measurements .................................................................. 77 126.96.36.199 Surface charge measurements .............................................................. 78 188.8.131.52 Absorbance and fluorescence spectroscopy ........................................ 78 3.2.4 Leaching Studies of Silica Coated Quantum Dot Nanoparticles (QDS NPs) .... 79 3.3 Challenges in QDSG NP Synthesis .................................................................................. 80 3.3.1 Optimization of Quantity of Gold and Thickness of Silica Coating .................. 81 3.3.2 Gold Speckle Conjugation in Microemulsion ...................................................... 82 3.3.3 Addition of Hydrazine ........................................................................................... 83 3.3.4 Attem pt at Direct Conjugation of Gold Spheres onto QDS NPs ........................ 83 3.3.5 Optimization of the Amount of APTS Added ..................................................... 84 3.3.6 Unsuccessful Attempt to Surface Functionalize the QDSG NPs ....................... 84 4 ENGINEERED NANOPARTICLES IN BIOLOGICAL SYSTEMS ..................................... 98 4.1 In Vitro Experiments with Fluorescent Silica Nanoparticles (FSNPs) .......................... 99 4.1.1 Cell Culture and Nanoparticle Incubation ............................................................ 99 4.1.2 Bioimaging Using Confocal Microscopy ........................................................... 100 4.1.3 Quantification of NP Uptake ............................................................................... 103 4.1.4 Cell Viability Studies ........................................................................................... 106 4.2 Gold Speckled Silica Coated Quantum Dot cored Nanoparticles (QDSG NPs) ......... 109 4.2.1 Bioimaging Studies in A549 Cells Using QDSG NPs ...................................... 109 4.2.2 Bioimaging Studies in Daphnia Using QDSG NPs ........................................... 110 4.2.3 Hyperthermic Characteristics of QDSG NPs ..................................................... 111
8 5 PARTICOKINETIC STUDY OF NANOPARTICLES IN RAT BLOOD ........................... 125 5.1 Experiments with Nanoparticles (NPs) in Rat Model ................................................... 125 5.1.1 Nanoparticles (NPs) used for Animal Study ...................................................... 125 5.1.2 Dosing the Rats .................................................................................................... 126 5.1.3 Nanoparticle (NP) Dispe rsion in Dosing Solution ............................................ 128 5.1.4 Analysis Protocol ................................................................................................. 130 5.1.5 Analysis of Blood Samples ................................................................................. 132 5.1.6 One Compartmental Model ................................................................................. 133 5.2 Establishing the Protocols for Animal Experiments ...................................................... 136 5.2.1 Effect of Background from Aqua Regia ............................................................. 136 5.2.2 Effect of Background from Other Components ................................................. 137 5.2.3 Digestion of Nanoparticles (NPs) ....................................................................... 138 5.2.4 Deposition of Nanoparticles (NPs) on Cannulae Tubes .................................... 138 5.3 Results from Blood Clearance Study.............................................................................. 142 5.3.1 Using Silica Coated Quantum Dots Nanoparticles (QDS NPs) ........................ 142 5.3.2 Using Gold Speckled Quantum Dot Nanoparticles (QDS G NPs) .................... 144 5.3.3 Using Bare Gold Nanoparticles (G NPs) .......................................................... 144 5.3.4 Using Aminated Gold Nanoparticles (G+ NPs) ................................................ 145 5.3.5 Using Pegylated Gold Nanoparticles (GP NPs) ................................................. 145 5.4 Differential Binding of Serum Proteins to Nanoparticles (NPs) .................................. 145 5.5 Discussions ....................................................................................................................... 148 6 CONCLUSIONS AND SUGGESTIONS FOR FUTURE WORK ....................................... 170 6.1 Conclusions ........................................................................................................................ 170 6.2 Suggestions for Future Work ............................................................................................ 174 LIST OF REFERENCES ................................................................................................................. 176 BIOGRAPHICAL SKETCH ........................................................................................................... 195
9 LIST OF TABLES Table page 5 1 Actual dose of nanoparticles administered to each rat ....................................................... 151 5 2 Amount (mass) of blood collected from rats dosed with nanoparticles ........................... 152 5 3 Observations from dispersion study .................................................................................... 155 5 4 Details of nanoparticles ....................................................................................................... 156 5 5 Nanoparticle deposition on polyurethane (PU) cannulae tube .......................................... 157 5 6 Nanoparticle deposition on polyethylene (PE) cannulae tube ........................................... 158 5 7 Quantitative data of analytical detection of blood samples collected from rats ............... 159 5 8 Preparation of nanoparticle stock solution ......................................................................... 160
10 LIST OF FIGURES Figure page 3 1 Synthesis of fluorescent silica nan oparticles ........................................................................ 85 3 2 Structure of folic acid............................................................................................................. 85 3 3 Folic acid conjugation on the fluorescent silica nanoparticles. ........................................... 86 3 4 Synthesis protocol for QDS nanoparticles ............................................................................ 87 3 5 Synthesis protocol for QDSG nanoparticles. ........................................................................ 88 3 6 Structure of QDSG nanoparticles .......................................................................................... 8 8 3 7 Transmission electron microscopic images of FSNPs of particle sizes .............................. 89 3 8 Coulter light scattering particle size measurements of FSNPs in DI water ........................ 90 3 9 Coulter light scattering particle size measurements of FSNPs in cell media ..................... 91 3 10 Absorption spectra ................................................................................................................. 92 3 11 Normalized fluorescence emission and excitation spectra of pure folate ........................... 93 3 12 Normalized fluorescence emission and fluorescence excitation spectra ............................ 93 3 13 Transmission electron microscopic image of gold nanoparticles. ...................................... 94 3 14 Absorbance spectra of gold nanoparticles in de ionized water. .......................................... 94 3 15 Transmission electron microscopic image of QDSG nanoparticles. .................................. 95 3 16 Absorbance spectra of QDSG nanoparticles in de ionized water. ...................................... 95 3 17 Fluorescence spectra of QDSG nanoparticles in deionized water. .................................... 96 3 18 Fluorescence spectra of QDS nanoparticles in de ionized water. ....................................... 96 3 19 Variation of absorbance spectra with increasing g old concentration. ................................ 97 3 20 Variation of fluorescence spectra with increasing gold concentration. .............................. 97 4 1 Fluorescence and transmi ssion image of A549 cells incubated with 100 nm. ................. 114 4 2 Fluorescence and transmission image of A549 cells incubated for 15 hours ................... 115 4 3 Fluorescence and transmission image of A549 cells incubated ........................................ 116
11 4 5 Typical protein concentration vs. absorbance readings curve. .......................................... 118 4 6 Quantification of cellular nanoparticle uptake. .................................................................. 119 4 7 Procedure for the cell viability test based on LDH membrane integrity assay. ............... 120 4 8 Percentage cytotoxicity of 100 nm and 190 nm aminated and folated FSNPs ................ 121 4 9 Percentage cytotoxicity of 100 nm and 190 nm aminated and folated FSNPs. ............... 122 4 10 Laser scanning confocal microscopic images of A549 cells ............................................. 122 4 11 QDSG NPs in daphnia. ........................................................................................................ 123 4 12 Hyperthermic characteristics of nanoparticles in A549 cells. ........................................... 124 5 1 Effect of increasing the aqua regia concentration in digested samples ............................ 161 5 2 Effect of increasing the aqua regia concentration with gold nanoparticles ...................... 162 5 3 Effect of increasing aqua regia concentration on the back ground signal ......................... 163 5 4 Standard curve for cadmium generated using spiked samples of QDS ............................ 164 5 5 Particle concentration of QDS nan oparticles in blood versus time of draw ..................... 164 5 6 Standard curve for cadmium generated using spiked samples of QDSG ......................... 165 5 7 Partic le concentration of QDSG nanoparticles in blood versus time of draw .................. 165 5 8 Standard curve of gold generated using spiked samples of G+ nanoparticles ................. 166 5 9 Particle concentration of G+ nanoparticles in blood versus time of draw ........................ 166 5 10 Standard curve for gold generated using spiked samples of GP nanoparticles ................ 167 5 11 Particle concentration of GP nanoparticles in blood versus time of draw ........................ 167 5 12 Histogram showing the half -life values of QD SG, QDS, G+, G and GP NPs ............... 168 5 13 Results from the one -dimensional gel electrophoresis experiment ................................... 169
12 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ENGINEERED NANOPARTICLES AS CONTRAST AGENTS: UPTAKE AND CLEARANCE STUDIES By DEBAMITRA DUTTA May 2009 Chair: Brij Moudgil M ajor: Materials Science and Engineering Nanoparticle (NP) based probes have shown tremendous potential as contrast agents. However, an added therapeutic modality on NPs can help in cancer detection and therapy. The objective of this work is to develop eng ineered NPs as contrast agents and conduct their uptake and clearance studies. Effect of physicochemical parameters (size, composition and surface charge) of NPs in A549 cells and rat blood was studied. 100 and 190 nm fluorescent silica nanoparticles (FSNP s), 15 nm silica coated quantum dots (QDS) and gold spec kled silica coated quantum dots (QDSG) and 15 nm gold NPs were synthesized using microemulsion and sol gel techniques. NPs were characterized for size, shape, surface charge, absorbance and fluorescen ce. Effect of two different FSNP -surface functionalizations: folate conjugation and amine modification was studied using A549 cells. Confocal microscopy and uptake studies showed folated NPs were uptaken more aggressively than aminated FSNPs and cell viabi lity was quantified using lactate dehydrogenase assay after FSNP uptake. Fluorescence from QD core and hyperthermic property of gold speckles on QDSG was utilized to image and kill cancer ( A549) cells. Synthesis protocol was established for QDSG NPs with optimal fluorescence and absorbance properties. Excess gold speckles could quench the QD-core fluorescence. Increased silica shell thickness be tween QD -core and gold speckles
13 prevent ed quenching. Bioimaging capability of QDSG was demonstrated in A549 cells and daphnia. Gold NPs with three different surface charges: bare gold G (negative), amine -modified gold G+, (positive) and pegylated-gold GP (neutral) NPs, QDS and QDSG were used for rat clearance studies. Blood samples collected at selected time points were analyzed for gold and cadmium u sing Inductively Coupled Plasma Mass Spectroscopy. A one -compartmental model was generated to calculate half life of NPs in rat blood. Half -life of QDS, QDSG and GP NPs were determined to be 12.59.7, 146.5 and 64015.5 minutes, respectively. G+ and G NPs were cleared rapidly (<5 minutes), QDSG and QDS NPs were cleared fast while PEG groups on GP led to delayed clearance and large half -life. Preliminary one -dimensional gel electrophoresis qualitatively showed that amount of adsorbed proteins on NPs were G+ and G > QDS and QDSG > GP and correlated directly with blood clearance behavior.
14 CHAPTER 1 INTRODUCTION AND SCIENTIFIC BACKGROUN D The field of nanotechnology has attracted a lot of attention over the last decade (De et al. 2008, Eijkel & van den Berg 2006, Grodzinski et al. 2006, Hiremath & Hota 1999, Juang & Bogy 2005, Nayak & Lyon 2005, Safarik & Safarikova 2002, Salata 2005) and can be easily recognized as one of the most critical re search endeavors of the early 21st century. It deals with research and technology development at an atomic, molecular and macromolecular scale by co ntrolled manipulations. The prefix nano is derived from the Greek word for dwarf as a nanometer (nm) is one thousand millionth of a meter (109 m). Literally, nanotechnology refers to a field of applied science and technology that deals with structures devices and systems with length scales ranging between 1 and 100 nm and possess novel properties characteristic of its dimensions. Firstly, nanomaterials with the same mass have a relatively larger surface area as compared to its larger counterparts. Thi s generally leads to an increased chemical activity due to the high surface energy. Secondly, the quantum size effects begin to dominate the material properties as the size is reduced to nanoscale dimensions, thus resulting in enhanced physical, chemical a nd biological properties. Therefore it is not surprising that research on nanotechnology has made a profound impact (Romig 2004) in various areas such as materials and manufacturing, energy, electronics, medicine and healthcare, biotechnology, information technology and national security (Cohen 2001, Eijkel & van den Berg 2006, Grodzinski et al. 2006, Juang & Bogy 2005, Nayak & Lyon 2005, Salata 2005, Silva 2004) By some estimates (Nel et al. 2006) nanotechnology is expected to far exceed the impact of the Industrial Revolution and projected to become a $1 trillion market by 2015 AD
15 1.1 Historical P erspective Contrary to popular belief, the first mention towards the conc ept of nanotechnology occurred in 1867 by James Clerk Maxwell, who proposed the concept of a tiny entity known as Maxwells Demon that can deal with individual molecules (Ivanitsky e t al. 1998) Initial experiments of nanoparticle observations and size measurement were reported in the first decade of the 20th century mostly from Richard Adolf Zsigmondys study of 10 nm gold sols and other nanosized materials (Zsigmondy 1916) Subsequently, in the 1920s Irving Langmuir and Katherine B.Blodge tts introduced th e concept of monolayer or one molecule thick layer of material (Blodgett & Langmuir 1932, Blodgett et al. 1932, Langmuir & Blodgett 1935) First surface force measurements by Derjaguin occurred in the early 1950s (Derjaguin et al. 1992) followed by a number of other research breakthroughs that served as the scientific basis to lay down the foundation for modern nanotechnology Late physicist and Nobel laureate Richard P. Feynmans clas sic lecture titled theres plenty of room at the bottom on December 29th 1959 at the annual meeting of the American Physical Society is considered an important landmark in the history of nanotechnology. It dealt with the future possibility and potential problems of manipulating and controlling things at the atomic scale (Feynman 1959) He visualized nanotechnology as the means to mimic nature and build nanosized objects, atom by atom and molecule by mole cule. He envisioned the possibility of using various sets of building tools as operating sets to manipulate the individual atoms and molecules up or down, tailoring it to specific applications. The term nanotechnology was actually coined in 1974, by the Japanese researcher Norio Tanigu chi (Taniguchi 1974) to mean precisi on machining with tolerances of a micrometer or less. Twelve years later in 1986, Eric Drexlers book on Engines of Creation and subsequent controversial study on Nanosystem s (discussion on the possibility of nanotechnology to
16 successfully replicate assemblers and lead to an exponential growth of productivity and personal wealth) is credited for creating public c onsciousness on nanotechnology (Drexler 1987) In recognition to the reality of Feynmans visio n in 1998, the National Science and Technology Council (NSTC) of White House created an interagency working group on nanoscience, engineering and technology. In January 2000, President Clinton in his State of the Union address, announced the $ 497 million federal National Nanotechnology Initiative for the fiscal year 2001 budget This initiative to form a broad -based coalition between academic institutions, private sectors, and local state and federal government actually laid the foundation towards establishing Feynmans nanotech dream. Since then, there have been innumerable related inventions and discoveries that is a testament to Feynmans extraordinary vision o n the future of nanotechnology (Bh ushan 2007) Today, it is envisioned that there shall be four generations of nanotechnology advancements. Currently, we have the first phase of passive nanostructural material developments to perform a single task, which shall be followed by the second ph ase of introducing active nanostructures for multi tasking. The third generation is expected to feature novel interactive nanosystems followed by the development of the first integrated nanosystem functioning much like a biological cell with hierarchical n ature (Nel et al. 2006) The use of materials with nanometric dimensions is not new. Commonly encountered in nature, some of the life-dependent biological molecules include proteins, enzymes and DNA. Silver and gold nanopar ticles (NPs) have been used to color ceramic glazes and stained g lass since the 10th century AD (Eichelbaum et al. 2007) Nanomaterials can be classified into three groups: natural, anthropogenic and engineered. The natural sources of NPs include forest fires and volcanic eruptions. Other biological moieties like viruses, magnetite (found in cells and
17 animals) and ferritin (protein storing excess iron in the body) is also a part of this group. The anthropogenic NPs are by -products of industrial activities like polish ing, simple combustion processes and internal combustion engines, power plants and welding fumes that release NPs into the environment. Even a simple process of food preparation like frying and grilling can be a source of NPs The last group of artificiall y generated or engineered nanomaterials refers to intentionally designed structures that exploit the technologically favorable properties that result as a consequen ce of particle size reduction. (Khler & Fritzsche 2007) 1.2 Engineered Nanomaterials Over the years, biological molecules occurring inside organisms and forming the basic units of life, served as a model for the development of engineered nanomaterials. Gener ally engineered nanomaterials are designed with specific physical and chemical properties to meet the requirements of a particular application (Decker 2006) They have some remarkable differences from their naturally occurring counterparts that are generally polydispersed in nature and possess chemically complex behavior. For example, nanosized natural products of combusti on processes may exhibit inconsistent composition, particle size and shape, and solubility characteristics. In contrast, the engineered structures are monodispersed and precisely regulated to have novel properties observable only at nanoscale dimensions. T oday, engineered nanomaterials have presented numerous new opportunities for completely new applications in the healthcare and automobile industries as well as shown a lot of potential to increase the performance of traditional products like cosmetics and food products. It is expected that there will be an exponential growth in the use of engineered submicroscopic scale materials over the next decade. However, with the ever increasing scope of nanoparticle (NP) applications over the last decade, the risk of exposure for human beings and the environment has become higher (Oberdorster et al. 1996, Oberdorster et al. 2005c) In contrast to the many efforts at exploiting the desirable
18 properties of NPs to improve human health, till very lately, there existed few systematic in vestigations into the specific properties of NPs that cause toxicity in living systems. This remains a challenge (Hett 2004, Nel et al. 2006) for the future growth of scope of nanotechnol o gy especially for its biological applications 1.3 Nanobiotechnology As the integration of nanotechnology into the physical and biological sciences continues to flourish, it is becoming increasingly evident that nanotechnology as applied to medicine w ill lead to significant advances in the diagnosis and treatment of various diseases (Holm et al. 2002) The ulti mate aim of medicine is to improve the quality of existence and it is anticipated that the use of engineered nanostructures and devices will help to monitor, control, construct, repair, defend and actually enhance human life. The use of particulates for na nomedicine will lead to a better understanding of the intricate mechanisms associated with various diseases. Certain applications in medicine that have been shown to make use of engineered nanostructures are: disease diagnostics, drug delivery, sensors, ge ne therapy, development of biocompatible prostheses and implants, and treatments for cancer and other diseases. As a result of the size dependent properties and dimensional similarity of nanomaterials to biomacromolecules, they are ideal for use in the ph ysiological environment, giving rise to the new field of nanobiotechnology. Nanobiotechnology is a young and rapidly evolving sub -field at the cross -road of two interdisciplinary areas biotechnology and nanoscience. It pertains to the utilization of bio logical systems to design functional nanostructures from organic and inorganic materials (Decker 2006) The topical areas of the field are still being defined towards a better understanding of living systems, developing new tools for medicine and better solutions for healthcare. Nanobiotechnology is expected to produce major advances in diagnostics, therapeutics and
19 preven t life threatening diseases like cancer and other neurodegenerative diseases like Parkinsons and alzheimers in the next decade (Goo dsell 2004) Some of the vast and diverse array of nanosized devices developed includes nanovectors for targeted delivery of cancer drugs and imaging contrast agents for the early detection of pre -cancerous and malignant lesions and polyps from biological fluids. These and many other similar nanodevices can provide the essential breakthrough in the fight against cancer in the near future (Kim et al. 2006) Consequently, it is not surprising that two years ago in 2006 there were nearly 250 nanotechnology -based medical products commercially available in the market an d many more were waiting in the pipeline (Dobr ovolskaia et al. 2008, Powers 2006) 1.4 Cancer Nanotechnology Despite nearly a quarter century of outstanding progress in fundamental cancer biology, there have been very little advances in the clinic regarding tumor detection. Two major roadblocks (Santra et al. 2005b) that account for this discrepancy are: (a) non availability of efficacious instrumentation for tumor detection at the very onset and (b) severe limitations of existing contrast agents for imaging a pplications. The scope of this thesis is limited to the latter problem and deals with the development of novel nanoparticle based contrast agents for optical imaging of cancer. The ultimate goal is to detect lesions at the very onset of transformational ev ents that leads towards malignancy. This may be possible through non -invasive routine screening from blood samples followed by more detailed molecular imaging of the contour profile of the growing lesions. Currently existing clinical cancer imaging technol ogies do not possess sufficient spatial resolution to identify lesions at the very onset (Sokolov et al. 2003a) Most imaging technologies require contrast agents, equipped with signal amplifying components attached to a molecular recognition and targeting ligand such as antibodies. Unfortunately, most contrast agents are somewhat limited to detecting abnormal ities at the microscopic level. In
20 order to combat th e formidable challenge of early cancer detection, it is important to identify suitable cancer biomarkers, understand their evolution over time and deploy new contrast agents especially engineered for early diagnosis. It is possible that nanotechnology (Ferrari 2005, Gao et al. 2004, Santra et al. 2005b, Wang et al. 2008, Wu et al. 2003) if properly integrated following thorough research on their environmental implications and health effects, could provide extraordinary opportunities to meet the challenges of bioimaging. Nanotechnology research is underway to estab lish suitable nanoparticle based contrast agents to detect lesions at the very onset by identifying molecular expressions in the microenvironment (Brigger et al. 2002, Rosi & Mirkin 2005, Sahoo & Labhasetwar 2003) There is a strong possibility of combining existing optical imaging technologies with the sophisticated nanoparticle based contrast agents for high resolution in vivo cancer imaging (Rosi & Mirkin 2005) 1.5 Optical Imaging [The following section has been reproduced from Santra & Dutta, 2007, with permission]. Optical imaging is a se nsitive, non invasive, nonionizing and relatively inexpensive technique that has shown a lot of potential for diagnostic imaging. Two major components associated with an optical imaging system are the imaging component and the optical contrast enhancing c omponent (i.e. contrast agent). Recent advances in optical imaging have utilized sophisticated laser technology, highly sensitive charged -coupled device (CCD) technology and powerful mathematical modeling of light propagation through the biological systems and all these developments have formed a solid basis for the imaging component. Molecular fluorescent probes have been successfully used as optical contrast agents for imaging a variety of cancer tissues in the past (Bremer et al 2003, Licha & Olbrich 2005) However, as mentioned before, the sensitivity of the contrast agent is a major obstacle in obtaining highresolution image. Again,
21 in vivo deep tissue optical imaging has been limited because of the low penetration depth of the light in the ultraviolet (UV) and visible spectral range (the approximate tissue penetration depth is about 1 2 mm). Near infrared (NIR) light in the spectral range between 650 nm and 900 nm is however capable of penetrating much deeper (up to several centimeters) into the tissue and skull (Bremer et al. 2003, Licha et al. 2002) This is due to the relatively low absorption of tissue components (water and hemoglobin) in the NIR spectral range. Therefore, the development of NIR based optical imaging system is attracting tremendous attention in recent years. For developing optical based imaging system, it is important to understand how light interacts with the biological tissues. U pon interacti ng with light in the UV and visible spectrum a ll biological tissues autofluoresce to a certain extent The autofluorescence originate s from the fluorescent molecules of tissues such as nicotinamide, flavins, collagen, and elastin (Bornhop et al. 2001) Simply, a tissue can interact with light photons by absorption, scattering and reflection. S ince biological tissue represents a complex system in terms of light propagation, it is expected that the optical image would be somewhat distorted. 1. 6 Optical Imaging Techniques [The following section has been reproduced from Santra & Dutta, 2007, with permission]. Optical based imaging methods such as confocal imaging, multiphoton imaging and many other techniques have been traditionally used to image fluorescence events that originate in vivo from surface and subsurface region. In recent years, advanc ed imaging technologies that use photographic systems with continuous or intensity-modulated light and tomographic systems has shown a great potential for deep tissue imaging. With the aid of highly sensitive contrast agents such as NPs (Santra & Dutta 2007) it may be possible to transfer optical imaging technology to human application.
22 1 .7 Optical C ontrast A gents [The following sec tion has been reproduced from Santra & Dutta, 2007, with permission]. The purpose of using an optical contr ast agent in the biological system is to enhance the optical contrast by virtue of their contrast enhancing properties for applications like fluoresc ence and scattering. Tissue contrast agents, for example, are capable of reducing the background signal and improving the image resolution. Fluorescent molecular contrast agents, mostly organic fluorescent compounds, possess high extinction coefficient and quantum yield and have potential in drastically suppressing tissue autofluorescence and hence background signal. The effective delivery (loading) of these contrast agents to the target tissue has also been realized to be an important factor for achieving better image contrast, other than its intrinsic fluorescent character istics (extinction coefficients and quantum yield) (Santra & Dut ta 2007, Santra et al. 2005b) The concentration of contrast agent per unit volume of target tissue would determine the signal strength. Therefore, higher loading of contrast agent is always desirable for better image resolution and hence in obtaining a s harp marginal contrast between the normal and the diseased tissue. A number of important features of an ideal contrast agent (Santra & Dutta 2007, Santra et al. 2005b) should be kept in mind prior to using or developing new contrast agents for diagnostic cancer imaging. Firstly, contrast agents should have minimum toxicity so that they can be administered safely. Secondly, contrast agents should have high extinction coefficient for effective absorption and high quantum yield for obtaining strong fluorescence signal. Thirdly, they should be photostable and should not have any photo-sensitizing effects (i.e. cause damage to cellular DNA and hence cell death; also termed as photosensitized cell death). Fourthly, contrast agents should be hydrophilic so that an aqueous based formulation can be easily made. Fifthly, contrast agents should preferably have an excitation and emission band maxim a in the
23 NIR range (650 nm to 900 nm) for deep tissue imaging. Lastly, for cancer imaging, contrast agents should be attachable to appropriate ca ncer specific delivery systems for example antibodies, peptides and folates for targeting. Organic fluorescent contrast agents, although studied extensively for a variety of bioimaging applications (Haugland 2003) starting from cellular to tissues to whole animal fluorescence imaging have several limitations for them to be considered as robust contrast agents (Santra et al. 2006) Firstly, organic fluorescent contrast agents (dyes) rapidly undergo degradation through photobleaching. As a result, fluorescence signal fades away when exposed to excitati on light source (particularly when laser is used for the excitation), limiting sensitive detection of the target. Secondly, fluorescent dyes are usually hydrophobic. In order to make aqueous based formulation, chemical modifications like the use of sodium salt are often required that sometimes compromises with their spectral characteristics. Thirdly, only a handfu l of fluorescent compounds possess low toxicity. Lastly, a very limited number of fluorescent dyes have excitation and emission band in the NIR s pectral range. 1 8 Nanoparticle Based O ptical C ontrast A gents [The following section has been reproduced from Santra & Dutta, 2007, with permission]. Nanoparticle (NP) based contrast agents present a whole new class of robust nanometer size (between 1 nm and 100 nm) particulate materials that has strong potential for optical imaging of cancer. There are several advantages of using NPs for bioimaging applications. Firstly, the sensitivity of the optical imaging could be greatly improved using nanoparticle bas ed contrast agents. A classic example is the fluorescent quantum dots (Q Ds ) and their applications in cancer imaging (Ben -Ari 2003, Gao et al. 2004, Medintz et al. 2005, Michalet et al. 2005, Stroh et al. 2005) The Q Ds are usually made of crystalline cadmium sulfide (CdS) and cadmium selenide (CdSe) based semiconductor particulate materials. They are small (<5 nm in
24 size) and bright havi ng a broad excitation band but a narrow emission band. Dye doped NPs such as dye doped silica (He et al. 2004, He et al. 2002, Santra et al. 2005c, Santra et al. 2001a, Santra et al. 2004b) dye doped polymer particles (Pan et al. 2004, Stsiapura et al. 2004) present another class of materials for sensitive cancer detection. In dye doped NPs, each particle carries thousands of dye m olecules, thus greatly enhancing the fluorescence signal (Santra et al. 2006, Santra et al. 2001a, Santra et al. 2001b, Sonvico et al. 2005) Dye doped NPs are usually smaller (about two to three orders of magnitude) than cells, which make them suitable for cellular application. Another well -studied particles are gold NPs (Copland et al. 2004, El -Sayed et al. 2005, Sokolov et al. 2003b, Sonvico et al. 2005) that have also been used for the sensitive cancer cell imaging. Gold NP s possess a strong surface plasmon band that originates from the efficient light scattering by the nanosize gold particles. Secondly, NP based contrast agents have better photostability in comparison to the traditional organic dye based contrast agents. This has tremendous potential for sensitive and real time monitoring of canc er progression like monitoring cancer growth and metastasis. Photostable NP s will allow non -invasive imaging of cancer tissue multiple times for monitoring tumor growth and also the effect of cancer drugs during cancer therapy. For example, Q Ds are extreme ly photostable. The effective surface passivation of Q Ds with a wide bandgap material such as zinc sulfide (ZnS) or zinc selenide (ZnSe) makes them photostable. In dye doped NPs, dye molecules remain encapsulated by the particle-matrix that protects them f rom photobleaching. This is because of the fact that particle -matrix is capable of somewhat preventing the penetration of oxygen molecules that cause dye degradation. Usually, Q D s are more photostable than the dye doped NPs. Gold NP s efficiently scatter light and do not fade away via photobleaching process.
25 Lastly, multiple imaging modalities (Santra et al. 2005a) can be integrated into NP based contrast agents (also called as multifunctional NP s), making them suitable for ima ging using multiple modalities such as fluorescence, X ray and MRI. This would have great importance for in vivo cancer imaging applications (Weissleder 2002, Weissleder et al. 1999) Once labeled with the multimodal contrast agents, tumors could be imaged noninvasively using a CT scan or MRI for th e pre -surgical assessment. During the surgical procedure tumor tissue could be directly visualized in real time by the optical property of the contrast agent (Santra et al. 2005a) This mode of tumor visualization would provide direct guidance to surgeons for the effective tumor resection, enablin g them to demarcate the boundary between the tumor and normal tissues. N P surface is usually modified to obtain multiple functional groups to improve aqueous dispersibility, specific targeting and biocompatibility (Gao et al. 2004, Levy et al. 2002, Pellegrino et al. 2005, Qhobosheane et al. 2001, Santra et al. 2005c, Santr a et al. 2001a, Santra et al. 2004a, Santra et al. 2004b, Santra et al. 2005d, Santra et al. 2005e, Santra et al. 2001b, Wang et al. 2005, Yang et al. 2004b) A robust NP design is the key step for the synthesis of highly sensitive optical contrast agent s. In a typical NP based optical contrast agent design, the optical core is encapsulated by an intermediate coating followed by an outermost layer containing appropriate functional groups for bioconjugation. NP s based contrast agents have strong potential for early cancer diagnosis since they are bright and photostable. In the following sections a variety of NP based contrast agents are discussed in considerable details. 1. 8 .1 Fluorescent Silica Nanoparticles (FS N P s ) Amorphous silica or silicon dioxid e NP s produced via Stobers sol gel method (Qhobosheane et al. 2001, Santra et al. 2005c, Stober et al. 1968) or by the microemulsion technique (He et al. 2004, Santra et al. 2001a, Santra et al. 2004a, Santra et al. 2004b, Santra et
26 al. 2001b) have rec ently found applications in bioimaging applications Unlike many other nanostructures, silica is not inherently fluorescent and hence cannot be exploited for sensitive imag ing applications by itself. However, fluorescent dye molecules can be incorporated ( dye doped) into the silica NP s to make them fluorescent (Santra et al. 2005c, Santra et al. 2001a, Santra et al. 2004a, Santra et al. 2001b) Another approach that has also been reported is to at tach fluorescent dye molecules via covalent binding into the silic a matrix For optical imaging applications (Santra et al. 2001b) it is preferable that dye molecules remain encapsulated by the silica matrix for various reasons. Several attracti ve features of silica based NP s include: water dispersibility, resistance to microbial attack and an optical transparent matrix that allows excitation and emission light to pass through efficiently. The silica NP s are resistant to swelling and remain uncha nged in a wide range of solvents that include aqueous based, neutral and acidic solutions. This property makes it especially attractive for use inside the physiological environment. Moreover, the process of doping fluorescent dyes inside the silica particl es is extremely simple and effective and the spectral characteristics of the dye molecules remain almost intact. Silica encapsulation provides a protective layer around the dye molecules, preventing the penetration of the reducing oxygen molecule that can photodegrade the dye molecules, bot h in air and in aqueous medium ( dissolved oxygen). As a result, photostability of dye molecules inside NPs increase s substantially, especially when compared to bare dyes in solution. The surface of silica NP can be easi ly modified to attach biomolecules such as proteins (Qhobosheane et al. 2001) peptides (Santra et al. 2004b) antibodies (Santra et al. 2001b) and oligonucleotides (Del Campo et al. 2005) using conventional silane based chemistry. A general synthesis s trategy of fluorescent silica NP s is the incorporation of organic or metallororganic dye
27 molecules inside the silica matrix. The dye doped silica based optical imaging probes are non isotopic, sensitive and relatively photostable in the physiological envir onment (Santra et al. 2001a, Santra et al. 2001b) Additionally, the interaction potential of the silica surface can be easily manipulated to facilitate the interaction with cells (Fang et al. 1999, Liu et al. 2005, Tan et al. 2000) Due to these novel features, fluorescent silica NP s (FSNPs) have found widespread applications in bioanalysis and bioimaging applications. Two routes for synthesizing dye doped silica nanoparticle have been reported in the literature, namely: (a) Stobers sol -gel method and (b) reverse microemulsion method (a) Stobers Method In a typical Stobers method, alkoxysilane compounds like tetraethyl orthosilicate (TEOS), tetramethylorthosilicate (TMOS), a variety of TEOS or TMOS derivatives etc.) un dergo base -catalysed hydrolysis and condensation reaction in ammonia -ethanol -water mixture, forming a stable alcosol. This method has been widely used for synthesizing both pure and hybrid (when more than one silane compound are used, such as dye doped sil ica particles) silica NPs with particle diameter ranging from a few tens of nanometers to several hundreds of nanometers (sub-micron size). Following the Stobers protocol (Stober et al. 1968) with a slight modification, the synthesis of fairly monodisperse organic dye doped fluorescent sili ca nanoparticles has been reported (Santra et al 2005c, Santra et al. 2001a, Santra et al. 2004b) Since organic dyes are normally hydrophobic, doping them inside the hydrophilic silica matrix is not straightforward. Typically, a reactive derivative of organic dye like amine reac tive fluorescein isoth iocyanate ( FITC ) is first reacted with an amine containing silane compound like 3 aminopropyltriethoxysilane (APTS) to form a stable thiourea linkage. Then FITC conjugated APTS and TEOS is allowed to hydrolyze and condense to form FITC -APTS conjugated sili ca particles. Note that particles so formed will have some amount of bare dye molecules on the
28 particle surface that is covalently attached. These bare dyes, due to their hydrophobic nature, will somewhat compromise the overall particle aqueous dispersibil ity and also be prone to photobleaching. Therefore, an additional coating with pure silica is usually applied around the dye -conjugated silica NPs (Santra et al. 2001b) Using Stober s method, bulk amount (kilograms) of silica particles can be easily produced in the laboratory. (b) Reverse microemulsion (W/O) method This method is used for the synthesis of pure silica, as well as inorganic and organic dye doped silica NPs. It is a ro bust technique to produce monodisperse particles in the nanometer size range (tens of nanometers to a few hundreds of nanometers). The W/O microemulsion is an isotropic, single -phase system that consists of surfactant, oil (as the bulk phase) and water (as nanosize droplets). Each surfactant -coated water droplets that are stabilized in the oil phase serve as an individual nanoreactor for the synthesis of silica NPs. The surfactant present at the oil and water interface is responsible for the thermodynamic s tability of the W/O microemulsion system. The nucleation and growth process occur inside the confined spherical volume of the nanoreactor. The size of the NPs can be controlled by varying the water to surfactant molar ratio of the microemulsion system. Th e fluorescence brightness of dye doped silica NPs can be improved by incorporating high -quantum yield organic dyes having large absorption coefficient values. In other words, brighter probes will improve the image resolution if encapsulated fluorescent dye s do not experience substantial photobleaching during imaging. 1.8.2 Quantum Dot (QD) Nano particles QDs are semi -conductor based nanocrystals t ypically in the size range of 1 12 nm. They are extremely bright, photostable, and considered a candidate mat erial to be an emerging probe for in vitro and in vivo molecular and cellular imaging (Dubertret et al. 2002, Gao et al. 2004, Gao et al. 2005, Medintz et al. 2005, Michalet et al. 2005, Rhyner et al. 2006, Wu et al. 2003) It
29 ha s been shown that QDs could improve tumor imaging sens itivity in vivo by at least 10 100 folds. The emission properties of QDs can be continuously tuned from 400 nm to 2000 nm by changing both the particle size and the chemical composition. These materials have a broad absorption band with narrow and symmetri c emission bands (full -width at half -maximum at 25 40 nm) which typically span from the visible to the NIR spectral range. The absorption properties are associated with the promotion of electrons from the conduction band to the valence band when the excita tion energy exceeds the semiconductor band gap resulting in the formation of an electron -hole pair. Some major optical features of QDs are described below: (i) Large molar extinction coefficient value. QDs are highly sensitive fluorescent agents (or fluor escent tags) for labeling cells and tissues. Unlike organic fluorescent compounds, QDs have very large molar extinction coefficient value (Leatherdale et al. ), typically in the order of 0.5 5 106 Mcm which means that QDs is c apable of absorbing excitation photons very efficiently (the absorption rate is approximately 10 50 times faster than organic dyes). The higher rate of absorption is directly correlated to the QD brightness and it has been found that QDs are approximately 10 20 times brighter than organic dyes (Bruchez et al. 1998, Chan & Nie 1998, Dabbousi et al. 1997) allowing highly sensiti ve fluorescence imaging. (ii) Excellent photostability. QDs are several thousand times more photostable than organic dyes. This feature allows real time monitoring of biological processes over a long period of time. (iii) Much longer lifetime. QDs are hi ghly suitable for time -correlated lifetime imaging spectroscopy. This is possible due to the longer excited state lifetime of QDs (about one order of magnitude longer than that of organic dyes), allowing effective separation of QD fluorescence from the background fluorescence. This will improve the image contrast by reducing the signal to -noise ratio (Jakobs et al. 2000, Pepperkok et al. 1999) in time -delayed data acquisition mode.
30 (iv) Larger Stokes shift value Unlike in organic dyes, the excitation and the emission spectrum of QDs are well separated which means there is a large S tokes shift value; typically 300 400 nm depending on the wavelength of the excitation light ). This allows further improvement of sensitivity in the detection by reducing the high background due to autofluorescence often seen in biological specimens (Gao et al. 2004) (v ) Multiple targeting capability The wavelength of QD emission is size dependent. This is a unique feature of QD materials in comparison to organic fluorescent dyes. The size dependent emission of QDs allows imaging and tracking of multiple targets simultaneously using a single excitation source. This feature is particularly important in tracking a pane l of disease specific molecular biomarkers simultaneously, allowing classification and differentiation of various complex human diseases (Gao & Nie 2003) Two commonly used methods for synthesizing QDs are (a) hot solution phase mediated and (b) reverse microemulsion mediated synthesis. (a) Hot solution phase mediated QD synthesis A popular technique of synthesizing QDs, which typically involves synthesis at elevated temperature near the high boiling point of nonpolar organic solvents. The Bawendi group (Murray et al. 1993) has reported the synthesis of highly crystalline and monodisperse (size distribution 8 11%) CdSe QDs u sing hightemperature growth solvents/ligands (mixture of trioctyl phosphine/trioctyl phosphine oxide, TOP/TOPO). A combination of tri n -octylphosphine oxide (TOPO) and hexadecylamine can also be used (Gao et al. 2005) There are primarily two reasons for using hydrophobic organic molecules as mixed s olvents or as a solvent / ligand mixture: the mixture serves as a robust reaction medium and also coordinates with unsaturated met al atoms on the QD surface to prevent the formation of bulk semiconductors. Following a simila r synthesis strategy Qu et al (Qu & Peng 2002) has reported
31 the formation of CdSe nanocrystals having fluorescence quantum yields as high as 85% at room temperature. (b) Reverse micelle mediated QD synthesis The before -mentioned reverse microemulsion technique can also be used for the synthesis of high quality core -shell QDs. Yang et al (Yang et al. 2004b) reported the synthesis of manganese (Mn) doped cadmium sulfide (CdS) core and zinc sulfide (ZnS) shell (CdS:Mn/ZnS) QDs using AOT (dioctylsulfosuccinate sodium salt) as a surfactant and heptane as the oil for the reverse microemulsion system. The bright yellow emitting CdS:Mn/ZnS QDs were reported to have an average size of 3.2 nm and highly photostable. A silica coating was applied inside the reverse microeulsion system to increase the photostability and decrease the possibility of cadmium leaching out into the physiological environment (Derfus et al. 2004b) 1.8 .3 Gold B ased Nanoparticles Recent advances in photonic technology have provided an alternative noninvasive technique to image cells in vivo. Metal -based NP s are especially very attractive because their optical properties are size and shape dependent. Use of gold and silver NP s for staining cells and tissue samples in electron microscopy has been in us e for over 30 years. Although nanosize metallic gold and silver do not fluoresce directly, they can effectively scatter light due a phenomenon called surface plasmon resonance (Santra et al. 2004a) This involves the collective oscillation of t he conduction electrons induced by the incident electric field (light). Colloidal particles can then exhibit a range of intense colors in the visible and NIR spectral regions. Various methods (Bajpai et al. 2007, Grzelczak et al. 2008, Guo & Wang 2007, Huo & Worden 2007, Perez Juste et al. 2005, Santra & Dutta 2007) have been reported in literature for the synthesis of colloidal gold nanospheres. Four major synthesis routes to chemically synthesize gold NPs are as follows (Santra & Dutta 2007, Santra et al. 2005b) :
32 (a) Reduction of gold precursors like gold tetrachloride (HAuCl4) using suitable reducing agents such as cit rate, sodium borohydrid e ascorbic acid and hydrazine (b) Microemulsions, copolymer micelles, reversed micelles, surfactants membranes and other amphiphilic compounds have been used for synthesizing stabilized gold NPs (c) Seed -mediated route using preformed metal lic seeds as nucleation centers. (d) Reduction of gold precursors using an appropriate combination of reducing agents and radiation techniques such as ultrasound and heat energy Gold NP s, because of their strong surface plasmon resonance (SPR) properties, have attrac ted considerable attention in bioimaging in recent years. The SPR signal originates from the collective oscillation of conduction electrons upon interaction with absorption photons The SPR frequency depends on various factors like particle size, shape, di electric properties aggregate morphology, surface functionalization and the refractive index of the surrounding medium Gold NP s have high absorption, and scattering cross section. Due to its excellent biocompatibility (Bright et al. 1996, Mann et al. 2000, Mrksich 2000) gold NP s have be en widely used in immunohistochemistry (gold based staining) and in ultra -sensitive DNA detection assays (Elghanian et al. 1997, Rosi & Mirkin 2005) A new class of molecular specific contrast agents for vital reflectance imaging based on gold NPs attached to probe molecules with high affinity for specific cellular biomarkers have been described (Sokolov et al. 2003b) The application of gold bioconjugates for vital imaging of precancers was demonstrated using cancer cell suspensions, threedimensional cell cultures, and norm al and neoplastic fresh cervical biopsies. They showed that gold conjugates could be delivered topically for imaging throughout the whole epithelium. Geoghegan et. al. (Geoghegan & Ackerman 1976, Geoghegan et al. 1978) used colloidal gold as an electron dense marker for the indirect detection of specific cell surface
33 molecules. Using gold labeled horseradish peroxidase, ovomucoid and rabbit anti goat IgG, they have successfully detected membrane bound concanavalin A, wheat germ agglutinin and goat anti -human IgM on blood lymphocytes respectively. Zhao et. al. (Zhao et al. 2003a, Zhao et al. 2003b) have established a simple and effective method for DNA immunization against Japanese encephalitis virus (JEV) infection with plasmids encoding the viral PrM and E proteins and colloidal gold. Inoculation of plasmids mixed with colloidal gold induced the production of specific anti JEV antibodies and a protective response against JEV challenge in BALB/c mice. 1 9 Surface Functionalization and Bioconjugation [The following section has been reproduced from Santra & Dutta, 2007, with permission]. For bioimaging applications, the NP based contrast agents have t o be appropriately surface modified with suitable targeting molecules. For example, in order to target cancer cells, the particles have to be attached to cancer specific antibodies or folic acid molecules that can be taken up by the tumor cells. This proce ss of surface modification and targeting generally involves a number of steps. Firstly, the particle surface is modified to obtain appropriate functional groups like amines, carboxyls and thiols. Next, NP s are bioconjugated or attached to the bio recogni ti on molecules like antibodies and folic acid molecules using suitable coupling reagents. Finally, bioconjugated particles are targeted to cancer cells. A few methods that have been frequently used for attaching specific functional groups on the surface o f various NP s are briefly described below (Santra & Dutta 2007) Bioconjugation with carboxylated particles. This is a common bioconjugation technique, especially used to immobilize protein molecules on the surface of silica NPs. The surface is modified to attach carboxyl groups ( COOH) by using carboxylated silane reagent. Biomolecules containing free amine functional groups (proteins a nd antibodies) are then covalently attached to the carboxyl functionalized NP using carbodiimide -coupling chemistry
34 Bioconjugation with aminated particles.It has been shown that many cancer cells over express folate receptors. Cancer targeting with fol ate -conjugated NPs has been recently reported Folates are chemically attached to aminated silica NPs using carbodiimide chemistry. Bioconjugation with avidin biotin binding. Avidin is a protein molecule that contains four specific binding pockets for biotin molecules. There exists a strong binding affinity between avidin and biotin molecules, which is comparable to covalent binding. Avidin coated NPs are typically attached to biotinalated molecules like antibodies and proteins. Bioconjugation via disulf ide bonding. Sulfohydryl -modified NPs are conjugated to disulfide linked oligonucleotides (like DNAs). In this method, di -sulfide bonding is used to attach the oligonucleotides to the NPs Bioconjugation using cyanogen bromide (CNBr) chemistry. NPs with hydroxyl groups can be activated by the reactive cyanogens bromide. Upon reaction with CNBr, a reactive OCN derivative of NPs is formed. This derivative then readily reacts with proteins via the amine group and forms a zero length bioconjugate with no s pacer in between the particles surface and protein molecule. 1.1 0 Bio distribution and Toxicity Studies of Engineered Nanoparticles It is evident that NPs have a lot of potential as contrast agents for applications in biology and medicine. According to GE Medical Systems the existing market for contrast agents in 2006 was estimated to be about USD 7 billion per year and there has been unprecedented growth in th e field in the last two years Consequently, the critical factor that will evaluate the utility o f these materials will be their potential toxicity inside the physiological environment. The manifested toxicity of the nanomaterial could be an outcome of the inherent chemical composition or its other physicochemical properties like particle size, shape, concentration, surface charge, surface functional group and mechanical stability (Hardman 2006, Nel et al. 2006, Oberdorster et al. 2005b) NPs as discussed abo ve, range from very simple compositions of pure gold and silver to complex engineered structures of quantum dots, especially where surface modifications have been applied. Some complicated nanostructures can be intentionally produced having special
35 charact eristics for therapeutic applications. This includes stealth properties that enable prolonged circulation in blood, escaping phagocytosis from macrophages, specific targeting to tissues and organs, translocating the blood brain barrier and sustained drug release capabilities. In contrast to the numerous efforts at exploiting the desirable properties of NPs in the literature there exist very few systematic investigations into the physicochemical properties of NPs that lead to adverse effects in living sys tems. The safety evaluations of the NPs cannot rely on the toxicological profiles of their larger counterparts. As yet, there are no paradigms to predict the biological responses of the engineered nanomaterials, l et al one the various functional groups and other surface modifications that frequently accompany them. The following sections contain a brief summary of the toxicity outcome from NPs used as contrast agents for this research work. Finally, the effect of various physicochemical parameters of NPs tha t can affect their biocompatibility and toxicity behavior is discussed in th e last section of this chapter. 1.1 0 .1 Amorphous Silica Nanoparticles Silica is the earths most abundant mineral, occurring in nature in several crystalline and amorphous forms The amorphous form of silica, which is used for bioimaging applications, is generally known to be comparatively non -toxic and biocompatible. It is used in the food paint, coatings and paper industries as a non -traditional biocompatible substance (Barnes et al. 2008) However, the crystalline forms of silica, especially -quartz, are generally associated with major health hazards like silicosis and cancer (Craighead 1988, Craighead 1992, Donaldson & Borm 1998, Driscoll & Guthrie 1997, Schins et al 2002) -quartz is the most thermodynamically stable crystalline form of silica at room temperature which is converted reversibly upon heating to quartz (another crystalline polymorph) (Dutta & Moudgil 2007) The different polymorphic forms are known to exhibit different levels of toxicity (Barnes et al. 2008) As the majority of the
36 population is exposed to -quartz, most toxicity experiments on silica are con ducted using this polymorph (IARC 1997) There are comparatively very few studies (Dutta et al. In Review for Publication, Renovanz 1984) reported on the toxicity aspects of dye doped amorphous silica NPs All bioimaging applications of silica essentially use the amorphous polymorph which appears to be biocompatible (Dutta et al. In Review for Publication, Rosi & Mirkin 2005) and have strong potential for biological applications (Santra & Dutta 2007, Santra et al. 2005b) 1.1 0 .2 Quantum Dots (QD) N anoparticles The semiconductor core of the QD nanocrystals is constituted of heavy toxic metallic comp onents. Although these metals contribute towards the unique optical and electronic properties of the QDs, they also make them hazardous for use inside the body. Most of the heavy metals like c admium, i ndium, and s elenium that constitute QDs are extremely t oxic to humans and can lead to renal, hepat ic and neurologic deficiencies (Cho et al. 2007, Derfus et al. 2004a, Hardman 2006, Maysinger et al. 2007) if they l each out. Hence for biological applications, it is critical to cap the QDs with a coating material in order to prevent heavy metal leaching. In fact, for decades these novel materials could not be used for biological applications, until protocols for modif ying the surface with appropriate c oatings were developed in 1998 (Bruchez et al. 1998, Chan & Nie 1998) to render the QDs water -soluble, less toxic and attachable to peptides, antibodies, nucleic acids and other low molecular weight biomolecules using covalent bonding. In fact, one of these studies demons trated for the first time in vivo that CdSe/ZnS QDs when coated with mercaptoacetic acid could bond onto blood transferrine (Chan & Nie 1998) These complexes were then taken up selectively by cancer cell (HeLa Cells). The controls particles with no transferrin could not be taken up as there was no re ceptor -mediated endocytosis.
37 Over the years, numerous studies have demonstrated the use of QDs coated with different substances for v arious biological applications (Akerman et al. 2002, Dubertret et al. 2002, Jiang et al. 2004a, Kirchner et al. 2005, Klostranec & Chan 2006, Lovric et al. 2005a, Lovric et al. 2005b, Medintz et al. 2005, Michalet et al. 2005, Shiohara et al. 2004, Smith 2002) However, there is not much information about the disposition and health consequences of QDs in animals and human beings. Most investigations on the QDs available in the literature have been carried out in vitro and report contradictory results regarding the toxicity aspects. A large number of these reports demonstrate that no toxicity was observed, while other investigators reveal a variety of cytotoxic symptoms like free radical generation, cell membrane integrity compromise and reduced mitochondrial activity due to incubation with quantum dots. The discrepancies in the literature can be attributed to various factors like lack of toxicologybased studies, differing QD dose and exposure, a wide range of physicochemical properties associat ed with each QD in the literature and the fact that most of these studies were performed by nanotechnology researchers rather than toxicologists or health professionals. In any case, these studies do provide an insight to the biological responses that coul d occur with the use of QDs inside the physiological environment. Existing in vitro studies in the literature seems to suggest that certain types of QDs may be cytotoxic. For instance, in an in vitro study to evaluate the cytotoxicity of CdSe QDs perform ed on liver cells it was shown (Derfus et al. 2004b) that the viability of hepatocytes incubated with the QDs declined depending on the particle concentration of the solution. T he reduction of cell viability was found to be related to particle concentration according to: 0.0625 < 0.25 < 1 mg/ mL and diminished significantly (6%) if the quantum dots were subjected to ultraviolet light for extended time (8 hours). Th is reduction of cell viability was attributed to the
38 cadmium io ns (Cd2+) that leached out of the quantum dots due to surface oxidation on exposure to ultraviolet light. Encapsulating the QDs in a layer of ZnS, a material that epitaxially matched the CdSe core of the QDs, could reduce this effect. But the Cd2+ ion leac hing could be actually reduced to zero when the QDs were encapsulated in 98% bovine serum albumen. Similarly, Shiohara et al. (Shiohara et al. 2004) reported a decrease in the cell viability as observed in three cells lines: kidney epithelial (Vero) cells, cervical c ancerous (HeLa) cells and primary human hepatocytes when incubated with mercaptoundecanoic acid (MUA) coated CdSe ZnS QDs with increasing particle concentration. Kirchner et al. (Kirchner et al. 2005) studied the cytotoxicity of colloidal CdSe and ZnS capped NP s, coated with various surface modifications and exposed to a number of tumor cells and human dermal fibroblasts cells (NRK fibroblasts, MDA -MB 435S breast cancer cells, CHO cells and RBL cells). In case of stable coatings, like applying a silica shell on the quantum dots followed by pegylation, the tox icity manifested was reduced as compared to unstable coatings like mercaptopropionic acid (MPA). Phosphosilicate coatings induced expedited Cd2+ leaching inside the shell. Interestingly, polymer coated gold NPs also displayed a cytotoxic effect compareable to the quantum dots. This seems to indicate that the toxic behavior could be due to the precipitated particles on the cells and not just due to the production of Cd2+ ions. Lovric et al. (Lovric et al. 2005a) reported the cytotoxicity of CdTe QDs coated with two different functional groups in rat pheochromocytoma cells (PC12) at particle concentrations of 10 g/ mL The two surface coatings applied were mercaptopropionic acid and cysteinamine. It was also reported that uncoated QDs were cytotoxic at 1 g/ mL Cell death occurred by chromatin condensation and membrane blebbing, both processes indicative of apoptosis in cells. Moreover, use of two different QD sizes showed a more pron ounced cytotoxicity for smaller and positively charged QDs as compared to their larger counterparts at
39 the same particle concentration. The exact mechanism of cell death was not known but was considered to be due to generation of cadmium ions, free radical formation or QD c ell interactions leading to loss of functions. On conducting further detailed analysis of the effect of QDinduced reactive oxygen species on the cell viability, the authors suggested that the toxicity could be partially induced by c admiu m. In another in vitro cum in vivo toxicity study of QDs, Hoshino et al. (Hoshino et al. 2004b) int ravenously injected QDs incorporated into El 4 cells into nude mice. As a control, they used MUA without any QDs for 12 hours and reported that severe cytotoxicity was observed in murine T -cell lymphoma (EL 4 cells) at 100 g/ mL Similarly, cells treated with cysteinamine showed weak genotoxicity at t he same particle concentration. The QDs could be detected in the kidney, lung, liver and spleen after 7 days with no apparent toxicity or damage to the mice. CdSe/ZnS QDs surface -functionalized with biotin we re also shown (Green & Howman 2005) to alter coiled double -stranded DNA by releasing sulphur dioxide (SO2) that gradually desorbed into solution to generate sulphite radicals. This was due to the surface oxidation of ZnS, which was accelerated in the presence of UV light. The proportion of DNA alt erations varied depending on the presence (56%) and absence (29%) of UV light. Compared to the number of in vitro studies, there are only few in vivo studies in the literature that report on quantum dot toxicity. In an intravenous mice study, Akermann et al. (Akerman et al. 2002) reported that th e nature of the coating could actually alter the distribution of the QDs in the various organs and tissues. They also demonstrated that QDs having PEG coating could actually reduce the liver and spleen capture by 95% and prolonged the half life in blood. Various other peptide coatings led to increased distribution in the lungs or in breast tumors in the study.
40 During in vivo imaging, Dubertret et al. (Dubertret et al. 2002) microinjected CdSe/ ZnS QDs coated with n -poly(ethylene glycol) phosphatidyl ethanolamine (PEG -PE) and phosphadylcholine (PC) into Xenopus frog embryo cells. They concluded that there was no significant toxicity from the Q D -micelles during embryogenesis in the study. 1.1 0 .3 Gold N anoparticle s Metallic NP s having a size range between 10 and 100 nm are known to exhibit the surface plasmon resonance ( SPR ) effect. This is a process by which the electrons in the metallic structure can resonate to incid ent radiation. It responds by a combination process of light scattering and absorption of electromagnetic radiation of wavelengths significantly larger than the particle itself. The effect is observed in the visible part of the spectrum for gold, silver an d copper NP s. For gold NP s, the plasmon resonance and consequently the absorption phenomena occurs at 520 nm, corresponding to green light in the spectrum. Although a proportion of the incoming light is scattered, the absorbed quantity of light is sufficient to generate localized heating or hyperthermia in the NP s. This behavior of gold, along with its oxidation resistance properties, can be exploited for therapeutic applications inside the physiological environment (Pissuwan et al. 2007a, Pissuwan et al. 2006, Pissuwan et al. 2007b, Pissuwan et al. 2007c) Localized application of heat has been utilized for anti tumor therapeutic applications for many centuries. Similarly, hyperthermic properties of gold NP s will be used to destroy the diseased tissues or for releasing the rapeutic payloads at target sites inside the body (Govorov & Richardson 2007, Govorov et al. 2006, Richardson et al. 2006, Richardson et al. 2007) Any energy source like infra red lamps, ultrasound or lasers can be used to generate the heat inside the particles. The only challenge is to restrict the NP s and the generated heat inside the target diseased tissues and allow healthy tissues to remain intact.
41 In recent years it has been demonstrated (Loo et al. 2004, O'Neal et al. 2004) that it is more convenie nt to shift the wavelength of the maximum absorption into the near infra red region (800 1200 nm) as the body tissues are comparatively more transparent in that region This will allow a higher resolution for deep tissue imaging and thereby enable detec tion of diseases at the onset. This has been demonstrated using high aspect ratio materials like gold nanorods (Huang et al. 2006, Huff et al. 2007a, Huff et al. 2007b, Maye et al. 2000, Niidome et al. 2006) which exhibited two plasmon absorption peaks (at 520 and 800 nm respectively) and amorphous silica particles encapsulated inside gold nanoshells (Diagaradjane et al. 2008, Hu et al. 2003, Loo et al. 2005a, Loo et al. 2004, Loo et al. 2005b) with varying opti cal properties over a broad range from near -UV to the mid IR. These nanoshells were shown to possess greater absorption efficiency than conventional near infra -red absorbing dyes by many orders of magnitude. Additionally, the gold nanoshells were less pron e to photobleaching. The early accounts of using hyperthermic properties of gold NP s for therapeutic applications were published in 2003 (Hirsch et al. 2003) and later (Loo et al. 2005a, Loo et al. 2004, Loo et al. 2005b) where nanoshells along with antibodies were used for actively targeting cancer ous cells. A follow up study, reported by the same group synthesized stealth particles by pegylating the gold nanoshells (O'Neal et al. 2004) and used it for passively targeting tumors in mice models. It was reported that NIR irradiation of the cancerous tissues led to an approximate temperature rise of 40 to 50 C which was used to preferentially kill the carcinoma. Colloidal gold NP s have also been used for therapeutic applications (Pitsillides et al. 2003) Specific antibodies were conjugated to the NP s and incubated in a co -culture of CD8+ and CD8 lymphocyte cells. On being irradiated with a laser at 532 nm, the NPs led to selective apoptosis in the CD 8+ cells. Moreover, certain experimental parameters were manipulated to
42 control conditions that led to preferential permeabilizing of the cell membrane in the target cells for upto two minutes. This showed that it is possible to use colloidal gold NP s for targeting and accessing intracellular organelles for therapeutic functions. 1.1 1 Physicochemical Parameters of N anoparticle s a ffecting their Bioactivity The unique physicochemical properties of nanosized particulate (NSP) systems can be attributed to the ir small size (Yin et al. 2005) shape, large specific surface area, chemical composition, surface activit y and interfacial interactions (Nel et al. 2006, Oberdorster et al. 2005b) However these novel characteristics that seem impressive from the material science perspectives could lead to adverse biological responses and develop into a hazard for human health and environment (Hoshino et al. 2004a, Karakoti et al. 2006, Ryman-Rasmussen et al. 2006) In fact, it has been suggested by researchers that NSPs even though sometimes not inherently benign, can affect the biological activities in cells, at the s ub cellular and protein levels (Nel et al. 2006) Additionally certain NSPs are able to travel th roughout the body and cross cell membranes to deposit in target organ s like the mitochondria to trigger injury and cell death (Xia et al. 2007) Therefore it is crucial that results from existing toxicity studies of NSP systems are assimilated in order to exactly identify the characteristics that po tentially pose a health risk and elucidate the underlying toxicity mechanism. It is indeed a challenge for scientists to design accurate experiments to specifically identify and reproduce these adverse biological interactions. The challenges can be ascrib ed to several issues (Teeguarden et al. 2007) : (a) dosimetry or t he correct method to express the dose of nanoparticulate systems (mass, dimensions, surface area, surface coating, state of aggregation etc) (b) confidence level regarding the exactly desired form of nanomaterial during dosage (c) difficulties in realtime detection and quantification of the nanomaterials inside the cells and tissues and (d) the need to characterize the NSPs at every stage of toxicological testing. The
4 3 following sections include an attempt to establish a correlation between the physicochemi cal properties of NSPs to their toxicological effects as reported in the literature database 1.1 1 1 Effect of Elemental C omposition The d ifferent compositions of NSPs lead to variations in the physical and chemical behavior of the material. It is an important parameter that can be correlated to the intrinsic properties (Donaldson et al. 2004) of the material to explain the variations in toxicity behavior of different nanomaterials. For instance in an initial nanotoxicity stud y (Ferin et al. 1990, Oberdorster et al. 1990) 20 nm alumina (Al2O3) and titania (TiO2) particles were compared to 500 nm Al2O3 and 250 nm TiO2 particl es. Although both materials in the nanosized dimensions induced significantly greater and more persistent inflammation than their respective larger counterparts, the inflammatory response in rats dosed with Al2O3 NP s was far more persistent as compared to rats with TiO2 NP s. Although the greater specific surface area of Al2O3 as compared to TiO2 played an important role, the material differences also contribu ted to the toxicity responses. The important role of particle chemistry in NP -induced toxicity was e vident from a subsequent inhalation study from the same research group (Oberdorster et al. 1995) using 20 nm polytetra fluoro ethylene (PTFE) fume particles in rats. They reported that inhalation of 50 g/m3 resulted in an acute pulmonary inflammation with high morbidity rates. The severe inflammatory responses of nano-sized PTFE fume particles contrast the minimal effects observed from 20 nm TiO2 and Al2O3 NPs Similarly, nanosized car bon black was found to be more severe than TiO2 NPs (Renwick et al. 2004, Renwick et al. 2 001) Again, both materials induced lung inflammation and damage in rat epithelial cells at greater extents than their larger counterparts. Several other different NP s (polyvinyl chloride, TiO2, S iO2, cobalt and nickel) ranging between a mean diameter of 14 nm to 120 nm were studied and only cobalt was found to induce toxicity in
44 endothelial cells and produce pro -inflammatory cytokine IL 8 (Peters et al. 2004) Amongst the other particles S iO2 induced minimal IL 8 release while TiO2 had a substantial one. Although no explanation for the differences in cytotoxic ity was presented, it could be due to both material and/or size differences of the various particles. A similar trend of composition dependence was also observed for ultra -fine (particles with mean diameter of less than 1 m) polymeric biomaterials (Tomazic Jezic et al. 2001) Two different polymers namely polystyrene (PS) and polymethyl methacrylate (PMMA) were delivered into rats by intraperitoneal administration The PMMA particles were recovered from the spleen while the PS particles, regardless of size were accumulated primarily in the rat adipose tissue of the peritoneal cavity with very few particles in the spleen. The response from cells in NP exposure studies ha s been mostly studied with regard to toxicity behavior. But remarkably enough, it has been shown that certain rare earth NP s like cerium oxide and yttrium oxide can protect nerve cells and retinal degeneration from various oxidative stresses. The neuroprot ective behavior was directly material structure dependent and was observed in NP s with single crystal structure with few twin boundaries or stacking faults and expanded lattice parameters relative to its bulk counterpart. The rare earth NP s act as free rad ical scavengers and can effectively reduce the amount of available reactive oxygen species which can kill the cells (Das et al. 2007, Schubert et al. 2006, Tarnuzzer et al. 2005) (a) Effect of Core constituent : Ce rtain nanomaterials are a combination of various components in a core -shell or other complex structures. Depending on the structure and surface chemistry in question, the particle may have reactive groups on the surface that can have an immediate signif icant effect on the biological responses. It is known that all materials are immediately covered by proteins, as soon as it comes in contact with the physiological environment. So most
45 of the instantaneous responses generated due to NP -cell interactions ar e determined by the nature of the deposited protein layer. But after a considerable amount of time, secondary events like material leaching through porous layers or surface layer dissolution may take over and the core constituent can have a more profound e ffect on the nanotoxicity profile of the material. For this reason, it is imperative that the biological behavior of the surface as well as the core constituent material is also understood while designing a core -shell NP for use in the physiological enviro nment. It has been reported that t he biodistribution behavior of QD s after 28 days from injection into the blood of rats starts to show a shift in the profile to a dual behavior, part of which resembles that of free cadmium (Yang et al. ). This similarity to free cadmium in the profile is indicative of heavy metal leaching which is extremely dangerous as cadmium is generally associated with various immune diseases. Moreover, possible gadolinium leaching from NP based magnetic contrast agents used for Magneti c Resonance Imaging (MRI) has been reported (Sharma et al. 2007) Thus, the release of the core material which is relea sed over a period of time and may have some potent toxicity can become a greater health concern as compared to the first -encountered functional group on the NP surface (b) Effect of Surface Composition: The surface composition will affect the uptake of th e NP by cells of the phagocytic system inside the physiological environment (Smith et al. 2006) This can be modula ted to a large extent by intentionally modifying or functionalizing the surface (Schellenberger et al. 2004) Us e of certain biomolecular linkers anchored on the surface or within vescicles or lip osomes (Nardin et al. 2000) has been shown to alleviate the biological responses. The affinity of the NP to a particular protein has thus been manipulated so that it can fit and thereby be used to target a certain biomolecular assembly on a membrane or within an
46 organelle or beneath the cell surface. The specificity of the surface can be used for analytical detection, optical labeling and other applications like drug or gene deliv ery to cells (Hood et al. 2002) In contrast, chemicals adsorbed on the surface may also adversely affect the reactivity of the NP s. Certain fractions from particulate air pollutants were observed to exert toxic effects on cells in vitro (Xia et al. 2006) (c) Effect of Purity : Presence of impurities like metal traces associated with the production of commercial nanotubes as catalysts have been shown to adversely affect biological responses and lead to the generation of free radicals and intracellular oxidative stress (Shvedova et al. 2003b) Especially, transition metals such as Fe, Ni and Cu have been shown to induce the formation of reactive oxy gen and nitrogen species through a Fenton -like reaction. The biological effect of SWCNTs before catalyst removal and containing 30% iron by mass has been studied in human keratinocyte cells (Shvedova et al. 2003a) A decrease in cell viability and glutathione depletion with a dose -dependent relationship was obs erved, which was considerably reversed by a metal chelator desferrioxamine. This indicates the significant role of the impurities present i.e. iron as a cause of some of the biological responses. Similarly, other investigators have reported on the dual rol e of e ssential transition metal ions, like copper and zinc, to act as cofactors as well as cataly sts for cytotoxic reactions (Finney & O'Halloran 2003) This implies that the biological responses of the same NSP will differ depending on the percentage purity of the sample in hand and the composition of the contamination in question. 1.1 1 2 Effect of P article S ize, S urface A rea and N umber The particle size, surface area and number of the NPs are closely related physical parameters that can influence the health eff ects of N P s to a large extent. Reducing the size to nanoscale dimensions results in a drastic increase of the surface to volume ratio, which means that there are more molecules of the chemicals are present at the surface. Although it is know n
47 that this wou ld lead to an increase in the intrinsic toxicity (Donaldson et al. 2004) the exact extent to which it contributes towards lung deposition, biopersistence is still not clearly understood. A small size of the NPs, with a corresponding large specific surface area makes them ideal for various applications. The ratio of the surface to total number of atoms or molecules increases exponentially with a decreasing particle size. This leads to increased surface reactivity and enhanced biological activity per unit mass as compared to larger particles. This increase in biological activity can be positive and desirable as for drug delivery and other anti oxidant applications or negative toxicity behavior as manifested through induce d oxidative stresses and cellular dysfunctions or a mix of both. In vivo studies have been reported using ultrafine particles t hat elicit more lung injury and pathology as compared to exposure to an equal mass of fine particles of the same material (Driscoll et al. 1996, Oberdorster 1993, Oberdorster et al. 1992, Oberdorster et al. 1994) D ue to the large specific surface area availa ble, th e particle size/surface area/ number are a more important determina nt of potential hazard than the mass of the material, provided the particle chemistry remains unchanged. On dosing rats by intratracheal instillation using 20 nm and 250 nm TiO2 particles with doses of 30 g and 2 mg, a much greater inflammatory response w as generated in the pulmonary system. But when plotted against the surface area of the particles, the neutrophil response fitted the same dose response curve (Oberdorster 2000) Similar results have been obtained by normalizing the lung weight to inflammatory responses in mice. Other investigators have also reported simil ar findings using carbon black (Brown et al. 2000, Stone et al. 2000) barium sulphate (Cullen et al. 2000, Tran et al. 2000a, Tran et al. 2000b) and polystyrene particles.
48 1.1 1 .3 Effect of Shape Nanomaterials have been synthesized in various shapes and structures such as spheres, rods, needles, tubes, pl ates and prisms. Recent evidences have indicated that the shape of the nanomaterials plays a crucial role in influencing the toxicity behavior. NSPs with high aspect ratio shapes like acicular or fiber like can obstruct phagocyte -mediated clearance mechani sms to induce enhanced toxicity as co mpared to isotropic structures (Brown et al. 2007) Moreover, it is very likely that the shape of the n anomaterial in question also affects the deposition kinetics and surface absorption. Champion et al. have used polystyrene particles of various shapes and sizes to study phagocytosis in alveolar macrophage cells (Champion & Mitragotri 2006) They reported that particles shape and not size play ed a dominant role in the phenomena of phagocytosis. The shapes, defined as the tangent angles at the point of initial contact, dictated whether the macrophages would initiate phagocytosis or would only spread over the particles. Surprisingly, par ticle size only regulated the completion of phagocytosis, especially in situations where the particle volume exceeded the cell volume. Genome expression array analysis were conducted on human fibroblast cells exposed to multi -walled carbon nano onions (MWC NO, aspect ratio = 1) and carbon nanotubes (MWCNTs, aspect ratio > 1). Results showed distinct differences in the gene expression profiles: MWCNTs exposure induced genes indicative of immune and imflammatory responses while MWCNOs in genes induced as respo nse to external stimuli (Ding et al. 2005) Similarly, Magrez et al. (Magrez et al. 2006) compared the cellular toxicity of carbon -based nanomaterials: MWCNTs, carbon nanofiber s (CNF) and carbon black (CB) N P s as a function of their aspect ratio on lung tumor cells. The number of viable cells decreased in the following order: CBN > CNF > CNT. They attributed the difference in toxicity behavior to the highly reactive sites present on the CBN due to a large number of dangling bonds compared to CNTs or CNFs where they only occur at lattice defects or at the end caps. Very
49 recently, Poland et al. have reported the asbestos -like pa thogenicity in rats due to exposure to MWCNTs that lead to inflammation and the formation of lesions, also known as granulomas (Poland et al. 2008) T he CNTs are hazardous fibers (i.e. thinner than 3 m, longer than 20 m and biopersistent in the lungs) and if the number of fibers reaches a sufficient level can chronically activate inflammatory cells, become genotoxic and lead to fibrosis and cancer in target cells and tissues. 1.1 1 .4 Effect of Crystal Structure Shrinkage in size from bulk to nanosized particles may lead to various changes in the material interactions that could indirectly affect toxicity behavior. A decrease in size may lead to an in crease in structural defects in the nanomaterial through the formation of discontinuous crystal planes and disrupted electronic configuration. This may lead to the formation of specific surface groups that may become reaction pockets for enhanced biologica l responses (Nel et al. 2006) For example, single -component transition metals or presence of transition metals on the surface can aid in electron capture that can lead to the formation of the superoxide radical. This would lead to the generation of reactive oxygen species (ROS) t hrough a process of dismutation, which is more commonly known as Fenton chemistry, and thus manifest toxicity due to induced oxidative stress. 1.1 1 .5 Effect of Surface Chemistry As mentioned be fore, NP s upon exposure to tissues and other biological fluids immediately adsorb macromolecules on their surface. The specificity of the adsorption process depends largely on surface characteristics like surface chemistry and surface energy of the NP s in question. Moreover, the degree of hydrophilicity and hydrophobicity of the surface regulates biological activities like cell-surface adhesion, protein denaturation at the interface and selective adsorption of materials at the interface. A variation in hydrophobicity leads to different
50 translocation routes in many biological compartments and differences in interfacial activities between nanomaterials and cells (Cauvel et al. 1997, Fubini 1997, Karakoti et al. 2006) 1.1 1 .6 Opsonization and Phagocytosis The clearance process of any foreign organism or particles that enters the blood generally occurs in three steps: opsonization, phagocyte opsonin protein binding and final ingestion of the foreign materials by the phagocytes (Owens & Peppas 2006) The process by which the foreign material gets covered by the opsonin proteins is known as o psonization. This process is followed by phagocytosis which involves engulfing and finally clear ing the foreign materials from the blood stream (Dobrovolskaia et al. 2008) These two clearance mechanisms inside the body make it possible to restrict undesirable elem ents larger than the renal threshold limit to reside in blood. Opsonization (Dobrovolskaia et al. 2008, Owens & Peppas 2006) generally occurs in the blood circulation and it can take anytime between a few seconds to several d ays for the process to complete. The precise mechanism by which this process (Moghimi & Hunter 2001, Moghimi & Szebeni 2003) is activated is very complex and not completely understood but components like immunoglobulins, C1, C2 and C3 are known to be the common opsonins. Other blood serum proteins like lamin in and fibronectin have been shown to play an important role in the clearance process using inherited and deficient animal models (Owens & Peppas 2006) The opsonon protein s, which are generally present throughout the blood, can come into contact with any introduced foreign nanoparticle system by random Brownian motion. However, once the proteins get close enough to the particle surface, they start interacting using several attractive forces like van der Waals, electrostatic, ionic, hydrophilic and hydrophobic, leading to opsonin binding on the particle surface. It is known that without the presence of the surface bound opsonin protein s it will not be possible for the phagoc ytic cells to recognize, bind and clear the foreign material.
51 The inactive opsonin protein present in the blood serum after encapsulating the foreign matter generally undergoes conformational changes to an activated protein structure that can be recogni zed by the phagocytes. There are several methods of phagocyte attachment onto the opsonin proteins. The phagocytic cells surfaces contain special receptors that function to interact with the modified conformation of the opsonins and acts to alert the phagocytes about the presence of foreign materials in the environment. Other non-specific attachment processes, especially between phagocytes and blood-serum proteins that have opsonized on hydrophobic materials have also been demonstrated (Moghimi & Hunter 2001, Moghimi & Szebeni 2003, Owens & Peppas 2006) The last and final step of ingestion involves endocytosis of the foreign material by the phagocyte. Secret enzymes and other oxidative reactive chemical factors (superoxides, peroxides and nitric oxide) are secreted to break down the phagocytosed material. As the initial opsonization of particles is the critical step towards phagocyte recognition and clearance from the blood -stream, most research towards drug delivery makes use of stealth NPs (Moghimi & Hunter 2001, Moghimi & Szebeni 2003) that can either avoid or de lay this process. Although, today there are no known techniques to block opsonization completely and effectively, research in the last few decades has successfully slowed the process, effectively increasing the blood circulation half -life by using stealth NPs for therapeutic applications. 1.1 1 7 Pegylation of Nanoparticles Several researchers have demonstrated a correlation between the particle surface charges to the induction of opsonin proteins. In general, it has been reported that the tendency of opso nization on hydrophobic surfaces is more and much quicker as compared to the hydrophilic surfaces. This is due to the enhanced adsorbability of blood serum proteins on the hydrophobic surfaces. Similarly, neutrally charged surfaces are known to have a lowe r rate of opsonization than their charged counterparts (Owens & Peppas 2006, Roser et al. 1998) Thus a widely used
52 method to prevent opsonization is to u se surface adsorbed or shielding groups that block any electrostatic and hydrophobic interactions that can aid the proteins to bind on the particle surfaces. Generally, these groups (Owens & Peppas 2006) are long hydrophilic polymer chains or non ionic surfactants like polyethylene glycol (PEG) polyacrylami d e and poly(vinyl) alcohol Other copolymers that contain PEG like poloxamers, polysorbates, poloxamines have also been us ed. The best stealth properties have been observed in PEG and PEG containing co -polymers (Moghimi & Hunter 2001, Moghimi & Szebeni 2003) P egylation refers to the molecular attachment of polyethylene glycols of various molecular weights (Moghimi 2006) to particles or active drug molecules to impart stealth functionality. Typically, these polymers are long, flexible and highly hydr ophilic and can effectively shield hydrophobic or charged particles from the blood protein (Carstensen et al. 1992, Muller et al. 1992, Norman et al. 1992) More importantly, they are neutral in charge and can reduce the electrostatic interactions (Owens & Peppas 2006)
53 CHAPTER 2 SPECIFIC OBJECTIVES AND RESEA RCH APPROACH 2.1 Gap Analysis The gap analysis outlined in this section is based on the critical literature review presented in the previous c hapter T wo major gaps were identified in the literature for this research effort: 1) absence of a NP system with multifunctional capability of bioimaging and therap y, and 2) absence of any clear correlation between the effects of physicochemical properties of nanoparticles (NPs) and the ir influence on the cellular uptake ( in vitro) and clearance behavior ( in vivo) and systematic particokinetic studies on NPs to comprehend their biodistribution behavior. Physicochemical characterization of NPs especially in biofluids and biological en vironment continues to be a challenge for developing reliable structure property performance correlation. The first gap relates to the fact that there currently exists limited research work on the develop ment of multifunctional NP s for bioimaging and thera p y It is known that quantum dots ( QDs ) possess the best fluorescent properties as compared to all other contrast agents. Gold NP s have been shown to have excellent absorbance properties that can be used for bioimaging applications. Additionally, gold has the potential for therapeutic applications due to its hyperthermic properties. Combining these two material propertie s provide an avenue to synthesize NP s with two different modes of contrast in the physiological enviro n ment as well as impart therapeutic propert y. There are numerous studies showing the bioimaging potential of QDs, but many of them also highlight the toxic aspect of these particles. However, not many attempts have been made to mitigate the toxic nature of QDs and study its blood clearance b ehavior in the physiological environment.
54 The second major gap identified for this research effort was the absence of adequate studies into the effect of phys icochemical p arameter s of nanosized partic u l at es on uptake and clearance Two of the major physicochemical properties of NP s that can influence their biological activity are material composition and surface chemistry The nature of the NP surface plays an important role in determining the toxicity of the material (Karakoti et al. 2006) The presence of different functional groups on the surface can potentially regulate the extent of cell -sur face interactions, protein adsorption and selective interaction of other components from the physiological environment thereby governing their biological activity in vitro and in vivo 2.2 Hypothesis Physicochemical parameters of NPs like material comp osition, particle size and surface functionalization can influence the cellular uptake and biodistribution and thereby determine its toxicity behavior. It is also known that QDs are extremely bright and photostable and have tremendous potential as bioimaging probes. However, due to the presence of heavy metals that can leach from the QDs they are considered toxic. Attempts are being made to use a morphous silica, a potential biocompatible material, to encompass the QDs and mitigate its toxicity. Further, d eposition of gold speckles on the outer silica shell can confer hyperthermic propert ies thereby impart ing therapeutic functionality to the resultant NP s Gold also imparts additional contrast capability due to its excellent absorbance properties. Additiona lly, modification of the NP surface can be u tilized for selective uptake which can be exploited for potential targeted therapeutic applications. 2.3 Specific Objective The overall objective of this research work is to develop engineered nanoparticula te systems for bioimaging applications and investigate the influence of their physicochemical
55 parameters on the cellular uptake (in vitro) and blood clearance ( in vivo ). T wo specific objective of th is research work were: D evelop ( synthesi s and characteri z ation ) NP systems for bioimaging applications and impart therapeutic functionality to the particles. S tudy the effect of various physicochemical parameters of the NPs like material composition, particle size, surface charge and functionality on the cellu lar uptake (in vitro ) and blood clearance ( in vivo ). 2.4 Research Approach As a first step towards developing engineered NPs fluorescent silica nanoparticles (FSNPs) were developed as bioimaging contrast agents by doping the amorphous silica matrix wi th organic fluorescent dyes. The cellular uptake was visually and qualitatively observed using confocal microscopy and quantita ti vely confirmed using protein assay technique s. Cell viability tests using conventional bioassay (LDH) were conducted to assess the biocompatibility of amorphous silica. CdS:Mn/ZnS QD NPs were then synthesized and coated with a biocompatible amorphous silica layer to form the silica coated quantum dot NPs (QDS) Leaching s tudies were performed on QDS NPs at different pH and temper atures to determine the amount of cadmium that could leach out. Gol d speckled silica coated quantum dots (QDSG) were developed by depositing gold speckles on QDS The QDSG NPs were used for imaging studies with daphni a and A549 cells. Heating studies were also conducted with the QDSG NPs to confirm the hyperthermic properties of the gold speckles. During this research effort, a ttempts to surface functionalize the QDSG NPs did not succeed. Therefore, colloidal gold NP s with positive, negative and near neutra l surface charge were synthesized as auxiliary particles to study the effect of surface charge on the biological responses of NPs In vivo studies were carried out to understand the behavior of
56 the engineered NP s in rat blood. For this study five different particles were used: a) bare colloidal gold particles (negative surface charge), b) gold particles conjugated with PEG (near neutral surface charge), c) Cysteine amine conjugated gold particles (positive surface charge), d) silica coated quantum dots and e) gold spec kled silica coated quantum dots. The NPs were intravenously injected into rats through the surgically attached cannulae tube on their jugular vein. Thereafter, blood was drawn out from each animal at predetermined time -points. The blood samples with NPs collected from the rats w ere digested and analyzed for gold and cadmium using inductively coupled plasma mass spectrometric ( ICP MS) technique A curve of NP concentration in blood versus time of draw was generated. Using this data and a one -co mpartmental model developed using equations from first -order reaction kinetics the half -lives (t) of the various NPs in rat blood were determined Protein adsorption studies were conducted to explain the blood clearance behavior of the different NPs. 2.5 Dissertation Outline The first chapter of this dissertation provides an introduction followed by a critical literature review to put the current research effort in perspective. In Chapter 2, (current chapter) the gap analysis from the literature review was presented, followed by the hypothesis and specific objectives of th e present study. T he research approach towards achieving these objectives was also outlined in Chapter 2 Chapter 3 presents the synthesis and characterization of all the NP s studied in the current research work. Chapter 4 discusse s the work related to in vitro studies conducted using the fluorescent dye doped silica NPs and quantum dot cored NP s. Chapter 5 describes the particokinetic studies of gold and quantum dot cored NP s in rat blo od. T he conclusions and the significance of this research work along with recommendations for future work are presented in Chapter 6.
57 CHAPTER 3 SYNTHESIS AND CHARAC TERIZATION OF ENGINE ERED NANOPARTICLES Th e synthesis and characterization of the various engineered nano particles (NPs) of interest for this research work is described in this chapter As outlined in the Research Approach, the various particles developed for this research effort are (i) fluorescent silica nanoparticles (FSNPs), (ii) c olloidal gol d spheres (G ), ( iii) silica coated quantum dot -core NPs (QDS) and (iv) gold spec kled silica coated quantum dot -core NPs (QDSG) In the first section, synthesis and bioconjugation protocols for these four NPs are presented. The second part of the chapter d eals with characterization of the NPs. This involved particle size and shape, surface charge, and spectroscopic ( absorbance and fluorescence ) studies The rationale was to choose the physicochemical properties of NPs that have been shown to influence their biological responses (Hardman 2006, Hoshino et al. 2004a, Nel et al. 2006, Oberdorster et al. 2005a, Oberdorster et al. 2005b, RymanRa smussen et al. 2006) The last section discusses the challenges faced during the research effort o f optimizing the QDSG synthesis protocol. 3.1 Synthesis and Bioconjugation Techniques Most applications in nanobiotechnology demand a homogenous morphology and monodispers ed particles, which generally lead s to a more consistent set of physical chemical and biological properties A wide range of techniques ha ve been developed in recent years a few can partially, while others effectively control the size and shape of the synthesized NPs (Sau et al. 2001) Sol gel processing, which is one of the most commonly used techniques can produce NPs of sizes 1 20 nm with consistent composition and stru cture but with a variable degree of monodispersity (Dutta et al. In Review for Publication) In fact, the size variations between the particles can even vary to 20%; however, for best nano -size related properties it is desirable that the variations are reduced to less than 5%.
58 Recently, t he use of a reverse or inverted micelle based system for NP synthesis has been developed for better control on the size and st ability of particles (Santra et al. 2001a, Santra et al. 2004b, Santra et al. 2001b) Reverse micelles, also called water in -oil (W/O) micr oemulsion (ME) system, are an isotopic, thermodynamically stable environment made of a homogenous mixture of oil, water and surfactant molecules. The surfactant capped water droplets remain uniformly dispersed in the bulk oil phase and the water droplets s erve as a tiny reactor for the synthesis of nanomaterials. Some of t he advantages of the reverse microemulsion technique are that it does not require extreme reaction conditions such as high temperature or high pressure and has a better control over the morphology and dispersity of the final product. But a prime drawback is the notably small volume of output from this synthesis technique which makes it difficult to scale up the synthesis process The size of the NP is controlled by varying the water to sur factant molar ratio ( Wd). Continuous collision between the water nano reactors due to Brownian motion leads to exchange between the short lived dimers that lead to the formation of droplets (Santra et al. 2001a) In th e present study we have used the sol gel technique to synthesize amorphous silica NPs (150 nm and above) and 15 nm colloidal gold NPs. The reverse micelle ME based technique was used for synthesizing amorphous silica NPs (below 150 nm), silica coated quantum dots (QDSS) and gold spec kled silica coated quantum dot cored NPs (QDGS). The synthesis protocols of all the above -mentioned NPs are described below For bioimaging, it is highly desirable that the NPs are appropriately surface modified with targeting molecule s. For example, for cancer imaging applications it is necessary that cancer specific antibodies and folates are attached to the surface of the NPs to achieve tumor targeting properties Usually the targeting molecules are not attached directly to the surf ace
59 of the NPs A n intermediate coupling molecule is used which is a surface -functional group on the NP and the other end of which is attached to the targeting molecule s such as folate and anti -bodies Firstly, the NP surface is functionalized with groups such as amines, carboxyls and thiols that can facilitate bioconjugation Next the bio -recognition molecules such as antibodies and folates are attached to the surface -functionalized NPs with the use of suitable chemistries like carbodiimide chemistry. The se steps are usually carried out in aqueous based solutions. Finally, the bio -conjugated NPs have to be dispersed in suitable biological solvents (depending on the specific bioimaging application) for practical applications. 3.1.1 Fluorescent Silica Na noparticles (FSNPs) As mentioned above, s ilica -based amorphous NPs for this research work were synthesized using two me thods: (a) Stobers method (sol gel technique) for 190 nm NPs (Stober et al. 1968) and (b) reverse ME technique for 1 0 0 nm NPs The Stobers method is generally used to pro duce large scale suspensions of sub-micron sized silica NPs while reverse microemulsion system yields monodisperse particles in the nano -sized regime. The particle diameter can be controll ed by manipulating the process parameters like temperature, reagent concentration and water to surf actant ratio for reverse microemulsion technique only Unlike QD s and gold NPs amorphous silica particles do not possess any inherent properties that can be exploited for imaging applications In order to overcome this limitation, amorphous silica NPs are frequently doped with organic and inorganic fluorescent dyes that can be tra ced using fluorescent microscopy (Santra et al. 2005c, Santra et al. 2001a, Santra et al. 2004a, Santra et al. 2004b, Santra et al. 2001b) Moreover, fluorescent silica NPs have some im portant features (Qhobosheane et al. 2001, Santra et al. 2001a, Santra et al. 2001b) that make them attract ive for bioimaging applications First, excellent sensitivity can be achieved by incorporating thousands of dye molecules into a single NP system for
60 example dye doped silica NPs. Second dye photostability is improved due to the protective encapsulation of fluorescent dyes by the host silica matrix. Third, the spectral characteristics of fluorophores remain intact in the host si lica matrix, making them suitable for both in vitro and in vivo applications. Finally, fluorescent amorphous silica NPs may be more biocompatible relative to other types of photostable labels such as QDs The synthesis protocols describe d in the followin g section provide details for the synthesi s of amorphous silica NPs with different types of dyes incorporated into the silica matrix. The two dyes used for this research work are (a) f luorescein -i sothiocyanate (FITC) which is an organic dye and (b) tris(2, 2' -bipyridyl)dichlororuthenium(II) (Rubpy) which is a metallorganic dye. FITC was chosen on the basis that organic dyes are inherently biocompatible. However, in order to overcome the limited photostability (Santra et al. 2006) of FITC the metallorganic dye, Rubpy, was used as an alternative fluorescent dye. 3.1.1. 1 Synthesis s trategies : FSNPs (a) Stobers method I n the typical Stobers method, alkoxysilane compounds (e.g. tetraethyl orthosilicate (TEOS), tetramethylorthosilicate (TMOS) and a variety of TEOS or TMOS derivatives ) undergo base -catalysed hydrolysis and condensation reactions in ammonia ethanol -water mi xture, forming a stable alcosol. Following the Stobers protocol (Stober et al. 1968) with a slight modification, fairly monodisperse organic dye doped fluorescent silica NPs have been synthesized for this research work. Since organic dyes are normally hydrophobic doping them inside the hyd rophilic silica matrix is not straightforward. Typically, a reactive derivative of FITC, amine reactive fluorescein isothiocyanate, is first reacted with an amine containing silane compound, 3 aminopropyltriethoxy silane (APTS), forming a stable thiourea l inkage. Then the FITC -APTS and TEOS are allowed to hydrolyze and condense to form FITC conjugated silica NPs.
61 Although t he positively charged amine functional group s on the surface of the silica NPs facilitate bio conjugation in the next step, it also shi fts the (negative) zeta potential towards zero. A zeta potential close to zero could lead to enhanced agglomeration. In order to reduce this possibili t y 60 l of a negatively charged silane agent, 3 -(trihydroxysilyl) propyl methylphosphonate (THPMP) (Gele st, Inc., Morrisville, PA) was added at the end, to increase the zeta potential of the silica s urface Using sol -gel techniques or Stobers method, bulk amount of silica NPs can be produced in a typical laboratory setup. Initially a stable 0.1M FITC -APT S conjugate is prepared by adding 69 mg APTS and 5.25 mg FITC and mix ed together in 1 mL of absolute ethanol in dry nitrogen and stirred magnetically for 24 hours. This FITC -APTS solution i s protected from light during reaction using an aluminum foil on an d around the beaker and later stor ed carefully in order to prevent photobleaching. The FITC molecules were covalently conjugated to the silica particles in order to m inim ize dye leaching from the NPs To prepare the 190 nm fluorescent silica NPs using Stobers Method 800 L ammonium hydroxide ( NH4OH) was combined with 10 mL absolute ethanol in a 20 mL round-bottom flask with magnetic stirring. After 5 minutes, 400 L TEOS and 200 L of the 0.1M FITC -APTS conjugate were added, followed by 60 L of THPMP af ter 30 min. Magnetic stirring was continued for 24 hours. All experiments were conducted at room temperature. After 24 hours, 10 mL of absolute ethanol was added to the solution and sonicated ( Branson 1510, Kell -Strom, Wethersfield, CT) for 5 minutes. Thes e amine modified NPs were washed thoroughly four times with ethanol and two times with DI water by ultra -centrifugation (Eppendorf Centrifuge 5804 R, Eppendorf North America, Westbury, NY) at 5000 rpm for 30 minutes per wash Vortex ing (Vortex Genie 2, A. Daigger and
62 Company, Vernon Hills, IL) and ultrasonication techniques were applied between two successive washes. The washed FSNPs were stored away from light till further use. In the case of Rubpy-doped particles, first a stock solution of 0.1 M Rubpy wa s prepared by adding 74.8 mg in 1 mL of de ionized water. To synthesize the amorphous silica NPs doped with Rubpy, everything else remained the same as above except 100 L of this stock solution and 100 L of APTS wa s added to the mixture (instead of 200 L of 0.1M FITC -APTS) followed by 60 L of THPMP after 30 min and stirred for 24 hours. The procedure for washing the particles remained the same as described above. (b) Reverse microemulsion method T he reverse or water in oil (W/O) microemulsion technique was used to synthesize 100 nm amorphous silica NPs, inorganic and organic dye doped as well as NPs with no dyes. A schematic representation of the synthesis of dye doped silica NP using reverse -microemulsion technique is shown in Figure 3 1 The W/O micro emulsion system was created by adding 0.44 g of de ionized water to 7.7 mL cyclohexane (Sigma -Aldrich, St Louis, MO) along with a 1.77g of the surfactant Triton X 100 (Sigma -Aldrich (St Louis, MO) and 1.6 mL of the co -surfactant n -hexanol (Sigma Aldrich (S t Louis, MO). This solution wa s magnetically stirred for 15 minutes resulting in the formation of nano-sized wat er droplets. After fifteen minutes, 50 L of the 0.1M FITC APTS conjugate wa s first adde d; followed by the addition of 100 L TEOS five minutes later. The hydrolysis of the silane reagents was then initiated by the addition of 100 L NH4OH. After 30 min, 15 L THPMP was added dropwise and stirring was continued for twenty -four hours The 100 nm dye doped amorphous silica NPs we re formed inside the water droplet in the W/O microemulsion. These particles we re then washed with a procedure similar to the one described above for NPs made by Stobers method.
63 T he synthesis of Rubpydoped FS NPs was carried out using the method described above, except 40 L of 0.1M Rubpy stock solution and 25 L of APTS wa s added to the mixture (instead of 50 L of 0.1M FITC -APTS ) followed by 15 L THPMP after 30 minutes and stirred for 24 hours. The procedure for washing the NPs also remained the same as described before The synthesis of FSNPs of both sizes using both techniques was repeated four times to verify the reproducibility of the synthe sis technique and consistency of the physicochemical parameters of the product during characterization. 184.108.40.206 Surface functionalization and b ioconjugation : FSNPs In order to facilitate the process of targeting cancer cells i n the present study, the amor phous silica NPs were conjugated to folic acid molecules For survival and proliferation, most animal cells require the vitamin folate. Folic acid is an essential component for nucleotide synthesis. As animal cells cannot synthesize folic acid by themsel ves, they express folate receptors on their surface for capturing exogenous folates (Antony 1992, Santra et al. 2005c) Neoplastic cells such as breast adenocarcinoma (Antony 1992, Ross et al. 1994, Santra et al. 2005c) lung adenocarcinoma (Franklin et al. 1994) oral carcinoma (Ross et al. 1994) and pituitary adenoma (Evans et al. 2003) overexpress folate receptors because they proliferate rapidly. Folic acid receptors are therefore considered as one of the significant tumor biomarkers for these neoplasms. Clinically challenging brain tumors such as malignant gliomas may also over express folate receptors, however adequate experimental evidence in this favor is currently unavailable. Folate enables a Trojan horse strategy, fooling tumor cells into taking up a folate molecule linked to an imaging or therapeutic agent (Antony 1992, Leamon & Low 1991) This is called folate -mediated up take (endocytosis). Covalently coupling folate to various
64 molecules yields a conjugate that can be endocytosed into tumor cells. Examples (Santra et al. 2005c) include radiopharmaceutical agents, chemotherapeutic agents, antisense oligonucleotides and ribozymes, proteins and protein toxins, immunotherapeutic agents, drug molecule loaded liposomes and plasmids, all of which have been successfully delivered to cancer cells via folate mediated transport mechanisms. Phase I and II clinical studies for the first folate -containing imaging agent were initiated in 1999, and clinical trials of folate conjugated therapeutic agents may soon fo llow. Folate -mediated macromolecule delivery, such as liposome DNA complexes (<200 nm) and monoclonal antibodies have been demonstrated in vivo suggesting that larger molecules can be targeted to tumors. Similarly, successful targeting of folate immobili zed NPs carrying contrast agents and drugs to tumor cells have been recently reported. Due to the presence of APTS and THPMP on the surface, the amorphous silica NPs have an amine -modified surface that is used to attach the folic acid molecules. The stru cture of folic acid contains two independent fluorescent moieties, p aminobenzoic acid (PABA) and methylpteridin (MTE) as shown in Figure 3 2 Folic acid was covalently attached to the positively charged amine functional groups on the surface of the silica NPs using conventional carbodiamide coupling chemistry as shown in Figure 3 3 As per published protocols (Santra et al. 2005c) two solutions were prepared separately. Solution I was prepared by adding the following and magnetically stirring for one hour: 1 10mM f olic acid (7.0 mg folate in 1.0 mL dimethyl sulphoxide (DMSO ) in a glass container, 2 4.1 mg of N Hydroxy Succinamide (NHS) in 1.5 mL de ionized water an d 3 34.2 mg of 1 -ethyl 3 (3 dimethylaminopropyl) carbodiimide hydrochloride ( EDC ) in 1.5 mL de ionized water.
65 Solution II contained 20 mg amine modified FSNPs in 1.0 mL DMSO to which 2 L triethylamine was added in a glass container. Both solutions wer e combined in a glass beaker and sonicated for five minutes, then stirred magnetically for one hour to obtain folate conjugated (folated) FSNPs. The NPs remained well dispersed in DI water for several hours without any noticeable settling. These particles were washed by centrifuging five times with DI water and stored and protected from light until further use. Following synthesis the NPs were first characterized and then used for in vitro experiments to show their potential as fluorescent probes for bioim aging applications. Results from characterization experiments conducted on these particl es have been described later ( Section 3.2.1) 3.1.2 Colloidal Gold Nanoparticles ( N P s ) Gold NP s of 15 nm particle diameter were prepared using s ol gel technique Thi s is a widely used method for the synthesis of gold NP s, or what are more popularly known as gold colloids and was chose n primarily for its simplicity and ease for relatively large -scale synthesis of gold NPs .. A detailed description of the synthesis prot ocol used is given below. 220.127.116.11 Synthesis s trategy : Gold NPs 10 mL of 2 mM Gold Tetrachloride (HAuCl4) solution (Sigma -Aldrich, St Louis, MO ) wa s added to 10 mL of nanopure de ionized (DI) water (NANOpure, Barnstead International, Dubuque, Iowa) in a 1 00 mL beaker. This solution wa s boiled on a hot plate while stirring using magnetic stir bar. When the solution start ed to boil, 2 mL of 1% sodium citrate solution ( Sigma -Aldrich, St Louis, MO ) was added. Over the next few minutes the color of the boiling solution progressively change d from light yellow to transparent to light blue or pale purple to deep purple to deep red. The color changes we re indicative of the nucleation and growth of colloidal gold particles (Huo & Worden 2007) Boiling wa s continued for ten additional minutes and the solution is then brought down to r oom
66 temperature This solut ion wa s left on a magnetic stirrer for 24 hours. This wa s followed by addition of de ionized water to bring the total volume of the solution to 20 mL Ultra c entrifugation of this solution wa s carried out at 6000 rpm f or 15 minutes. The supernatant wa s car efully removed with a micro -pipette and the centrifugation process wa s repeated. The resulting product a dark colored solution containing 15 nm gold spher es is stored till further use. 3.1.2. 2 Surface functionalization and bioconjugation: Gold NPs Zeta potential measurements showed that the 15 nm bare gold spher es (G ) exhibit a negative charge on the NP surface. As mentioned in the Research Approach, one of the objectives of this research effort is to study the effect of surface functionaliz ation of th e gold NPs on the blood clearance behavior in rats. For this purpose, the negatively charged gold colloids were surface-modified with appropriate functional groups to obtain positively and neutrally charged gold particles. The bare gold colloids were used as the negatively charged particles in the studies. N ear n eutral charges on the gold NP s was obtained by p eg ylation and a positive ly charged surface was obtained by using cysteine amine chemistry to conjugat e amine functional group s on bare gold NPs P eg y lation is a process of attaching polyethylene glycol (PEG) on the surface of a particle. In order to synthesize PEG -conjugated gold NPs (GP) thiol terminated methoxypoly (ethylene glycol) (mPEG -SH) of 5000 molecular weight was used (Sigma Aldrich, St Lou is, MO ). The solution containing the bare 15 nm colloidal gold NPs was increased in concentration to 200 mg/ L by centrifugation. 15 mL of this solution wa s added to 75 L of 1 mM mPEG SH 5000 in a 20 mL beaker. The solution was magnetically stirred for 12 hours after which it was washed twice using de -ionized water by centrifugation at
67 6000 rpm for 15 minutes the supernatant was removed and the pegylated gold particles collected and stored in bottles inside the refrigerator To synthesize positively char ged gold NPs (G+), first, a stock solution of 0.00001N cysteine amine in aqueous solution was prepared. 1 mL of this stock solution was added to 8 mL of the bare colloidal NP solution at a concentration of 1000 mg/ mL It was magnetically stirred for 12 hrs followed by washing with de -ionized water by centrifugation at 6000 rpm for 15 minutes. The supernatant wa s carefully removed and the positively charged amine modified gold NPs collected and stored till further use. T he protocol s for the synthesis of all three gold NPs (G G+ and GP) were repeated four times to confirm the r eproducibility of the technique and the consistency of the physicochemical parameters of the NPs 3 .1.3 Silica Coated Quantum Dot Nanoparticles (QDS N P s ) A variety of techniques hav e been reported in the literature to synthesize QD s of different elemental compositions and sizes Some of these techniques have been described in c hapter one In this research work, a mo dified version of Yang et al. s (Yang & Holloway 2004, Yang et al. 2004a, Yang et al. 2004b) synthesis protocol u sing the reverse microemulsion technique, was employed for the synthesi s of silica coated quantum dots (QDS). The modified synthesis protocol is described briefly below. 18.104.22.168 Synthesis s trateg y : QDS NPs Reverse micelle mediated CdS:Mn/ZnS QD synthesis. The reverse -microemulsion ( ME ) technique describe d at the beginning of the chapter can also be used to synthesize QDs as reported by Yang et al. (Yang et al. 2004b) This is a robust method that has been used at room temperature and normal atmospher ic pressure for the synthesis of manganese (Mn) doped cadmium sulfide (CdS) core and zinc sulfide (ZnS) shell (CdS:Mn/ZnS) quantum dots. The ME system employed in the present study, consist ed of AOT (dioctylsulfosuccinate
68 sodium salt) as a surfactant and h eptane as the oil. The bright orange emitting CdS:Mn/ZnS quantum dots had an average quantum dot size of 10 nm, and were determined to be highly photostable. The protocol was modified to apply a post coating of amorphous silica layer to prevent the possibi lity of any cadmium leaching (Derfus et al. 2004b) into the physiological environment A schematic for the QDS synthesis process is shown in Figure 3.4. Briefly, cadmium acetate or Cd(CH3COO)2.2H2O, manganese acetate or Mn( CH3COO)2, zinc acetate or Zn(CH3COO)2, dioctyl sulfosuccinate, sodium salt (AOT), heptane, tetraethyl orthosilicate (TEOS) and NH4OH (30% by wt.) (Sigma Aldrich, St Louis, MO) were used to prepare three aqueous solutions in 20 mL vials. Solution I containe d 0.24 g of cadmium acetate and 0.0031 g of manganese acetate in 9 mL of de -ionized water. Solution II contained 0.2812 g of sodium sulphide in 5.4 mL of de ionized water. Solution III contained 0.2642 g zinc acetate in 5.4 mL of de ionized water. All thre e solutions were magnetically stirred for fifteen minutes to dissolve all solutes. Three ME systems we re then prepared in three different conical flasks. Flask A contain ed 4.46 g of AOT in 100 mL of heptane. One -fifth of the solution I wa s added to Flask A and left to magnetically stir for 30 minutes. Flask B contain ed 13.38 g of AOT in 300 mL of heptane. All the contents of solution II we re added to Flask B and left to stir for 30 minutes. After 30 minutes, the contents of Flask A and Flask B we re mixed together and stirred for three hours. The color of the solution changes to yellow and the flask wa s enclosed in an aluminum foil to shield it from light. Flask C contain ed 13.38 g of AOT in 300 mL of heptane. The contents of solution III we re added to Flas k C and left to stir for 3 hours. The contents of Flask C we re introduced into the beaker containing the mixture of Flask A and
69 Flask B, using a variable flow mini -pump at 1.5 mL per minute. After this addition, the resulting solution wa s left to stir for 30 minutes to obtain the CdS: Mn / ZnS core -shell QDs In order to apply an amorphous silica coating on the QDs 2.5 mL of TEOS wa s added to the solution and allowed to stir for fifteen minutes. 1.5 mL of NH4OH wa s added to a ME system made of 3.685 g of AOT in 82.5 mL of hept ane. This ME system wa s added to the QD TEOS solution and left to stir for 24 hours. NH4OH initiate d the hydrolysis of TEOS and condensation reaction to form a silica coating over the QDs Since the reactions take place in a ME system a relatively long reaction time of 24 hours wa s required for achieving the desired coating. In order to aid the bioconjugation process, the outer silica surface needs to be functionalized with amine functional groups. C onsequently, 1.25 mL of TEOS and 1 mL APTS wa s added to the silica coated QD solution after twenty -four hours. This wa s followed by the addition of another ME system of NH4OH, which wa s composed of 1.5 mL of NH4OH added to 3.685 g of AOT in 82.5 mL of heptane. This mixture wa s left to stir for 24 hours. The amine -modified silica coated quantum dots (QDS) we re precipitated by the addition of a 25 mL methanol per 25 mL of the QDS solution. The particles we re then washed by centrifugation at 6000 rpm for 30 minutes. This wa s followed by two additional washes in methanol and 2 washes in de ionized water with vortexing and sonication in between each wash. The particles we re then stored away from light till further use. The QDS NPs were used as control particles in this research work. In addition to characterizing the QDS NPs, further preliminary leaching studies were carried out in cell media under normal (25C and pH 7.4), acidic (25C and pH 3) conditions, and at elevated temperature (57C and pH 7.4) to emulate different environments inside th e cells like the
70 acidic pH 5 of lysosome (Kobayashi et al. 2008, Yamamoto 2004) and elevated temperature (Levine & Robbins 1970) Analytical detection techniques were used to check the possibility of any cadmi um leaching from the quantum dots inside the biological media. An Inductively Coupled Plasma Mass Spectrometer was used for cadmium detection and quantifying in the supernatant of A549 cells The results of this leaching study have been included later in t his chapter. 3.1.4 Gold S pe ckled Silica C oated Quantum Dot Nanoparticles (QD S G N P s ) In order to conjugate gold spec kles on the QDS NPs, two separate ME systems are prepared. ME I wa s prepared by adding 4.8 mL of 5% HAuCl4 solution (in de -ionized water) to 13.38 g of AOT in 300 mL of heptane. ME II wa s prepared by adding 2.4 mL of 0.05M hydrazine to a solution of 13.38 g of AOT in 300 mL of heptane The QDS NPs we re first prepared using the exact same protocol described above except that they we re allow ed to remain in the ME system at the end. This means, that the NPs we re not precipitated using methanol and the washing steps described in the previous section we re not carried out. In order to conjugate the gold speckles on the QDS NPs, 200 mL of this QD ME solution is added to 200 mL of ME I and stirred for 3 hours. After this, 200 mL of ME II wa s added and the set up is left on a magnetic stir plate for 48 hours. This leads to the formation of gold spec kles on the amine -modified silica coated QDS NP s. Th e particles we re then precipitated using methanol, washed three times with methanol and two times with de ionized water, as described for the QDS NPs, and stored away from light till further use. A schematic for the QDSG synthesis process is summarized in the flowchart shown in Figure 3.5. A schematic representing the QDSG NPs structure is shown in Figure 3.6.
71 3.2 Characterization of N anoparticles (NPs) The NPs synthesized for this research effort and described in the previous section were characterized fo r particle size and shape, surface area and charge, absorbance and fluorescence spectroscopy The following section describes the characterization results of each set of NPs. 3.2.1 Fluorescent Silica Nanoparticles (FSNPs) The FSNPs were characterized for particle size, shape, surface charge, absorbance and fluorescence spectroscopic properties. 3.2 .1.1 Particle s ize m easurements Particle size measurements for FSNPs in the dry state were conducted using transmission electron microscopic (TEM) imaging using a Hitachi H 7000 microscope (Japan). For TEM sample preparation, a drop of the FSNPs dispersed in denatured ethanol solution was directly placed onto a carbon -coated copper grid (200 mesh). The average particle size was measured using Image Tool sof tware (Wilcox et al. 1995) The average size was obtained based on the diam eter measurement of 100 particles. P article size of FSNPs synthesized using Stobers method was measured to be 1 90 3 0 nm whereas FSNPs synthesized using reverse ME method were 10010 nm in size, ( Figure 3.7a and b respectively) Particle size in de ionize d water was also measured using Coulter light scattering technique (Coulter LS 13 320), as this would reflect the state of particle dispersion in biological solutions such as cell media. The samples were dispersed in de -ionized water by five minutes each o f sonication and vortexing. Based on Coulter data, ME mediated FSNPs were found to be monodispersed and the particle size was calculated to be 104 nm, Figure 3.8b, in DI water. Coulter data also showed that NP s produced using Stobers method were
72 monodispe rse with average particle size of 221 nm, Figure 3.8a, in DI water. Both TEM and Coulter data were in good agreement, suggesting that particles remained well dispersed in DI water. Coulter data also confirmed the effectiveness of the surface modification p rotocol using THPMP as a dispersant to improve NP dispersibility in water. For the in vitro studies (Chapter 4), the FSNPs were pre -conditioned with cell media (RPMI 1640) before dosing the cells. It was therefore important to measure particle size in th e cell media. The samples were dispersed in cell media by five minutes each of sonication and vortexing. Coulter measurements showed that particles tend to agglomerate in cell media (Figure 3.9) The larger 1 9 0 nm NP s ( Figure 3.9a) exhibited more aggregation as compared to the smaller 1 0 0 nm NP s ( Figure 3.9 b) The apparent size of the smaller particles in the cell media was found to be in the range of 40 nm to 20 microns and the larger particles exhi bited an apparent size range of 2 to 200 microns. It was fo und that the NP s can be usually dispersed by increasing the time of sonication and vortexing prior to particle size measurements. However, excessive application of these processes could lead to disintegration of the proteins constituting the cell media. Th is would amount to changing the basic physical, chemical and/or biological characteristics of the media. Thus, for this research work the NP dispersion was attempted by limiting sonication and vortexing each to 5 minutes prior to particle size measurements using Coulter light scattering technique. 3.2 .1.2 Surface c harge measurements The surface charge of a particle in a suspension determines the dispersibility of the particles and the stability of the suspension. Zeta potential is a measure of the surfa ce charge of the particles in a particular suspension. Thus the magnitude of zeta potential of the suspension is an indication of its potential stability. If the particles have an effective positive or negative surface charge, they tend to repel each other and thus preclude the possibility of
73 agglomeration. Generally, particles with zeta potential values greater than +20 mV or lesser than 20mV are expected to form stable suspensions. Non amine -modified (bare) silica NPs and amine -modified silica NPs with and without THPMP were suspended in aqueous solutions at 200g/ mL and characterized for surface charge in Brookhaven Zeta PALS The bare silica NPs had a zeta potential value of 35.73 6.3 mV and, as expected, and remained well dispersed in water. The amine -modified silica NPs without THPMP were found to have a zeta potential of 0.39 0.11 mV and this caused the particles to agglomerate in water. In order to prevent agglomeration, THPMP was added to these particles, as discussed before This caused the zeta potential of the particles to increase to a value of 3 1.27 mV and prevent particle agglomeration. 3.2 .1.3 Absorbance and fluorescence s pectroscopy The ultraviolet -visible (UV -Vis) absorption and steady-state fluorescence spectra for pure folic aci d, aminated FSNPs and folated FSNPs were recorded in de ionized water. The absorption spectra were recorded on a spectrophotometer (Shimadzu UV -Vis model 2410 PC) in de ionized water. Steady -state fluorescence excitation and fluorescence emission spectra w ere recorded on a spectrofluorometer ( Fluorolog Tau 3, Jobin Yvon Spex Instruments, S.A. Inc. equipped with a 450 W xenon lamp as an excitation source) to confirm the presence of both folic acid and FITC in aminated FSNPs All measurements were performed at room temperature and all experimental solutions were prepared in de ionized water. Folic acid contains two independent fluorescent moieties, p aminobenzoic acid (PABA) and methylpteridin (MTE ) (Figure 3.2). PABA absorbs at 265 nm and emits at 336 nm, a nd its emission is highly pH dependent (Tanojo et al. 1997) MTE has two absorption bands at 275 nm and 352 nm and it emits at 447 nm (Espinosa -Mansilla et al. 1998) In the
74 following sections, spectroscopic characterization of folate, amine FSNPs and folate FSNPs will be discussed. The a bsorption spectra of folate, amine -FSNPs and folate -FSNPs are shown in Figure 3.10. Folate showed a combined absorption from PABA and MTE moieties. The absorption band maxima appeared at 280 nm and 355 nm for folate (Figure 3.10a). Folate immobilized FSNPs (Figure 3.10c) showed the absorption maximum at 280 nm with two shoulders at longer wavelengths (355 nm and 490 nm). The absorption band at 280 nm and the appearance of shoulder at 355 nm confirmed the presence of folate on folate -FSNPs. As expected, amine FSNPs did not show any folate characteristic peaks or shoulder in this region but a shoulder at around 490 nm (Figure 3.10b). The appearance of 490 nm shoulder is due to the absorption of FITC molecules that are doped in FSNPs. The f luorescence excitation and emission spectra of folate are shown in Figure 3.11. Upon excitation at 280 nm, folate showed dual emission at 355 nm and 447 nm, which corresponds to the emission from PABA and MTE, respectively ( Figure 3.11b ). When excited at 355 nm, folate showed the only emission from the MTE moiety at 447 nm, which is similar to what was reported in the literature (Santra et al. 2005c) (Figure 3.11a ). Fluorescence excitation spectra of folate, when recorded at 447 nm emission, showed two excitation peaks at 280 nm and 355 nm due to the MTE moiety ( Figure 3 .11d ). Similarly, folate excitation spectra recorded at 355 nm emission showed an excitation peak at ~289 nm (Figure 3.11c ), which is due to the PABA moiety. Folate emission band at 447 nm was found stronger than the 355 nm emission band (Espinosa -Mansilla et al. 1998, Santra et al. 2005c) We, therefore expect to see the 447 nm emission band in folate -FSNP.
75 Multiple fluorescence bands ap peared from folate FSNPs due to the presence of both FITC and folate. When excited at 492 nm, folate -FSNPs showed emission band maximum at 512 nm, which is the characteristic emission of FITC molecule (Figure 3.12a). The excitation spectra recorded at 51 2 nm emission showed a sharp peak at 492 nm and a broad band at 360 nm (Figure 3.12c). The appearance of 360 nm emission is due to the MTE moiety of folate. When excited at 355 nm, folate FSNPs showed dual emission at 450 nm and 509 nm (Figure 3.12b). This is due to the fact that the excitation at 355 nm caused excitation of both folate and fluorescein molecules. The 450 nm and 509 nm emission bands, which are close to 447 nm and 512 nm emission bands, we re therefore assigned to folate and FITC emission, r espectively. The observation of folate emission from folate -FSNPs at 450 nm confirmed the successful folate immobilization onto the FSNPs The excitation spectra recorded at 447 nm emission showed band maxima at 375 nm (Figure 3.12d). The 375 nm excitation band is assigned to the folate. The spectral shift of characteristic folate excitation and emission band maxima in folate -FSNPs is due to the folate immobilization, confirming the fact that the microenvironment of immobilized folate is different from free folate molecules present in the solution 3. 2 .2 Colloidal Gold N anoparticles (N P s ) The results of particle characterization carried out for all three gold particles: (a) bare gold (G ), (b) aminated gold (G+) and (c) pegylated gold (GP) are discussed in this section. As before, the particles are characterized for size, shape, surface charge (zeta potential), absorbance and fluorescence spectroscopy. 22.214.171.124 Particle s ize measurements Gold NP s were synthesized for a target particle size of 15 nm. The samples were dispersed in de ionized water using five minutes each of sonication and vortexing. The
76 particle size measurements were carried out using a Differential Sedimentation CPS Disc CentrifugeTM. T he parti cle size measurements for the gold NP s in de -ionized water show ed a narrow size distribution range of 155 nm It indicate d that the particles were well dispersed without any significant agglomeration. The samples were dispersed in cell media by five minutes each of sonication and vortexing. The particle size measurements in cell -media show ed a wide range of size distribution of 15 nm to 350 m. I n the context of the size distribution in de ionized water, it can be concluded that the particles tend to agglomerate in cell media. This could possibly be due to change in the surface charge of the NPs as a result of adsorbed proteins from the biological media onto the particle surface. The shape of the gold NP s was visualized using Transmission Electron Microscopy (JEOL TEM JEM 200CX). The TEM image in Figure 3.13 indicate d that although not very monodisperse, the particles are spherical in shape. 3.2 2 2 S urface c harge measurements The zeta potential values of the three particles were measured using a Brookhaven Zeta PALS. The values obtained were 34.5 7.8 mV for G -, +24 5.8 mV for G+ and 5 1.9 mV for GP particles. These values clearly confirmed the fact that the surface functionalization of the bare gold NPs achieved the desired results. 3.2 2 3 Absorbance and fluorescence s pectroscopy The ultraviolet -visible (UV -Vis) absorption and steady-state fluorescence spectra for gold NPs dispersed in de -ionized water were recorded at room temperature on a fluorescent plate reader using the Softmax Pro V5 software The results of absorbance spect roscopy of the particles are shown in Figures 3.14 It has been reported that 15 nm gold NP s absorb light at 5 2 0 nm (Maye et al. 2000) and there is a peak in the region in the absorbance spectra in Figure 3.14 which confirms that the particles are indeed 15 nm in size. Gold NPs do not
77 possess any inherent fluorescent property. The refore, as expected the fluorescent spectra of gold NP s (not shown) only showed a peak at 470 nm which was identified as the first harmonic peak of the Raman spectra of water (Frost et al. 2006) 3.2.3 Silica Coated Quantum Dot Core Nanoparticles (QDS and QDSG NPs) The silica coated quantum dot (QDS) and gold spec kled silica coated quantum dot (QDSG) NPs were characterized for size, shape, surface area, surface charge (zeta potential), absorbance and fluorescence spectroscopy. 3. 2.3.1 Particle s ize m easurements The QDSG and QDS NPs were synthesized for a target particle size of 15 nm. In order to determine their actual particle size, the particle size measurement s were carried out using the Differential Sedimentation CPS Disc CentrifugeTM. T he QDSG and QDS samples were dispersed in de ionized water by sonication and vortexing for five minutes each before th e particle size measurement s were conducted. It wa s eviden t that there wa s no variation in the particle size due to the conjugation of gold specks on the QDS particles. TEM image of QDSG NPs in the dry state is shown in Figure 3.15 The sample for TEM imag es w as prepared in the same way as described earlier for F S NPs. 126.96.36.199 Surface a rea m easurements The BET (Brunauer, Emmett and Teller) method was used to measure the specific surface area of QDS and QDSG NP s using the Quantachrome Autosorb IC -MS. In order to measure the specific surface area, 1 mg of each of the NPs as dispersed in ethanol were placed inside a vacuum chamber and dried. The values obtained for QDS and QDSG were 151.2 and 229.7 m2/gm respectively.
78 3.2 .3.3 Surface c harge m easurements The zeta potential values for the QDS and QDSG NPs were measured using a Brookhaven Zeta PALS. The values of zeta potential measurements were 6.3 2.9 mV for QDS and 4.1 1.7 mV for QDSG particles respectively. Therefore, it was not possible to draw any conclusive evidence regarding the effect of additin g the g old speckles on the positively charged aminated silica coating on the QDS particles. As bare gold has a negative surface charge, the addition of the specks may have caused a slight drop in the zeta potential value in the QDSG NP s. 3.2 .3.4 Absorbance and fluorescence s pectroscopy The ultraviolet -visible (UV -Vis) absorption and fluorescence spectra for QDSG and QDS NPs in de ionized water were recorded at room temperature on a fluorescent plate reader using the Softmax Pro V5 software The absorbance and the fluorescence spectra of QDSG and QDS are shown in Figure 3. 16 to 3. 18. The absorption spectra of bare gold NPs (Figure 3.14 ) show a peak at 530 nm, near the characteristic wavelength of 520 nm of gold NP s. From the literature (Hu ff et al. 2007a, Huff et al. 2007b, Tong et al. 2007) it is known that gold nanorods have a characteristic peak maxima at 790 nm. The absorbance spectra for QDSG (Figure 3. 16) show a shoulder at 530 nm and another absorption maximum at 790 nm. The first shoulder at 530 nm and the absorption peak at 79 0 nm belong to the gold specks with possibl e nanosphere and nanorod s hapes on the surface of the particle. The fluorescence excitation and emission spectra of QDSG and QDS are shown in Figure 3. 17 and Figure 3. 18 respectively. Both NP s display the quantum core spectral characteristics when excited at 355 nm, emission spectra of both NP s showed a peak
79 maxima at 590 nm from the QDs with a small shoulder at 430 nm which is due to the excitation wavelength and the first harmonic peak in the Raman spectra of water. The absorbance spectra taken between 300 nm and 800 nm confirmed the absence and presence of gold specks on the QDS and QDSG NPs respectively. On the other hand, the fluorescence spectra (emission spec tra taken at the excitation wavelength of QDS at 355 nm and excitation spectra taken at the emission wavelength of QDS at 590 nm) confirmed the fluorescent properties of the quantum dot core for both the particles. The absorbance and fluorescence measurem ents on the particles also confirmed that neither the gold specks nor the quantum dots had quenched one another and the particle synthesis had the desirable optical properties 3.2.4 Leaching Studies of Silica C oated Quantum Dot Nanoparticles (QDS NPs) As cadmium and most of the heavy metal constituents of QDs are extremely toxic and undesirable to the physiological environment, any possibility of metal leaching is one of the biggest challenges for the use of QDs for bioimaging applications. As mentioned before, preliminary leaching studies of QDS NPs were carried out to determine whether or not cadmium is leaching out through the silica coating of the NPs. Phosphate buffer saline (PBS) solution is a biological medium frequently used to inject test sample s in animal studies. The leaching studies were carried out by dispersing the particles in PBS solution at two different pH values, 3 and 7.4, and two different temperatures, 25C and 57C. The low pH value of 3 and a high temperature of 57C were chosen to simulate the highly acidic and elevated temperatures inside an abnormal cellular environment Stock solutions of the QDS NPs were prepared in PBS solution at concentrations of 100 and 1000 g/ mL Four six -well plates were used for the experiment. Plate A contained
80 two wells each with 2 mL of 100 g/ mL QDS solution, another two wells each with 2 mL of 1000 g/ mL QDS solution and the remaining two wells contained 2 mL of pure PBS solution (as controls). The PBS solution maintains a pH value of 7.4 and Plate A was maintained at room temperature (25C). Plate B has exactly the same contents as Plate A except it was maintained at 57C. Plate C initially had identical contents as Plate A. The acidi ty of all the wells was decreased to pH value of 3 by the addition of a few drops of hydrochloric acid. The content of Plate D was identical to that of Plate C but was maintained at 57C. All four plates were placed in an incubator for 72 hours. The sampl es were taken out after 72 hours and centrifuged at 4000 rpm for 30 minutes. The supernatant from each well was then collected and acid -digested by adding concentrated nitric acid. These samples were then diluted with de ionized water and made up to 5 mL These samples were then analy zed in ICP MS to detect cadmium. The ICP MS analysis results for the samples from Plate A, B and C yielded cadmium levels of less than 1 part per billion (ppb) and the sample from Plate D showed a cadmium level of 10 ppb. The normal level of cadmium in human body is 0.5 ppb (Godt et al. 2006) The detected amount of cadmium in the samples w as assumed to be within the biological error with minimum cadmium leaching from the QDS NP s in PBS solution incubated for 72 hours. 3.3 Challenges i n QD S G NP Synthesis This following section presents the challenges that were encountered during the development of th e synthesis protocol for the novel QDSG NPs. Most challenges were overcome except for one, which has been listed as a part of the future work in Chapter 6.
81 3.3.1 Optimization of Q uantity of G old and T hickness of S ilica C oating The synthesis protocol for QDSG and QDS NPs described ( Sections 3.1.3 and 3.1.4) was a novel route developed as part of this research work. During synthesis of QDSG NPs, the amount of silica and gold had to be optimized so as to obtain the right fluorescence and absorbance charact eristics for the NPs. As will be shown in this section, excess of gold speckles on the surface of silica in QDSG NPs could lead to quenching of the fluorescence properties of the particle. But reducing the gold speckles too much would make the hyperthermic aspect of the particles less effective. Therefore, an optimized amount of gold speckles need to be conjugated to the silica surface of the QDS NPs so that the resultant QDSG NPs possess both fluorescence and hyperthermic properties. Two different methods were conceived to obtain optimized fluorescence and hyperthermic properties. The first method was to vary the amount of gold on the silica coating. The second method was to increase the spacing between the QD -core and gold speckles. The rationale behind t he choice of the second method is that the quenching of the fluorescence properties by gold can be reduced by increasing the spacing between the QD core and gold speckles (Santra et al. 2006) In the first method, the amount of gold was varied by adding different concentrations of HAuCl4 solution, while keeping all other parameters constant. The different concentratio ns of HAuCl4 solutions that were used for the study were 0.1, 0.2, 0.3, 1.0, 2.0, 3.0, 6.0, 10.0%. The NP s thus obtained were characterized for absorbance, (Figure 3. 19), and fluorescence (Figure 3.2 0 ) spectroscopy. From the absorbance spectra in Figure 3. 19, it can be concluded that an increasing concentration of HAuCl4 leads to an increased deposition of gold speckles on the silica coating to form the resulting QDSG NPs. The intensity of absorbance was low for HAuCl4 concentration values of 1% and below and
82 increases considerably for values 3, 5 and 10%. The fluorescence spectra in Figure 3.20 show the quenching characteristics of increased amount of gold speckles on the QDSG NPs. The fluorescence intensity of the QD -core is highest for 0.1% of HA uCl4 and was lowest for 10%. By comparing the two curves the 3 5% range was chosen as the optimized concentration of HAuCl4 solution. In the second method, the thickness of the silica shell on the QD -core can be varied by varying the amount of TEOS added. Two different amounts of TEOS were used for this study: 1.25 mL and 2.5 mL All other synthesis parameters were kept constant. From the fluorescence spectra it was found that the fluorescence intensity was higher for the particles synthesized with 2.5 mL TEOS than with 1.25 mL The absorbance spectra for these same particles showed no significant difference. These observations support the conjecture that increasing the spacing between QD -core and gold speckles leads to improved fluorescence properties. H owever, increasing the thickness of silica coating makes the particles larger in size. Therefore, the 2.5 mL TEOS addition was accepted for the synthesis of the particles. The absorbance spectra in Figure 3. 16 and the fluorescence spectra in Figure 3. 17 for QDSG (that were discussed in Section 3.2.3) are for particles that were synthesized with an optimum HAuCl4 concentration of 5% and TEOS volume of 2.5 mL 3.3.2 Gold S peckle C onjugation in M icroemulsion The protocol for gold speckles conjugation on the outer silica coating was described in Section 3.1.4. Briefly, the two reagents for gold speckle formation namely HAuCl4 and hydrazine were prepared in two different microemulsion systems and introduced to the quantum dots, which were suspended in another microemulsion system. This process produced the QDSG NPs with the best fluorescence and absorbance properties. However, the final process was settled upon after trying out three other processing routes. In the first trial,
83 no microemulsion systems were employed. Both the reagents were added directly to the washed quantum dot NPs that were dispersed in de -ionized water. In the second trial, the two reagents were added to quantum dot that were suspended in the microemulsion system (in which they were synth esized). In the third trial, HAuCl4 was in a microemulsion system while hydrazine was added directly to quantum dots in microemulsion system. It was observed from the fluorescence and the absorbance spectra (not shown) that with each trial both the propert ies improved considerably. The improvement is probably due to the better control of the reactions occurring in a microemulsion system instead of direct addition (Zhang et al. 2007) 3.3.3 Addition of H ydrazine Earlier in this chapter, it was mentioned that the addition of hydrazine (using a ME system) was carried out after thr ee hours after the addition of HAuCl4. In some of the early trials, hydrazine was added immediately after the addition of HAuCl4. From the fluorescence and absorbance spectra (not shown) of the particles synthesized with these two different times of hydraz ine addition, it was found that the delayed addition resulted in superior absorbance properties. The fluorescence properties remained unchanged for the NPs at both times of hydrazine additions. A possible explanation for this observation could be that the delay in addition allowed more time for the HAuCl4 to enter the nanoreactor (water droplets). This led to an improved deposition of gold speckles on the silica outer layer, thus, resulting in enhanced absorbance properties. 3.3.4 Attempt at D irect C onjugation of G old S pheres onto QDS NPs An attempt was made to directly conjugate already formed 2 nm gold spheres on to the silica outer layer of the QDS NPs using thiol chemistry in de ionized water instead of carrying out the gold speckle conjugation in a microemulsion system. The rationale behind
84 this attempt was that in this method the gold conjugation can be carried out at a much larger scale than when compared to the use of microemulsion system. Unfortunately, this route did not produce the desired gold conjugation. The TEM images showed inconsistent gold speckles on the QDSG NPs. It also resulted in the presence of a large amount of unreacted gold nanospheres in the solution. Therefore, this method of gold conjugation was discarded. 3.3.5 Optimization of the A mount of APTS A dded The addition of APTS at the time of coating the QD -surface with silica leads to creation of positively charged amine functional groups on the outer surface of the silica coating. These amine groups are used subsequently to aid in the conjugation of gold speckles. However, if there is unreacted APTS in the solution there is a possibility of formation of a gold -APTS complex. This resulted in the formation of gold nanospheres in addition to QDSG NPs. This formation of gold nanosph ere was confirmed via analysis of TEM images. In order to avoid this undesirable result, the amount of APTS addition was optimized to values stated earlier in the optimized established protocol. 3.3.6 Unsuccessful A ttempt to S urface F unctionalize the QDS G NPs One of the initial objectives was to study the effect of surface functionalization of QDSG NPs for biodistribution studies. The as synthesized QDSG NPs had a slight positive surface charge (4.1 1.7 mV) Conjugation with thiol polyethylene glycol m olecules was successfully carried out to produce QDSG NPs with a nearly zero surface charge. However, attempts to produce particles with large positive surface charge by conjugating cysteinamine molecules or large negative surface charge by conjugating thi ol propionic acid were unsuccessful. In both cases, the particles started agglomerating immediately after the addition of the reagents and soon formed a clear solution with black precipitates.
85 Figure 3 1. S ynthesis of f luorescent silica nanoparticle s us ing reverse -microemulsion technique Figure 3 2 Structure of folic acid [Figure reproduced co authored publication with permission from Journal of Nanoscience and Nanotechnology]
86 Figure 3 3 F olic acid conjugation on the fluorescent silica nanopart icles
87 Figure 3 4. Synthesis p rotocol for QDS nanoparticles 30 minutes stir ring Cd Ac Dehydrate: 0.048 g (1.8 X 10-4 mole) Mn Acetate: 0.00062 g (2mol %) Water: 1.8 ml Multiply by five for synthesis and take one fifths of the mixture into Flask A FLASK A AOT: 4.46 g Sodium Sulphide: 0.2812 g (4 X 9) 10-4 mole) Mn Acetate: 0.00062 g Water: 5.4 ml FLASK B AOT: 13.38 g Heptane: 300 ml Zn Acetate: 0.2642 g (2 X7.2 X 10-4 mole) Water: 5.4 ml 10 minutes mixing FLASK C AOT: 13.38 g Heptane: 300 ml 3 hours stirring Mixing and Stirring for 30 minutes CdS: Mn Micelle Solution TEOS = 2.5 ml NH 4 OH = 1.5 ml AOT = 3.685 g Heptane = 82.5 ml 15 minutes stirring 24 hours stirring TEOS = 1.25 ml APTS = 0.25 ml NH 4 OH = 0.9 ml AOT = 2.21 g Heptane = 50 ml water = 3.6 ml AOT = 4.42 g Heptan e = 50 ml 24 hours stirring
88 Figure 3 5 S ynthesis protocol for QDSG nanoparticles Figure 3 6 Structure of QDSG n ano particle s 12 hours stirring 24 hours stirring 30 minutes stirring Cd Ac Dehydrate: 0.048 g (1.8 X 104 mole) Mn Acetate: 0.00062 g (2mol %) Water: 1.8 ml Multiply by five for synthesis and take one fifths of the mixture into Flask A FLASK A AOT: 4.46 g Heptane: 100 ml Sodium Sulphide: 0.2812 g (4 X 9) 104 mole) Mn Acetate: 0.00062 g Water: 5.4 ml FLASK B AOT: 13.38 g Heptane: 300 ml Zn Acetate: 0.2642 g (2 X7.2 X 104 mole) Water: 5.4 ml 10 minutes mixing FLASK C AOT: 13.38 g Heptane: 300 ml 3 hours stirring Mixing and Stirring for 30 minutes CdS: Mn Micelle Solution TEOS = 2.5 ml NH 4 OH = 1.5 ml AOT = 3.685 g Heptane = 82.5 ml 15 minutes stirring 24 hours stirring TEOS = 1.25 ml APTS = 0.25 ml NH 4 OH = 0.9 ml AOT = 2.21 g Heptane = 50 ml water = 3.6 ml AOT = 4.42 g Heptane = 50 ml 5 % HAuCl 4 =7.5 ml 0.005M Hydrazine = 6 24 hours stirring Intermediate ZnS Layer Innermost CdS Core Gold Speckles Outer Silica Shell Silica shell thickness was optimized to prevent quenching of fluorescence core due to presence of gold speckles Amount of amine groups and # of gold speckles on the silica shell were optimized Silica Shell Thickness
89 Figure 3 7 T ransmission electron microscopic images of FSNPs of particle sizes (a ) 190 nm and (b ) 100 nm. In both images the scale bar represents 500 nm.
90 Figure 3 8 Coulter light scattering particle size measurements of FS NPs in DI water (a) 190 nm and (b) 100 nm ( b ) (a)
91 Figure 3 9 Coulter light scattering particle size measurements of FSNPs in cell media (a) 190 nm a nd (b) 100 nm. (b) (a)
92 Figure 3 10. A bsorption spectra of (a) pure folate (b) 190 nm FITC -doped aminated FSNPs and (c) FITC -doped 190 nm folated FSNPs. [Figure reproduced with permission from Journal of Nanoscience and Nanotechnology] (a ) a (b ) (c ) Absorbance (Arbitrary Units) Wavel ength (nm)
93 Figure 3 11. Normalized fluorescence emission and excitation spectra of pure folate in DI water. Emission spectra (a) and (b) at 355 nm and 280 nm excitation wavelengths, respectively and excitation spectra (c) and (d) at 355 nm and 447 nm emission wavelengths, respectively. (e) Absorption spectrum of folate. [Figure reproduced with permission from Journal of Nanoscience and Nanotechnology] Figure 3 12. Normalized fluorescence emission and fluoresce nce excitation spectra of folate FSNPs in water. Emission spectra (a) and (b) at 492 nm and 355 nm excitation wavelengths, respectively and excitation spectra (c) and (d) at 512 nm and 447 nm emission wavelengths, respectively. Absorption spectrum (e) of folate [Figure reproduced with permission from Journal of Nanoscience and Nanotechnology]
94 0 0.05 0.1 0.15 0.2 0.25 0.3 300 350 400 450 500 550 600 650 700 750 800 Wavelength [nm] Normalized Intensity Figure 3 13. T ransmission electron microscopic image of gold nanoparticles. Figure 3 14. Absorbance spectra of gold nanoparticles in de ioniz ed water.
95 Figure 3 15. T ransmission electron microscopic image of QDSG nanoparticles 0 0.1 0.2 0.3 0.4 0.5 0.6 300 400 500 600 700 800 Wavelength [nm] Normalized Intensity Figure 3 16. Absorbance spectra of QDSG nanoparticle s in de -ionized water.
96 0 20 40 60 80 100 120 430 480 530 580 630 680 730 780 830 Wavelength [nm] Normalized Intensity Figure 3 17. Fluorescence spectra of QDSG nanoparticles in de ionized water. 0 50 100 150 200 250 300 350 400 450 430 480 530 580 630 680 730 780 830 Wavelength [nm] Normalized Intensity Fig ure 3 18. Fluorescence spectra of QDS nanoparticles in de ionized water.
97 Figure 3 19. Variation of absorbance spectra with increasing gold concentration. Figure 3 20. Variation of fluorescence spectra with increasing gold concentration.
98 CHAPTER 4 EN GINEERED NANOPARTICL ES IN BIOLOGICAL SYSTEM S Th e nanoparticles ( NPs ) that have been discussed in the previous chapter were developed as a part of this research effort for use in biological systems as contrast agents with multi -functional therapeutic proper ties. This chapter presents the use of these NPs in biological systems to demonstrate their bioimaging and therapeutic potential, and identify the different physicochemical parameters of the NPs that influence their uptake behavior and biocompatibility in cells The first part of the chapter presents the cellular uptake and viability studies carried out with fluorescent silica NP s (FSNPs) Previous investigators have established that the biological responses of NP s are largely governed by their physicochem ical properties like particle size (Carstensen et al. 1992, Hamoir et al. 2003, Warheit et al. 2005) shape (Brown et al. 2007, Chithrani & Chan 2007, Chithrani et al. 2006) concentration (Dutta et al. In Review for Publication) surfac e chemistry (Hoshino et al. 2004a, Karakoti et al. 2006) and state of agglomeration (Borm et al. 2006, Teeguarden et al. 2007) In vitro experiments using FSNP s were carried out to understand the effect of particle size, concentration, incubation time and surface functionalization on particle uptake in cells. Cell viability tests were conducted using conventional biological assays to confirm the biocompatibility of the FSNPs The second section deals with bioimaging studies using the engineered quantum dot (QD) cored NPs. Preliminary cellular uptake studies were conducted using gold spec kled silica coated quantum dot cored nanoparticles (QDSG NPs ) i n cells and daphnia The hyperthermic property of gold speckles on QDSG was confirmed using polymeric stearates and A549 cells.
99 4.1 In Vitro Exper iments with Fluorescent Silica Nanoparticles (FS NPs ) Amorphous silica NPs with two different surface modification s amine -modif i ed (aminated) and folate -conjugated (folated) were synthesized as described before In order to develop a bioimaging agent, it is important to establish the various parameters that can influence particle uptake and also by quantifying the amount of uptake by the cells Subsequently, it is important to study any potential toxic effects of the NPs inside the cells. Bioimaging an d bio compatibility studies of the surface modifi ed FSNPs were carried out using microscop ic and spectroscopic techniques. Laser scanning c onfocal m icroscopy (LSCM) along with transmission electron microscopic images (TEM) allowed the visual observation o f NP s uptake n by cells Fluorescence techniques and conventional protein assay s were used for quantifying NP s that had been taken up or were associated with the cells. Finally, toxicity assessment using lactate dehydrogenase (LDH) homogenous membrane integri ty assay was conducted to determine whether the cell membrane integrity had been compromised du ring NP exposure. The e ffect of particle size, particle concentration and incubation time were studi ed In order to test this possibility that folic acid conjuga ted NPs can be used to selectively target tumor cells confocal microscopic imaging, uptake quantification studies and toxicity assessments were conducted using amorphous silica NPs with (folated) and without folic acid (aminated). 4.1.1 Cell Culture and Nanoparticle Incubation Human lung carcinoma A549 cells (a model cancer cell line) and normal human dermal fibroblast cells (both from American Type Culture Collection, Rockville, MD) were grown in RPMI 1640 cell media supplemented with 10% fetal bovine s erum, 2 mM L Glutamine, and an antibiotic antimycotic mixture (Cellgro, Mediatech, Inc.). For confocal imaging, trypsinized cells (200 L of a 2.0x104 cells/ mL suspension that had been treated
100 with 0.25% w/v Trypsin/ 0.53 mM EDTA solution for detachment, C ellgro, Mediatech, Inc.) were plated in four 16 -well glass slides (Lab Tek, Nalge Nunc Intenational, IL). Cells were placed in the cell culture chamber for at least 24 hours to allow them to adhere to the slide surface and begin logarithmic growth. At thi s point, cells were generally 50% confluent and ready for nanoparticle exposure. Both 100 nm and 190 nm size aminated and folated amorphous silica NPs were first conditioned for 15 minutes in cell media at particle concentrations of 50, 100, 200 and 400 g/ mL The particle concentrations for dosing the cells were selected from preliminary in vitro studies that showed NP uptake at these concentrations after five hours In preparation for NP treatment, expent cell media was removed from e ach 16 -well slide with cells and replaced by media containing NPs Exposure was carried out for 5, 15, 24 and 48 hours in a humidified cell culture chamber containing 5 % CO2 at 37oC. After incubation, cells were rapidly washed 6 7 times with cold phosphate buffer saline ( PB S ) solution for pH 7.4. Care was taken to ensure that all unbound NPs have been washed out during washing process. Cell morphology remained unaltered before and after PBS washing, indicating no noticeable effect of PBS during washing. 4 1.2 Bioimaging U sing Confocal Microscopy L aser scanning c onfocal microscopy (LSCM ) has been employed to obtain optical images with high resolution In LSCM, a laser beam passes through a light source aperture which is then focused into a small focal volume within a fluor escent specimen by an objective lens A mixture of emitted fluorescent light and reflected laser light is obtained from the focal volume. This light mixture is recollected by the objective lens. The laser light is separated from the light mixture by a beam splitter, which allows the laser light to pass through while reflecting the fluorescent light into the detection apparatus. A photodetection
101 device detects the fluorescent light and records it on a computer. The laser scans over the plane of interest crea ting an image of the whole area. The brightness of the resulting image depends on the relative intensity of emitted fluorescent light. Images can be collected at different focal planes by raising or lowering the microscope stage. The computer can generate a three-dimensional picture of a specimen by assembling a stack of these twodimensional images from successive focal planes. The use of confocal microscopy enables noninvasive, optical sectioning of intact, thick, living specimens and it requires a minimu m of sample preparation. As LSCM depends on fluorescence of the specimens, a sample is usually treated with fluorescent dyes to make the objects visible. Olympus FluoView 500 (Olympus America, Center Valley, PA) laser scanning confocal microscope was used for imaging cells for this research work The data was analyzed using FluoView software ( MicroSuite FIVE Version 5, Olympus America, Center Valley, PA) that was integrated with the microscope. In addition to the fluorescent photodetection device, this mi croscope has another detector for collecting the reflected laser light. In the fluorescent images described below, images generated by both these detectors are shown adjacent to one another. The excitation and emission wavelengths for fluorescent dye (FITC) were 490 nm and 515 nm, respectively. The effect of both 100 nm and 190 nm size aminated and folated NPs on the cellular uptake process was investigated at particle concentrations of 50, 100, 200 and 400 mL The studies were conducted for incubation t imes of 5, 15, 24 and 48 hours. The qualitative effect s of increasing the incubation time from 5 and 24 hours on the particle uptake proces s for both aminated and folated amorphous silica NPs are shown in Figure 4.1. The microscopic images s how ed that sig nals from the images of cells dosed with folated NPs (Figures 4.1c and 4.1d ) exceed ed that of the ir
102 aminated counterparts (Figures 4.1a and 4.1b ) even after 24 hours. From this data, it wa s evident that on keeping the particle concentrations and incubation time constant and comparing between the folated and aminated FSNPs, a much high er quantity of the former are internalized by the A549 cells as compared to the latter. The differences in the particle uptake between the folated and aminated NPs was expected as folic acid molecules are preferentially uptaken by cancer cells that overexpress folate receptors (Wang & Low 1998) The confocal images did not provide conclusive data to differentiate the particle uptake at different incubation times. The figures for 5 hours and 24 hours do not show a discernable difference. It appears that the particles are internalized by the cells within five hours and there is no substantial increase in uptake with time for folated FSNPs and slight increase s for aminated FSNPs. T he mL ) on the uptake process for both aminated and folated FSNPs is shown in Figure 4.2. It can be observed that the uptake of aminated FSNPs (Figures 4.2a and 4.2b ) increased with increasing particle concentration. However, similar conclusion could not be drawn for the folated FSNPs (Figures 4.2c and 4.2d ) as they were being aggressively uptaken by the cells, irrespective of particle concentration. It appears that cells that overexpress folate recepto rs can uptake as many folated NPs as possible to the extent of saturating the receptors (Sa ntra et al. 2005c) The effect of particle size on the cell uptake is shown in Figure 4.3. Larger FSNPs seemed to result in relatively more fluorescence signal intensity for both aminated (Figures 4.3a and 4.3b) and folated (Figures 4.3c and 4.3d) NPs Th is could be either due to increased particle uptake by the cells or an increased number of fluorescent dye molecules inside the larger particles which can emit a higher intensity of fluorescence. There was no conclusive evidence to relate the effect of par ticle size to the cellular uptake from these results.
103 4.1.3 Quantification of N P U ptake The confocal microscop ic study provide d a visual method to observe the cellular uptake of the FSNP s. As shown in the previous section, the effect of some parameters such as size and particle concentration was not clearly understood with visual observations. Th us a method to carry out a semi -quantitative analysis of the NP uptake was developed Since the NP s are fluorescent, an assay was developed that uses NP fluore scence intensity as a marker to quantify the number of NP s uptaken by the cells. For this purpose, calibration curves of fluorescence intensity versus particle concentration, (Figure 4.4), and absorbance intensity versus cell protein concentration, (Figure 4.5), were generated As per the established protocols (Dutta et al. In Review for Publication) a mount of cell associated particles w as estimated by calculating the fluorescent intensity and the corresponding amount of protein in the cell lysate from each well in a ninety -six well microtiter plate. For total protein assays, A549 cells were plated at a density of 7000 cells per well in four 96 well Fluoro NuncTM t issue culture plates (Fisher Scientific, Pittsburg, PA ) and cultured for 24 hours (37oC, 5% CO2) to obtain an adherent monolayer of cells. Cell media from tissue culture plates was removed just before addition of NP s solutions. Stock solutions of folated a nd aminated FSNPs were freshly prepared in cell media (pH 7.2) at a concentration of 800 g per mL for each solution. Serial dilutions from NP s' stock solutions were performed and added to cells in 96 well plates so that the final concentrations were 400, 200, 100, 50, and 0 g/ mL (200 L /well). Each a ssay point w as conducted in quadruplicates. The incubation time for this study was fixed at five hours as it was established that a higher incubation time seemed to have a minor effect on the particle uptake. Cells were washed five times with ice -cold sterile PBS (pH 7.4) to remove unbound NP s and lysed in 75 L lysis
104 solution (1% w/v SDS, 1% v/v NP40, 150 mM NaCl, 50 mM Tris, pH 7.5, and 0.125 U/ L benzonase, Novagen, San Diego, CA). Twenty -five L of cell lys ate from each well was used to determine the total cell protein content using a Micro BCA protein assay kit (Pierce Chemical Company, Rockford, IL). The remainder of the cell lysate was used for the quantification of cell associated FSNPs at various conce ntrations and incubation times. A fluorescent plate reader (Safire, Tecan Group Ltd., Mannedorf, Switzerland ) was calibrated with standard solutions containing varying concentrations of the FSNPs between 0.0122 g/ mL to 6.25 g/ mL in cell lysate solution (generated by homogenizing 10,000 untreated A549 cells in 200 L of the above mentioned lysis solution) The excitation and emission wavelengths of FITC, 490 nm and 515 nm respectively were used for the study. A ccording to established protocols (Dutta et al. In Review for Publication) the quantified protein was expressed as milligram of cellular protein and the correspon ding NP uptake was expressed as micrograms of FSNPs per milligram of cellular protein (mg of particles/mg of protein). The results of the uptake quantification study are plotted in Figure 4.6. It can be observed that for all particles the uptake steadily increases with particle concentration. Comparing the uptake of the folated and the aminated NP s it can be seen that, in general, the uptake for folated particles is higher than the aminated particles at all particle concentration except for the 400 g/ mL value where the two values are almost identical It i s known that particles can enter the cells through diffusion process (Limbach et al. 2005, Zhu et al. 2006) Further cells that overexpress folate receptors are known to aggressively uptake folic acid molecules through receptor mediated en docytosis process (Santra et al. 2005c) Based on the fact that cancer cells do overexpres s folate receptors, it is perhaps not surprising that we
105 observed receptor -mediated endocytosis of folated NP s by A549 cells. However, it is unlikely that aminated FSNPs would be internalized in a similar fashion as folated FSNPs. In the case of folated NP s, both the phenomena of passive process of diffusion and the active process of receptor mediated uptake complement each other to increase the particle uptake as compared to the aminated NP s in which only the passive diffusion process governs the uptake. H owever, at the high concentration of 400 g/ mL the diffusion process itself is sufficient to saturate the particle uptake by the cells, which is indicated by the approximately same mass of particles uptaken for the two sizes (100 nm and 190 nm) of folated NP s and the larger (190 nm) aminated NPs It is therefore clear that particle concentration has a direct correlation with NP uptake by cells. However, increase in particle size due to agglomeration could not be ruled out as this would also result in higher fluorescent signal intensity. It can be clearly seen that the particle uptake is anomalously low in the case of 100 nm aminated NP s as compared to the others. There is an explanation to rationalize this observation. It was observed from the particle size measurements using Coulter Light Scatter ing results (Figures 3.7a and 3.7b) that the particles agglomerated resulting in larger sizes in cell media as compared to the same measurements in de -ionized water Tests were conducted to measure the effective p article size of 100 and 190 nm FSNP s in ce ll media As previously shown in Figure 3.8b, particle size measurements of 100 nm FSNPs indicate d an apparent size range of 40 nm to 20 microns whereas 190 nm FSNPs in cell media, (Figure 3.8a ) indicate d an apparent size range of 2 to 200 microns. P articl es in this size range are suspected to appear to cells as a foreign material leading to non -specific phagocytosis (a cellular process to engulf bacteria, virus and other non -viable cells ) (Dutta et al. In Review for Publication) The 190 nm particles are in a higher size range compared to
106 the 100 nm particles and, therefore, could be more actively uptaken by the cells through the non -specific phagocytosis process. T his is a possible explanation to the comparatively low uptake values of the 100 nm aminated particles. However, for folated NP s of 100 nm particle size, the receptor mediated endocytosis dominates the process of uptake and thus is much higher when compared to its aminated counterpart. 4 1.4 Cell V iability Studies The confocal imaging and NP uptake quantification studies provide insight into cellular uptake of the folated and aminated NPs After observ ing particle internalization the next step was to carr y out cell viability studies to assess the cytotoxic ity of the FS NP s and thereby determine their biocompatibility Potential cytotoxicity of A549 cells when treated with either aminated or folated FSNPs was evaluated using the CytoTox ONE Homogenous Memb rane Integrity Assay Kit from Promega (Madison, WI). If the cell membrane integrity is compromised as a result of cytotoxic agents, the intracellular lactate dehydrogenase ( LDH ) will leak out of the cell membrane and will be found in the extra -cellular med ia. The assay detects and quantifies the amount of LDH that was released into the cell media. This is a cell lysis (cell death) quantification method based on the measurement of LDH activity released from the cytosol of damaged cells into the supernatant. Cytotoxicity is expressed according to the following expression: cells untreated cells treated cells untreated cells treatedLDH Max LDH Max LDH LDH (%) ty Cytotoxici (4 1) In the enzymatic assay, a resazurin compound is bioreduced to a soluble resorufin in the presence of LDH. Resazurin is a non-fluorescent compound and becomes fl uorescent when it is reduced to resorufin ( excitation and emission wavelengths of 560 and 590 nm, respectively). The amount of released LDH during nanoparticle exposure was measured at
107 various particle concentration and incubation times. The details of the procedure are given below as we have modified the original assay protocol to adapt NP s as testing materials. Cells were seeded into 96 well microtiter plates at approximately 4 5 x 103 cells/200 L The plates were incubated overnight at 37 oC in a humidi fied cell culture chamber at 5 % CO2, to allow cells to adhere to the bottom of the wells. Stock solutions of either aminated or mL particle concentration (as described in previous section) were used for cell dosing. N P s were added to wells so that final concentrations tested were 400, mL in quadruplicates (200 L /well). The p lates were then incubated at 37oC for different periods of incubation (5, 15, 24 and 48 hours), akin to the NP uptake experiments. A set of wells without cells was included in the assay for background controls. After incubation L of the media from each well (well A1 in Figure 4.7 ) of the original 96 well assay plate (referred to as plate I) was transferred to a fresh 96 well plate (referred to as plate II). Rest of well contents was termed as pellets. Cells in each pellet were then lysed L of diluted (1:5) lysis solution (Promega, Madison, WI). This modification of the Promega kit procedure allowed us to determine the total releasable LDH from each well and account for possible pipetting errors during cell plating. Both plates I and II were centrifuged at 3500 rpm at 25oC for 4 minutes. The purpose of centrifugation was to separate NP L of supernatant from each well of Plate I and Plate II was transferred into two fresh 9 6 well plates (referred to as Plates III and IV, respectively). Following assay protocol, 50 L of the CytoTox ONETM reagent was then added to each well of Plates III and IV, and then incubated for 30 minutes. The stop solution was added after incubation a nd plates were read using a fluorescence plate reader (Model Safire, Tecan, NC).
108 LDH assay data were compiled and presented in Figure 4.8 at three different incubation times, 5 hours ( Figure 4.8a ), 24 hours ( Figure 4.8b ) and 48 hours ( Figure 4.8c ). In thes e experiments, cells with no NP s and quartz were treated as the negative and positive controls respectively. The LDH value of cells with no NP s was established as the baseline at 0% and that of quartz was found to be 24%. It was observed that both aminated and folated NP were relati vely non -cytotoxic to A549 cells as compared to quartz particles The maximum percentage of cytotoxicity value was calculated to be 8% when aminated FSNP s were pre conditioned with cell media containing serum proteins and inc ubat ed with cells for 48 hours at a particle concentration of 200 g/ mL Considering all experimental parameters such as particle size, concentration and incubation time, data from the present LDH study showed that both pre -conditioned aminated and folated FSNPs appeared to be non -cytotoxic to A549 cells. The percentage of toxicity was about 8% for the highest nanoparticle concentration and longest incubation times tested. One can argue that preconditioned particles appeared to be non -cytotoxic because they are already coated with the serum proteins that are present in the cell media. Therefore, pre conditioned FSNPs for all practical purposes should be considered as protein coated particles rather than aminated or folated particles. In order to resolve the effect of pre -conditioning the NPs on their cytotoxicity furth er experiments were performed where A549 cells were incubated with either aminated or folated NPs without pre -conditioning with cell media. These experiments were performed similar to those with preconditioned NPs by including parameters such as incubation time, particle size and particle concentration. The results from the LDH experiment s in which A549 cells were incubated with non pre -conditioned 100 nm and 190 nm aminated and
109 folated FSNPs for 2 hours are shown in Figure 4.9. A n increas e in the cytotoxic ity values for all NPs as compared to their pre conditioned counterparts was observed T he average percentage of cytotoxicity for non pre -conditioned aminated and folated FSNPs was observed to be 20%. This suggest ed the possibility of surface passivation o f FSNPs by serum proteins when pre -conditioned in cell media. However, it was also clear that pre -conditioning did not completely passivate particle surface because of the fact that folated FSNPs were more aggressively uptaken than the aminated FSNPs. Wh ether or not FSNPs were pre -conditioned, there was no significant difference between the aminated and folated FSNPs in terms of percentage cytotoxicity. It can be concluded that within the limits of biological variation (10%) pre -conditioned FSNPs in gen eral whether aminated or folated, were no n -cytotoxic to A549 cells However, FSNPs that were not pre -conditioned manifested toxicity behavior that became comparable to quartz 4.2 Gold Spec kled Silica Coated Q uantum D ot cored Nanoparticles (QDS G NPs) Bioimaging studies were carried out with QDSG NP s using A549 cells and daphnia Further, heating studies were carried out both in polymeric stearates and A549 cells to confirm the hyperthermic properties of the gold speckles. These three studies are describ ed in the subsequent subsections. 4.2.1 Bioimaging Studies in A549 Cells Using QDSG NPs The cell culture and incubation procedure for A549 cells is similar to the description in Section 4.1.1. A volume of 1 mL of A549 cells at a concentration of 2.0x104 cells/ mL were plated in six -well plates. QDSG & QDS NP s (particle sizes of 15 nm) were pre conditioned in cell media for 30 minutes to prepare a stock solution of 1000 g/ mL This stock was used to prepare QDSG solutions of concentrations 100, 200 and 250 g/ mL These solutions were then added to the six -well plates containing A549 cells in duplicate wells and
110 incubated for 24 hours. After incubation, the supernatant was removed and the cells were washed with cold PBS to remove all non associated particles thereby only keeping back NPs that have been either internalized or are attached to the cell surface. Using the same process as described in Section 4.1.2, confocal microscopy studies were conducted on QDSG NP s. The excitation and emission wavelengths us ed for imaging these NPs we re 355 nm and 590 nm, respectively The results of LSCM studies on amine -modified QDSG (Figure 4.10) show that the NP s were uptaken by the A549 cells in 24 hours The QDSG NPs were not aggressively internalized and behaved simil ar to the aminated FSNPs (described in Section 4.1.2). Most likely the QDSG NPs are taken up by the A549cells by an active process of diffusion, similar to the aminated FSNPs As before, the amount of NP uptake in cells would depend on the range of partic le size encountered by the cells after any particles agglomerat ion that might occur in cell media. 4.2.2 Bioimaging Studies in Daphnia Using QDSG NPs In order to demonstrate the bioimaging functionality of QDSG NP s in living systems, a very simple anim al model of d aphni a was used for this study. The use of daphnia for NP uptake studies has been shown by previous investigators (Lovern et al. 2008) A brief description of the experiment on daphnia for bioimaging using QDSG NPs is given below. A glass beaker of one liter volume was filled with fresh water and QDSG NP s were added to make a stock solution of 8 mM cadmium concentration. A large number of d aphni a was introduced into this aqueous stock solution and left undisturbed for 24 hours. The d aphn i a wa s then taken out of the water, washed several times with cold PBS and depurated to remove all non associated particles from the organism and leave behind only the internalized NP s inside The d aphn i a w as then imaged with a fluorescent microscop e using
111 the excitation and emission wavelengths of the QD -core in the QD SG for bioimaging T he microscopic images of d aphn i a with orange colored (region of interest has been marked with arrows) QDSG NP s in the gastrointestinal tract are shown in figures 4. 11a and 4.11b The control daphni a with no NP s are shown in Figure 4.11 c and 4.11d which have no fluorescence signal in the images. Similarly, the control daphni as with amorphous silica NPs, without any dyes, showed no fluorescence. This experiment demonst rate d the bioimaging capabilit y of the QDSG NP s in simple models of living organisms. 4.2.3 H yperthermic Characteristics of QDSG NPs T he gold speckles on the silica coated quantum dots were expected to impart hyperthermic properties to QDSG NP s. Previou s investigators have reported on hyperthermic properties of gold nanostructures (Govorov & Richardson 2007, Govorov et al. 2006, Huff et al. 2007b, Richardson et al. 2006) In order to confirm this hypothesis, heating studies were conducted using the QDSG NPs First, the particles were tested with polymeric stearate, which began to melt easily with heat generated from the NPs Next, these NPs were incubated with A549 cells and tested for hyperthermic properties. In both cases, the excitation of gold was carried out using a laser source at 785 nm wavelength. This arrangement was used for real time observation of the effect of laser on the NPs i n polymeric stearates and cells. Stock solutions of QDSG and QDS NPs at a concentration of 1 mg/ mL was prepared in ethanol, which dries up rapidly when exposed to atmosphere. 500 mg of commercially available polymeric stearate ( Sigma -Aldrich, St Louis, MO ) was measured and added to each well in a six -well plate. 1 mL of the stock solution of QDS was added to 2 wells and 1 mL of QDSG stock solution to two other wells. The other two wells contained only the polymeric stearate and were used as controls for the experiment The plates were exposed to atmosphere
1 12 (in a hood) for one hour to al low complete evaporation of ethanol from the wells. Following this, the six -well plates were exposed to the laser source that produced laser beams at 785 nm wavelength in the Bio Raman Spectroscope It was observed that the polymeric stearate in the wells with QDSG NP s beg an to heat up and melt after approximately five minutes. After further heating for five minutes, smoke start ed to emanate from the wells and the polymeric stearate turn ed black locally in the area where the beam wa s focused. At this point, the experiment wa s stopped. This same p rocedure when applied to the control wells and wells containing QDS NP s showed no visible heating even after 30 minute of exposure to the laser beam. These observations clearly confirm the hypothesis that the gold speckles impart hyperthermic properties to QDSG NPs T he hyperthermic experiments were conducted in A549 cells, which were plated in six -well plates in a s imilar manner as described before ( Section 4.2.1). A stock solution of QDSG and QDS NP s were prepared in cell media at a concentration of 1 mg / mL 1 mL of the QDS stock solution was added to two wells each containing cells ( 2.0x104 cells/ mL ) in 1 mL of cell media. This br ought the effective concentration of the solution in the wells to 500 g/ mL The same procedure was repeated in two other well s using QDSG stock solution. The last two wells were left with cells and no particles to be used as controls. The cells were incubated with the NP s for 24 hours. After incubation, the supernatant was removed from all six wells and washed three times with cold PBS in order to remove the NP s that are not associated with the cells. The six -well plate wa s introduced into the BioRaman Spectroscope and each well is exposed to the laser beam at 785 nm C ells incubated with QDSG NP s bega n to change their shape and eventually disintegrate. During the process of disintegration, initially it appear ed that the cells swelled up like a bubble and then the bubble burst leaving
113 behind a black hole in the area were the beam was focused with cellular debris strewn around i t. This disintegration process was not observed in the control wells and the wells with QDS NPs T his confirm ed that the hyperthermic property of the gold speckles on the QDSG NP s w as active in vitro The effect of exposing the A549 cells dosed with QDSG and QDS NPs, to the laser source can be seen in Figure 4.12 Figures 4.12 (a) and (b) shows the A549 cells dosed with QDSG NPs before and after exposure to laser. A small black hole and other changes due to heating ( marked with black circle s in the figure ) c ould be observed in the cells after laser exposure. Similar changes c ould not be seen in the cells dosed with QDS NPs as seen in Figures 4.12 (c) and (d), before and after exposure to laser and in control cells with no NPs as seen in Figures 4.12 (e) a nd (f) before and after exposure to laser T he bioimaging and therapeutic functionalities of the engineered NPs developed for this research work has been successfully demonstrated in this chapter. The effect of various physicochemical parameters of the NPs on the cellular uptake was studied qualitatively and semi -quantitatively using confocal microscopy and protein assays. Biocompatibility assessments on NPs were carried out to study the effects of pre -conditioning using cell media. As mentioned in Chapt er one, in order to establish any NP based contrast agent, it is important to understand the biodistribution behavior of the particles inside living systems. Consequently, the use of the NPs developed for this research work, in rat model for blood clearanc e studies shall be discussed in the following chapter.
114 Figure 4 1 Fluoresce nce and transmission image of A 549 cells incubated with 100 nm aminated FSNPs for time of incubation (a) 5 hours and (b) 24 hours and 100 nm folated FS NPs for time of incubation (c) 5 hours and (d) 24 hours at particle concentration 100 g / mL Presence of folic acid has led to the uptake of a substantial amount of particles by the cells. (b) (c) (d) ( a ) (d) ( b ) ( c )
115 Figure 4 2 Fluorescence and transmission image of A 549 cells i ncubated for 15 hours with 100 nm aminated FSNPs at particle concentration (a) 50 g/ mL and (b) 200 g/ mL and 100 nm folated FSNPs at particle concentration (c) 50 g/ mL and (d) 200 g/ mL There is a reduced amount of aminated nanoparticles uptake by the c ells.
116 Figure 4 3 Fluorescence an d transmission image of A 549 cells incubated for 15 hours at particle concentration 200 g/ mL with (a) 100 nm and (b) 190 nm aminated FSNPs and (c) 100 nm and (d) 190 nm folated FSNPs. Although there is an increase in the fluorescence intensity for the larger nanoparticles it does not necessarily imply that this is due to an increase in the nanoparticle uptake, as the larger nanoparticles can enclose a greater amount of dye molecules. ( a ) (d) ( b ) ( c )
117 y = 171.2x + 41.305 R2 = 0.9935 0 250 500 750 1000 1250 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 Concentration (microgm/ml) Fluorescent Readings 50 nm amine modified Silica NP Linear (50 nm amine modified Silica NP) Figure 4 4 Typical particle concentration vs. fluorescence readings curve used as for calibration to compare and determine unknown particle concentrations. The curve has been plotted for 100 nm aminated amorphous silica nanoparticles.
118 y = 0.0013x + 0.1427 R2 = 0.9774 0.000 0.500 1.000 1.500 2.000 2.500 3.000 0 250 500 750 1000 1250 1500 1750 2000 2250 Concentration (microgm/ml) Absorbance Standard Curve for Protein Assay Linear (Standard Curve for Protein Assay) Figure 4 5 Typical protein concentration vs. absorbance readings curve used as for calibration to compare and determine unknown protein concentrations. The curve has been plotted for 100 nm aminated amorphous silica nanoparticles.
119 Figure 4 6 Q uantification of cellular nanoparticle uptake.
120 A B C D E F G H Figure 4 7 P rocedure for the cell viability test based on LDH membrane integrity assay. Well A1 of Plate I 200 L cells + cell media + FSNPs Well A1 of Plate II 100 L supernatant Well A1 of Plate 1 ~ 100 L cell lysate + FSNPs Add 10 L (1: 5) lysis solution Centrifuge @ 3500 rpm for 4 min and collect 50 L of supernatant Collect 100 L of supernatant Centrifuge @ 3500 rpm for 4 min and collect 50 l of supernatant Well A1 of Plate III 50 L supernatant Well A1 of Plate IV 50 L supernatant Add 50 L CytoTox ONE TM reagent Incubate for 10 min and add stop solution Incubate for 10 min and add stop solutio n Read on fluorescence plate reader at excitation and emission wavelengths 560 nm and 590 nm, respectively. Add 50 L CytoToxONETM reagent Read on fluorescence plate reader at excitation and emission wavelengths 560 nm and 590 nm, respectivel y.
121 0 0.5 1 1.5 2 2.5 % cytotoxicity 0 2 4 6 8 % cytotoxicity 0 2 4 6 8 10 % cytotoxicity 100 nm aminated FSNPs 100 nm folated FSNPs 190 nm aminated FSNPs 190 nm folated FSNPs Figure 4 8 Percentage cytotoxicity of 100 nm and 190 nm aminated and folated FSNPs preconditioned in media and incubated for (a) 5 (b) 24 and (c) 48 hours (a ) (b) (c) 50 g / m L 100 g / mL 200 g / m L 400 g / m L 50 g / m L 100 g / mL 200 g / m L 400 g / m L 50 g / m L 100 g / mL 200 g / m L 400 g / m L
122 Figure 4 9 Percentage cytotoxicity of 100 nm and 190 nm aminated and folated FSNPs that were not preconditioned in media and incubated in A549 cells for 2 hours. Figure 4 10. Laser s canning c onfocal microscopic images of A549 cells incubated with QDSG nanoparticles at 200 g/ mL for 24 hours in (a) transmission, (b) fluorescence and (c) combined transmission and fluorescence modes. 50 g / m L 100 g / mL 200 g / m L
123 Figure 4 11. QDSG NP s in d aphn i a. (a) a nd (b) QDSG NP s uptaken by d aphn i a after 24 hours as observed with an orange coloration in the gastrointestinal tract. The arrows in (a) and (b) point to QDSG NPs. (c) and (d) Control d aphn i a with no NP s ( c ) ( d ) ( a ) ( b )
124 Figure 4 12. H yperthermic c haracteristics of nanoparticle s in A549 cells incubated for 24 hours and exposed to laser at 785 nm using QDSG nanoparticles (a) before and (b) after exposure, using QDS nanoparticles (c) before and (d) after exposure and using control cells with no nanoparticles (e) before and (f) after exposure ( c ) ( a ) ( b ) ( d ) ( e ) ( f )
125 CHAPTER 5 PARTICOKINETIC STUDY OF NANOPARTICLES IN RAT BLOOD The re have been numerous in vitro and in vivo studies on the toxicity outcome of quantum dots (QDs). However, inconsistencies regarding various iss ues like nanoparticle (NP) dosimetry, routes and duration of exposure and choice of animal models exist in many of the se in vivo studies (Alexis et al. 2008, Harper et al. 2008, H e et al. 2008, Huang et al. 2008, Jain et al. 2008, Khan et al. 2005, Li & Huang 2008, Semmler Behnke et al. 2008, Sonavane et al. 2008, Yang et al. 2007) Similarly, uncertainties in particle concentrations used for dosing cells, state of agglomeration a nd effects of surface functionalizing the nanoparticles (NPs) are some of the challenges encountered for the in vitro studies (Teeguarden et al. 2007) Moreover, most of these studies do not provide reliable correlation between the physicochemical properties to the NP disposition (Committee 2004) In this study, a systematic investigation of the blood clearance behavior of NPs in rat was carried out using analytical detection technique. It is anticipated that results from this present study will clarify and eliminate the discrepancies in the literature relating to the effect of material composition and surface charge on the blood clearance behavior of NPs. Th e goal wa s to develop a predictable and reliable methodology to elucidate the clearance behavior from the half life values of the NPs The final section describes a preliminary protein adsorption study to visually observe the adsorbed proteins on the various NPs. It was conducted using one -dimensional gel electrophoresis using QDS, QDSG and gold NPs with three surface charges : positive, negative and near neutral. 5.1 Experiment s with Nanoparticles ( NPs ) in Rat Model 5.1.1 N anoparticles (N Ps ) used for A ni mal S tudy The synthesis and characterization procedures of the NPs used for the animal studies have been discussed in details in Chapter 3 Briefly, five NPs belonging to two different classes
126 gold and quantum dot based NPs were used for the rat b lood c learance studies. The five different NPs were: 1) bare gold NPs, G (negatively charged surface), 2) amine-modified gold NPs, G+ (positively charged surface), 3) pegylated gold NPs, GP ( near neutral surface charge), 4) silica coated CdS: Mn/ZnS quantum dot s (QDS) and 5) gold spec kled silica coated CdS: Mn/ZnS quantum dots (QDSG). As per established practice (Teeguarden et al. 2007) a preliminary one -compartmental model was first developed using half -life (t) data of various NP systems reported in the literature (Alexis et al. 2008, Banerjee et al. 2002, Guo et al. 2007, He et al. 2008, Lee et al. 2007, Li & Huang 2008, Liu et al. 2008, Sonavane et al. 2008, Wang et al. 2004, Yang et al. 2007) This model was then used to study the effect of various NP parameters (percentag e composition of constituent element, particle concentration and rat weight) on blood clearance and half -life behavior in rat blood. An optimal dose of NPs to be injected into the rats was selected and the model was then modified using experimental data fr om analytical detection of the blood samples collected from the animal study. The half life value of each NP was calculated from the one -compartment al model to further refine the model necessary to determine the particokinetic behavior of the NPs in rat bl ood 5.1.2 Dosing the Rats Twenty two Sprague Dawley rats were purchased from Charles River Laboratories International Inc, (Wilmington, MA) and acclimated for one week in the animal facility at (Teeguarden 2008) All animal tr eatments and experimental protocol for this study was subjected to the review and approval from the Institutional Animal Care and Use Committee (IACUC). Therefore, the animals were handled according to standard animal husbandry practices and given humane treatments with regard to alleviation of suffering during the entire animal study. The
127 animals were housed in facilities having less than 12 hours of light/dark cycle, temperature controlled to room temperature with ad libitum water and food availability. Fifteen rats were used as experimental animals and dosed with NPs and seven were used as control animals. The experimental rats were labeled 1 to 15, with three rats dedicated to be dosed with each NP. Two of the three rats for each NP were housed in a me tabolism cage, in order to facilitate the collection of urine and feces at pre determined time -points while the third rat was housed in a normal animal cage. The control rats were labeled 16 to 22 and blood samples from the control rats were used to prepar e the spiked samples to generate the reference curves for the animal study. All animals were provided by the supplier with surgically implanted cannulae tubes made of polyurethane (PU) attached to the jugular vein of the rats. All five NPs, as dispersed in biological media, were injected intravenously through the cannulae tubes into the jugular vein of the rats over two minutes. For the rats inside the metabolism cages (first two rats for each NP), dosing was done through the PU tube and drawing of blood was carried out through an eight inch long polyethylene (PE) tube attached to the cannulae of the rats. For the third rat for each NP, kept in a normal animal cage, dosing as well as drawing out blood was conducted through the PU tube provided by the suppl iers. At the start of the experiment, the rats were weighed on a laboratory scale and noted to weigh between 240 and 275 grams (Table 5 1). The mass of NP s used for dosing the rats were calculated such that a rat weighing 225 grams was dosed with approxi mately 0.5 mg (0.5 mL stock solution of NP at 1000 g/ mL in 0.45% bacteriostatic solution) of each NP solution by intravenous injection through the surgically implanted PU cannulae tubes attached to the jugular vein. The syringes used for dosing the NPs we re pre and post -weighed in order to ascertain the
128 exact dose of NPs delivered to each individual rat (Table 5 1). Similarly, the glass tubes used for blood collection and storage were pre and post -weighed to determine the exact mass of blood collected f rom each animal (Table 5 2). Blood samples of approximately 400 L volume was drawn from each of the 15 rats just prior to dosing i.e. at time -point 0 minutes, using the pre heparinized syringes and luers and placed in pre -heparinized borosilicate glass tubes. Similarly, post dosing, 400 L of blood was drawn out from the rats at every pre-determined time -point of 5, 15, 30, 60, 180, 360 and 1440 minutes. Serial sacrifices of the animals were conducted using carbon dioxide atmosphere at the end of the study i.e. after 1440 minutes (24 hours) following dosing of the rats Tissue samples, including liver, lungs, kidneys, heart, spleen, brain, muscles and fat were collected after the sacrifice. Urine and feces were collected from 2 of the 3 rats dosed with each NP i.e. the rats placed in the metabolism cages. All tissues, b lood plasma, urine and feces were labeled and stored in freezers at 80C for future analysis. The carcass of all animals were labeled and stored in freezers at 80 C for possible further investigations. The blood samples collected from the rats at diffe rent time -points were analyzed for gold (molecular weight = 196.97) in rats that were dosed with G GP and G+ NPs. Rats dosed with QDS and QDSG were analyzed for cadmium (molecular weight = 112.41). All analyses of blood samples were conducted at standard conditions (room temperature and pressure) utilizing inductively coupled plasma mass spectroscopy ( ICP MS) techniques. 5. 1 3 Nanoparticle ( NP ) Di s persion in D osing S olution Gold NPs have been shown to exhibit variations in color with a change in size (Jiang et al. 2004b) During the process of NP administration into the rats, it was noticed that the stock solution of gold NPs on contact with biological media displayed a gradual change in color from
129 dark red to bluishred, purple and finally formation of black precipitates that settled down in a clear solution, These visua l observations indicated the formation of gold NP agglomerates in the bacteriostatic solution used as the biological media. In order, to study the effect of the different dispersion media (blood -plasma, heparin lock flush solution and bacteriostatic solu tion), a stock solution of gold NPs was prepared at a concentration of 1000 g/ mL in de -ionized water. The stock solution was then added in increasing amounts (with infinitesimal increments) to different vials containing blood -plasma, heparin and bacterios tatic solutions respectively and visually observed for any changes in color of the solution. Generally, heparin lock flush solution (Emergency Medical Products Inc, Waukesha, WI ) is used in an animal study to prevent blood coagulation while 0.9% bacteriostatic solution (Med Tech Resource Inc., Eugene, OR) is used as a buffer to maintain a pH 7.4 in the rat blood after dosing the animal with NPs. It was observed that the addition of blood turned the color of the stock solution to dark red. Thereafter, it w as difficult to visualize any observable color changes in the red colored gold NPs that can be attributed to the formation of agglomerates in the presence of blood. An addition of an equal amount of 100% (by mass) heparin solution (or more ) to gold NP solu tion changed its color from red to bluish -red, purple and finally after ten minutes led to the formation of black precipitates in a clear solution. For example, an addition of 0.5 mL heparin solution or more to 0.5 mL gold NP solution resulted in the descr ibed color changes of the solution. Finally, addition of bacteriostatic solution to gold colloids at a ratio of 7:10 led to the agglomeration of the negatively charged colloidal gold NPs. For example, an addition of 0.35 mL of bacteriostatic solution or mo re to 0.5 mL of gold colloids led to the a fore -mentioned color changes. This simple experiment, using visual inspection, provided an insight into the maximum amount and
130 concentration of heparin lock flush solution and bacteriostatic solution that could be used to disperse the particles to prepare the stock solution. As per the IACUC protocols (Teeguarden 2008) at the national laboratory, the acceptable range for composition of the bacteriostatic solution (buffer) for an animal study was between 0.3 to 0.9%. Howe ver, it was impossible to disperse the NPs in the 0.9% bacteriostatic solution, which is the preferred biological media for animal studies. There was an immediate change in color o f the gold NP stock solution in the 0.9% bact eriostatic solution which indicated the formation of micron -sized aggregates. F ormation of aggregates was avoided by reducing the electrolytic concentration of the media used for dispersion to 0.45% bacteriostatic solution. T he present study, although near ing the lower electrolytic limit, was valid. The stock solution of NPs for the animal study was prepared in 0.45% bacteriostatic solution with trace quantities of heparin in it to emulate the conclusions from the observations of the above study. The result s of the visual observations from this study are summarized in Table 5 3. The bare gold (G ) and pegylated gold (GP) NP samples remained dispersed in 0.45% bacteriostatic solution at their primary particle sizes for 24 hours. The positively charged gold N Ps, QDS and QDSG NPs remained dispersed for 12 hours only. The actual dosing solutions of the five NPs for the animal study were prepared the previous night, eight hours before inject ion into the rats in the morning. 5.1.4 Analysis Protocol As mention ed above, 400 L of actual blood samples were collected from the fifteen experimental rats at different time points. The blood was collected in pre -heparinized disposable borosilicate glass culture tubes 16 x 150 mm (Fisher Scientific, Pittsburg, PA) The blood samples were treated with 2 mL of concentrated (70%) nitric acid (Sigma -Aldrich, St. Louis,
131 MO) to digest all organic components present in the samples. The mixture of blood, NPs and acid in the glass tube was then heated in a dry bath using aluminum blocks at 125C for 15 minutes. The bath was then cooled to 110C, followed by the addition of 1.6 mL of pure aqua regia (Hydrochloric Acid: Nitric Acid = 3:1) into the glass tubes to allow acid digestion of gold and quantum dot NPs. Further heating was c ontinued for 15 minutes before the samples were evaluated for completeness of digestion. The high effective concentration of aqua regia (40%) and nitric acid (50%) used was sufficient to completely digest all NPs. The glass tubes were then removed from the dry bath, allowed to cool to room temperature and the contents were filtered through Acrodisc 0.45 m PTFE syringe filters (Acrodisc: Catalog Number 4219, Pall Life Sciences, Ann Arbor, MI) to remove all tissue debris from the samples. Considering complete digestion of all NPs, the possibility of any loss of analyte during passage through the filters wa s negligible. This was verified from the background level signals for gold and cadmium detection in samples prepared by scraping and digesting the filters (post -filtration of NPs) and analyzed using the ICP MS Post -filtration, the acid -digested blood samples from the animal study were then stored into 15 mL polystyrene centrifuge tubes for further dilution and analysis using ICP MS In summary, the following acid-digested protocol was used for the subsequent studies. Addition of 1.5 mL concentrated HNO3 ( 70%) to the blood-plasma samples in glass tubes samples are heated in aluminum blocks in a dry bath at 125C for 15 minutes The samples should be completely digested and clear at this point with no tissue remaining, if not, the temperature of the dry bath is carefully increased to 140C and heating is continued for another 15 minutes. The dry bath is now cooled to 110C and the tubes are removed and allowed to cool Addition of 0.5 mL hydrogen peroxide (30%) to the samples and the glass tube is put back into the dry bath at 110C and digestion is continued for another 60 minutes to ensure complete digestion of all biological molecules in the samples.
132 Next 1 mL of aqua regia (HNO3: HCl = 1:3) is added and the samples are put back into dry bath at 110C a nd heating is continued for another 15 minutes. Samples are filter ed through a 0.45 micron and put into 15mL polystyrene centrifuge tube s Samples are stored in 15 mL centrifuge tubes for future analys is. 5.1.5 Analy sis of Blood Samples A n Agilent 7 500CE series ICP MS ( Agilent Technologies, Inc, Sa n ta Clara CA ) with an octapole reaction system (ORS) was used for analytical detection of gold and cadmium in blood samples in the present study. The theoretical detection limit of an ICP MS for gold and c admium was reported (Lewen et al. 2004) to be 1 and 3 nanograms per liter respectively. Generally, nanograms per liter is more commonly expressed as parts per trillion (ppt) for analytical detection techni ques. However, it is to be noted that the reported detection limits have been determined using elemental standards in dilute aqueous solutions with an expressed 98% confidence level. However, detection limits inside the biological matrix was suspected to b e much lower due to the presence of high backgrounds and have to be individually determined for the samples and the particular an alyte in the matrix in question The ORS technology of the Agilent 7500 CE series ICP MS uses helium (He) collision mode for t he predictable removal of all matrix interferences and allows full -scan semi quantitative analysis. Moreover, there is optional hydrogen (H2) and argon (Ar) reaction modes for the sensitive (single digit ppt) detection limits for various additional elements. The analytical technique employed in the present study utilized the argon mode for detecting gold and the helium mode for detecting cadmium for the quantum dot NPs respectively. Two separate standard reference curves were generated using known ( spiked) samples of gold and QD NPs respectively. The first set of gold spiked samples consisted of increasing gold concentration of 0, 1, 2, 4, 8, 16, 32, 64 and 128 parts per billion in control rat blood. The second
133 set of samples generated using known amounts of QDS NPs contained increasing cadmium concentration of 0, 1, 2, 4, 8, 16, 32, 64, 128 and 256 parts per billion in control rat blood. All samples were prepared in triplicates and the average values of the three readings obtained from the ICP MS was used t o generate the standard curve. The standard deviations of the three readings along with the value of R2 of the linear line gave an indication to the accuracy of the curve fit for the standard curve. The reference curves were used to quantify the amount of gold and QD cored NPs present in the blood samples. Finally, a concentration time profile curve was generated for each of the five NPs. This curve along with equations in the one compartmental model was used to calculate the half -life value of each NP in rat blood. More d etails of this procedure are given later in this chapter. 5.1.6 One Compartmental Model A common approach, especially used by researchers in the pharmaceutical field is to develop pharmacokinetic models that can describe the time -de pendent distribution and disposition of a substance in a living system. Towards a medicinal end-point, these models are used to estimate an optimum drug dosage and schedule protocol. For the industrial end -point, such models help to characterize the toxins and assess the transport and metabolism characteristics of the substance in vivo. Various pharmacokinetic models consider the animal body as a system of compartments. The rate of transfer of the drug between one compartment to another and the rate of eli mination from the various compartments is assumed to follow first order or linear kinetics. The one compartmental model, which is the simplest model, presumes that the whole body is a single homogenous unit for kinetics considerations. The one compartmen tal model has been shown to be especially relevant for the pharmacokinetic analysis of drugs that can distribute extremely rapidly throughout the body. The blood plasma or serum is almost invariably the anatomical
134 reference for the one compartmental model It assumes that the rate of change of drug concentration in the blood plasma quantitatively reflects the rate of change in drug concentration throughout the body. Investigators (Teeguarden et al. 2007) have shown that similar studies for NPs using one compartmental models can be conducted. Accordingly, a one compartmental model was developed in this research, to assess the clearance of NPs from rat blood. These results are anticipated to provide some insight to NP clearance from the blood without the considerations of other competitive and complicated processes li ke tissue uptake and presence of opsonins, which may be incorporated into subsequent research studies. First Order Kinetics. In the one comp artmental model, the NPs, following intravenous injection into the body, distribute rapidly and are eliminated accor ding to first order kinetics i.e. the rate of particle loss from the body is given by: KX dt dX (5 1) where X is the mass of the particles in the body at time t after injection and K is the first order (elimination) rate constant for the NP (negative sign implies that the NP mass in the body reduces with time). In order to describe the time course of the NPs after injection into the body, Equation 5 1 can be integrated to obtain Kt X X exp0 (5 2) where X0 is the initial mass of the particles. Applying natural logarithm on both sides of Equation 5 2 Kt X X 0ln ln (5 3) Converting Equation 5 3 in terms of Log results in
135 303 2 log log0Kt X X (5 4) The relationships expressed by Equations 5 3 and 5 4 can be written in terms of the concentration of the particles per unit volume of blood (instead of mass of particles ) as follows. kt C Ct exp0 (5 5) 303 2 log log0kt C C (5 6) where C0 is the initial NP concentration in blood immediately after injection th at is at t = 0 Ct is concentration after time t and k is the first order rate constant for concentration -time relationship. Equation 5.6 indicates that a plot of log C versus t is a linear curve for the assumed first order kinetics. C0 can be obtained by the extrapolation of the log C versus t plot to time zero. This value of C0 is used in calculating the apparent volume of distribution, V given by the relationship 0 0C X V (5 7) The slope of the line generated from Equation 5 6 is equal to k/2.303 and the value of k is obtained from the value of this slope. Alternatively, setting C = C0 /2 and t = t1/2 gives another relationship between the (elimination) rate constant k and half -life t1/2 as k t 693 02 / 1 (5 8) where t1/2 is the biological half life of the NPs in the rat blood. The biological half -life of a ny substance is defined as the time it takes to lose half of its activity i.e. expressed by a drop in concentration to half its initial value. An important aspect of a first -order process is that the t1/2 is independent of the initial concentration of the substance. E quations 5 1 to 5 -8 were used for
136 developing the one compartmental model The model was initially used to calculate the particle concentration of the dosing solut ions at 1000 g/ mL for the animal study to ensure detectability on the ICP MS. Relevant information (composition, surface area, density) for each NP was used as input parameters for the model to calculate the half -life of the NPs. For example, the relevant particle inf ormation for the QDSG NPs is listed in Table 5 4. 5.2 Establishing the P rotocols for A nimal E xperiments Before conducting the actual animal experiments, it was necessary to develop protocols for analytically detecting the analytes (gold and cadmium) in the blood sample collected after NP injection into the rats. During the process of establishing the protocol, the effect of a number of different variables on the experimental data was encountered This section describes the effect of the different variab les and the optimized protocol established for the animal study. 5.2.1 Effect of B ackground from Aqua Regia Preliminary observations from the ICP MS demonstrated that high concentrations of aqua regia in solutions present a background signal even withou t any presence of gold in the solution. Therefore, it was necessary to determine an upper limit of the aqua regia concentration below which the background effect was insignificant. In order to minimize the background signal from aqua regia, the effect of v arying concentration of aqua regia on the ICP MS readings was studied. A series of spiked samples having concentrations of aqua regia 0, 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5 and 10% of the as prepared aq ua regia concentration (1:3 HCl :HNO3) were prepar ed. An increasing ICP MS background signal (detecting for gold) as a function of increasing aqua regia concentration for samples dispersed in water when no NPs were present can be seen in Figure 5 1 It can be observed that for aqua regia concentrations of 0.625% and above, there is a background contribution to the instrument signal due to the high concentration of acid. Moreover, above concentrations of 1.5% it was observed that the background effects far
137 supercede the signal intensity of gold solutions c ontaining 32 ppb of gold. Therefore, the aqua regia concentration used for digesting the samples was maintained at 5% and below for all subsequent studies. 5.2.2 Effect of B ackground from O ther C omponents The experiment in the preceding section was conducted with aqua regia in water (matrix). A similar experiment was conducted with aqua regia in a matrix that would imitate the experimental conditions of the animal study. This matrix contains control rat blood, nitric acid and water. First, six glass test tubes were each filled with 1.0 mL of control rat blood and 1.0 mL of nitric acid. To these 6 test tubes 1 mL of 0%, 1%, 2%, 3%, 4%, 5% and 6.25% of aqua regia were added. The volume of the solution in each glass tube was then increased to 10 mL by the ad dition of 7 mL of water to each tube. This results in an effective concentration of aqua regia in the test tubes of 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5% and 0.625%, respectively. The results from ICP MS analysis of these s olutions are shown in Figure 5 1 It c an be observed that the background reading is constant over the range of 0% to 0.625% aqua regia concentration. This trend is similar to the above experiment of aqua regia in water. However, the magnitude of background signal is uniformly higher in the sec ond case. It could be easily inferred that this background is due to the ot her constituents of the matrix-blood and nitric acid. It is imperative to reduce the background effect of blood and nitric acid to a minimum value, which can be accomplished by di luting the above solutions with water. It was foun d that diluting the above solution in blood and nitric acid matrix containing 0.5% aqua regia by 4 times with water reduced the background signal to the levels observed with pure water. In all subsequent ex periments prior to the animal study, the blood samples were digested using 5% aqua regia, followed by dilution to forty times with de ionized water before analysis to reduce background effects and generate best results.
138 5.2.3 D igestion of Nanoparticles ( NPs ) Experiments were carried out using a series of spiked samples containing a constant amount of 50 ppb (a central point in the reference curves) of gold NPs in blood. These NPs were digested by adding the following concentrations of aqua regia: 0, 0.78 1.56, 3.13 and 6.25% and continued heating in an aluminum block at 125C in the dry bath for 15 minutes. Thereafter, the samples were diluted by ten times with de ionized water to reduce the aqua regia concentration to less than 0. 625% before being analyzed in the ICP MS. The concentration of gold as a function of aqua regia concentration is shown in Figures 5 2 It can be seen that the curve increases initially and then flattens in the region of 3% to 6.25% aqua regia concentration. At concentrations low er than 3%, the particles were not digested completely and the undigested particles were not analyzed by the ICP MS resulting in a lower concentration. The fact that the curve flattens out in the 3% to 6.25% region indicates that the particles have been completely digested. The effect of increasing aqua regia concentration on samples in acid and blood as the matrix are compared in Figure 5 2 Moreover, similar to previous observations (Figure 5 1 ) there was a noticeable higher background effect even for s amples with gold NPs in the presence of blood as compared to water at higher aqua regia concentrations The cumulative effects of all the above findings are shown in Figure 5 3. The blue line in the background shows the effect of increasing aqua regia conc entration on the background while the red line shows the extent of NP digestion with increasing aqua regia concentration. 5.2.4 Deposition of Nanoparticles ( NPs ) on Cannulae Tubes It h as been previously mentioned that the dosing of rats was carried out by injecting the NPs through the cannulae tubes made of PU. However, to draw the blood out, the rats in the metabolism cages had an 8 inch PE tube attached to the already existing PU tubes. It was found,
139 during preliminary studies, that the particle concen tration in the blood drawn through the PE tubes was consistently lower than the particle concentration in the blood drawn through only the PU tubes. In order to quantify possible loss of gold or QD NPs through adsorption onto the PE or PU tube surface duri ng intravenous injection and blood collection, two experiments were conducted. The rats were provided by the supplier with PU cannulae tubes, 3 inches in length, 0.025 inches internal diameter and dead volume of 0.05 mL surgically attached to the jugular veins. Additionally, for the two rats placed in the metabolism cages, PE tubes, 8 inches in length, 0.023 inches internal diameter and dead volume of 0.1 mL were attached to facilitate the drawing of blood collection through the metabolism cages. For eac h NP, only one of the 3 rats was placed outside the metabolism cage. Stock solutions for all five NPs were prepared in bacteriostatic solution, sonicated for 15 minutes and subsequently vortexed for five minutes and then added to control blood collected from rats prior to the experiments to bring the final concentration to 1000 g/ mL in blood. Four 15 mL centrifuge tubes for each NP were pre -weighed and kept aside. Sample PU tubes having 0.025 inches internal diameter and 3 inches in length were pre -weighed and 500 L NP dosing solution is passed through the tubes using a syringe The exiting solution from the tube is collected in the first pre -weighed 15 mL centrifuge tube, Tube A, which is then weighed to calculate the mass of sample. The PU tube is also re-weighed after rushing the sample through it. Thereafter, 500 L phosphat e buffered saline (PBS) solution is then passed through the tube and collected in the second pre -weighed centrifuge tube, Tube B, which is again weighed to calculate the mass of dead volume + PBS solution. The PU tube is also re -weighed to calculate the ma ss of PBS solution remaining back in it. Then, 500 L of 5% aqua regia is passed through the tube.
140 The exiting solution from the tube is collected in the third pre -weighed centrifuge tube, Tube C, which is again weighed as before along with the PU tube. For the gold NPs, a final 500 L of 5% aqua regia was passed through the tube and for the QD -based NPs, this final step was carried out with 2% nitric acid. The exiting solution from the tube is collected in the fourth pre -weighed centrifuge tube, Tube D, which is again weighed as before along with the PU tube. All sample collected in Tubes A to D were then digested using 1 mL of 5% aqua regia and heated at 125 C for 15 minutes. This was followed by addition of 1 mL of 2% nitric acid and heated at 110 C for 15 minutes. The samples were then dialyzed through 0.45 m filters and the volume of the samples is brought to 10 mL by adding de ionized water. The samples were then analytically quantified for gold and cadmium using the ICP MS (a) Using Polyurethane Tubes. The above experiment was first conducted usin g PU cannulae tubes and the results are given in Table 5 5. The a mount of analyte detected by the ICP MS in T ube B for all 5 NPs after PBS rinse was less than 10%. It is important to note that the d ead v olume of the PU cannulae tube is 0.05 mL which is 10% of the total volume injected Therefore, it can be concluded that a t otal amount of NP loss of les than 10% after dosing, which was recovered by the PBS rinse is within the expected margin. The a mount of analyte detected in tube C after the first 0.5% A qua Regia rinse was less than 2% for all the NPs and the a mount of analyte detected in tube D after the last rinse with 0.5% Aqua Regia for all NPs was less than 1% This confirmed a minimal adsorption of NPs from both the gold and the quantum dot solution inside the PU cannulae tube during dosing the rats. (b) Using Polyethylene (PE) Tubes A similar experiment was conducted using PE cannulae tubes, similar to the ones used for drawing blood from the rats in the metabolism cages in the animal study. Three pieces of PE tubings (8 inches in length, 0.023 inches internal diameter and
141 dead volume = 0.05 mL ) were used for each NP dosing solution. The experiment was conducted in a way to imitate the process of drawing blood from rats in metabolism cages to analy ze for any possibility of NP adsorption on the PE tube material. The results from the experiment conducted using the PE tubes are shown in Table 5 6. Contrary to the previous experiment using PU tube, in this case the loss of NPs due to adsorption on the P E tube was substantial for all particles except for GP. The t otal amount of NP loss after dosing as recovered by the PBS rinse was greater than 30%. The dead volume of the PE tube was 0.075 mL which is 15% of the total volume. Thus the NP loss due to adsorption on the PE tube was much higher than the expected margin of dead volume. The a mount of analyte detected in T ube C after the first 0.5% Aqua Regia rinse was greater than 8 % for all the NPs and the a mount of analyte detected in Tube D after the last rinse with 0.5% Aqua Regia for all NPs was greater than 2 % This experiment confirmed that PE cannot be used as a cannulae tube material for further animal studies, as there would be NP loss during dosing and blood draw due to NP adsorption on the surface of the tube. The observations from the experiment agreed with the results of the animal studies as shown for the QDSG NPs in Table 5 7 A high value of each analyte (gold and cadmium) was detected in Rat 3 placed in the normal cage and not attached to the PE tube as against a much lower value of the analyte in the blood of Rats 1 and 2 in the metabolism cages. Moreover, the discrepanc y in the value f o r Rat 3 kept in the normal cage was only ob served for the blood samples drawn out at the 5 minute time -point. The differences in the analyte detected between the three rats reduced remarkably for the 15 minute time -point. Final ly, there was negligible differ ence for blood samples drawn at and after the 30 minute time -point for all three rats. It can be explaine d that the available sites on the PE tube were partially covered by the adsorbing NPs after the blood draw at the 5 minute time -point and completely covered after the blood draw at
142 the 15 minute time -point. Thereafter, the adsorption sight could have been saturated and hence no variations were observed in the detected analyte for blood drawn after the 15 minute time period. These observations were similar for the other three NPs namely QDS, G+ and G NPs For GP NPs no significant differences were obser ved between Rat 3 and Rats 1 and 2 at all time points. GP NPs are known to have stealth characteristics due to the presence of polyehtyleneglycol (PEG) that projects hydroxyl functional groups on the surface of the NPs. These functional groups make the NPs hydrophilic in nature and thus the proteins in the physiological environment do not get adsorbed onto the particle surface. However, this is not the case for the other four NPs and, thus, a distinctly different behavior can be observed in the remaining for NPs when compared to GP. Therefore, it can be surmised that surface functionality of the NPs is a crucial factor in determining the adsorption of NPs on the PE surface. 5.3 Results from Blood Clearance Study In th e following section s the results of the rat blood clearance study will be presented The stock solutions were prepared with NPs dispersed in bacteriostatic solution s at varying concentration in such a way that the final NP concentration was 1000 g/ mL in 0.45% bacteriostatic solution. The stoichiometric calculations for preparing the dosing solutions of each NP at the target concentration of 1000 g/ mL are shown in Table 5 8. The blood samples containing the NPs were digested, analyzed and the hal flife values were finally calculated 5.3.1 U sing S ilica C oated Q uantum D ots Nanoparticles (QDS NPs ) As per dispersion protocol established in Section 5.1. 3 s tock solution s of silica coated quantum dot (QDS) NPs at a particle concentration of 1000 g / mL in bacteriostatic solution was prepared. The solution was vortexed (Vortex Genie 2, A. Daigger and Company, Vernon Hills, IL) and sonicated (Branson 1510, Kell Strom, Wethersfield, CT) respectively for five minutes
143 for better NP dispersion. 500 L of Q DS in 0.45 % bacteriostatic solution was administered into the cannulae tube of three rats (labeled 13 15) and the solution was rinsed up and down to ensure that all QDS NPs in the dose including the dead volume had been injected into the rat blood. Subsequently, 400 L of blood was drawn from each rat at the pre -determined time points, collected in pre -weighed glass tubes and acid digested with the established digestion protocol described before. The concentrated QDS samples had the following ratios: blood -plasma: H2O2: nitric acid: aqua regia = 4: 5: 15: 16 (total volume of 4 mL ). 300 L of the above -mentioned concentrated QDS samples wa s mixed with 2.7 mL nanopure de -ionized water and vortexed and sonicated for 15 minutes respectively for homogeniz ation The diluted samples (C/10 of the actual concentration of the acid digested samples) we re now ready to be analyzed for cadmium on the ICP MS in the helium gas mode. A reference curve of cadmium was generated using spiked samples of QDS NPs containing 0 256 ppb of cadm ium and digested using the same acid composition and ratios used for the pilot study blood samples. The reference curve for cadmium wa s f irst generated ( Figure 5 4 ), followed by blood sample analysis using the same instrument settings. Cadmium wa s detecte d and quantified in the blood samples by the ICP MS analysis, from which the actual amount of NPs present in the blood at the time of draw is back calculated using the percentage composition values of cadmium in the QDS NP s and the sample dilutions made be fore the analysis. A graph of particle concentration in blood versus time profile was plotted for the QDS NPS (Figure 5 5 ) and the half -life wa s calculated using the one -compartmental model. The half life of the QDS NPs was found to be 12.5 + 9.7 minutes.
144 5.3.2 U sing G old S peckled Q uantum D ot Nanoparticles (QDSG NPs ) The QDSG samples were treated exactly in the same way as the QDS samples mentioned above in Section 5.3.1 for particle and blood digestion. The samples were analyzed for cadmium as the quan tity of gold in the specks was extremely low (Cd =21.4%, Au = 4.39% by weight). The reference curve generated for Cadmium is shown in Figure 5 6 while the PK data of the QDSG samples as obtained from the ICP MS from cadmium quantification and back calculat ions (to make up for dilutions) are plotted in Figure 5 7 QDSG NPs were determined to have a half life of 14 + 6.5 minutes which was similar to the value for QDS NPs. 5.3.3 U sing B are G old Nanoparticles (G NPs ) For the gold based NPs, ICP MS analysis was done to detect gold as an analyte. The acid digestion protocol for the NPs was exactly the same as the one used for the silica coated quantum dot (QDS) samples (Section 5.3.1). 300 L of the G+ samples was mixed with 2.7 mL nanopure de -ionized water (C/10 of the actual concentration of the acid digested samples) and vortexed and sonicated for 15 minutes respectively to homogenize the samples. The diluted samples were then analyzed fo r gold on the ICP MS in the normal argon gas mode. A reference curve (similar to Figure 5 8) of gold was generated using spiked sample s of bare gold NPs containing 0 128 ppb of gold that were digested using the same acid protocol used for blood s amples fro m the animal study. The PK data of the bare gold samples showed the presence of a very low amount of gold in the blood even at the 5 minute time -point, (similar to Figure 5 9 ). The level of gold NP concentration level remains unchanged and there is no di fference in the gold detected and quantified between blood draw at any of the various time points mentioned before.
145 5.3.4 U sing Aminated G old Nanoparticles (G+ NPs ) The digestion protocol for G+ NPs was same as described in previous section. The refere nce curve generated using gold NPs wa s similar to that shown in Figure 5 8 The PK data of the blood samples containing amine -modified gold NPs wa s also similar to that shown in Figure 5 9 The G+ and G NPs had a similar blood clearance behavior and appea red to have been removed even before the first blood draw at the five minute time -point after NP injection into the rats The half life values of G+ and G NPs was < 5 minutes. 5.3.5 U sing Pegylated G old Nanoparticles (GP NPs ) As before, the same protoc ol for particle and tissue digestion was followed for the pegylated gold NPs (GP). A reference curve for gold NPs was generated (Figure 5 1 0 ). The PK data obtained from the ICP MS from gold quantification is shown in Figure 51 1 The PK data of the blood s amples showed significant amount of gold in the blood even after 1440 minutes. The GP NPs had a half -life of 640 + 15.5 minutes The half -life values for all five NPs used for this study are shown in Figure 5 12. 5.4 Differential Binding of Serum Protei ns to Nanoparticles ( NPs ) Recently, it has been reported (Cedervall et al. 2007, Lundqvist et al. 2008, Lynch et al. 2007, Lynch & Dawson 2008) th at when particles enter the physiological environment, they are immediately covered by proteins that play a critical role in determining biodistribution, clearance and inflammatory processes. A s mentioned before, many such proteins known as opsonins can facilitate particle recognition and trigger phagocytosis by the cells of the reticuloendothelial system (RES) leading to particle removal from the blood often within seconds. This may be followed by either a relatively benign response or stimulation of the inflammatory system that can eventually lead to various particle -induced diseases. In order to assess the biocompatibility of NPs, it is important to understand the proteins that bind to the sur face of the NPs. The
146 determination of the proteins that bind (Dutta et al. 2007) to the particle surface can be achieved using one dimensional (1 -d) and two -dim ensional (2 d) gel electrophoresis and isotope -coded affinity tags (ICAT). Various investigators have reported the used of 2 d electrophoresis of adsorbed proteins to characterize the pattern protein NP binding and the ICAT technique to identify and quanti fy human serum proteins adsorbed onto particles (Wasdo et al. 2008) Mo st of these techniques are extremely complex and are beyond the scope of this dissertation. Therefore, the 1 d gel electrophoresis process was selected to get a preliminary idea of the molecular weight of the proteins that bind to the surface of the QDS, Q DSG and the gold NPs dispersed in rat serum. The results of the 1 d gel electrophoresis are shown in Figure 5.12. Stock solutions for QDS and QDSG NPs were prepared according to published protocols (Wasdo et al. 2008) 100 mgs of QDS, QDSG, G+, G and GP NPs were placed in 50 mL centrifuge tubes along with 1 mL of PBS. The t ubes are sonicated and vortexed for 5 minutes respectively and the NP suspensions of QDS and QDSG were transferred to the reservoir of 300 mL 0.22 m polysulfone centrifuge filters (Whatman Catalog No. 66107169). The spin filters were centrifuged down usi ng a swinging bucket centrifuge at 42 X g for 30 minutes, till the entire PBS solution had passed completely through the filter. The NPs were trapped inside the filter reservoir to form a column of approximately 5 mm in height. A 0.5 mL aliquot of pooled rat serum (Innovative Research, Novi, MI) was transferred to the NP column and the spin filter was centrifuged at 100 X g for 30 minutes until the serum had completely passed through the filter. After the first pass, the serum filtrate was recovered and mad e to pass through the column for a second time. The column was subsequently rinsed four times with PBS to remove all residual serum. Following this, the NPs were taken to a 1.5 mL centrifuge tube using three 0.5
147 mL volumes of PBS. The particles were then p elleted by centrifuging at 4000 X g and the supernatant was discarded. As the G+, G and GP NPs were reasonably well dispersed and stable in water, they could not be retained in the 0.22 m centrifuge filters In order to make the NP column, the gold NPs were taken in a 1.5 mL centrifuge tube, and centrifuged at 8000 X g, and the supernatant was carefully discarded. The pelleted NPs were rinsed by resuspending them in PBS, re centrifugatin g at 8000 X g, and discarding the supernatant. This process is repeated for four times, followed by the addition of 0.5 mL filtered pooled serum to the NPs, and final re suspension of the NPs in the serum by vortexing. The centrifuge tubes are placed on a shaker (Innova 2000 oscillating platform shaker) and agitated for 30 minutes at 200 rpm. Following this, the tube was centrifuged at 8000 X g, and the supernatant was discarded. The NPs were suspended in 0.5 mL of fresh PBS and centrifuged again at 8000 X g, and the supernatant was discarded. This rinsing is repeated four times to a point that there should be no more protein left in the supernatant, as verified by using the bichrinninc acid (BCA) protein assay. For protein desorption, all NPs are resuspen ded in 0.3 mL of 1% sodium dodecyl sulphate (SDS) in 0.1 M tris buffer, pH 8.5 and sonicated and vortexed for 5 minutes respectively a fter the final rinse Following this, the NPs are centrifuged down at 8000 X g for 30 minutes and the supernatant was tran sferred to another fresh 1.5 mL with an automatic pippetter. Acidified acetone was added to the supernatant and the tube was left undisturbed for 20C. The precipitated proteins were centrifuged and removed and washed with 1 mL of acetone and stored at 80C, until using it for loading the gels to conducting the 1 d gel electrophoresis. Thereafter, the gels were loaded with the protein ladder, QDS, G+, G and QDSG NPs as shown in Figure 5 .13. The set up was left undisturbed for the next 8 hours until the completion of electrophoresis.
148 Finally, the gels were removed from the set up, scanned and analyzed. It is observed from Figure 5 .13, that a comparatively higher amount of proteins are located in lanes (c) and (d) that were loaded with samples containing adsorbed proteins on G+ and G NPs respectively. A significantly lesser quantity of proteins were observed in lanes containing the QDS and QDSG NPs as seen in lanes (b) and (d). The amount of adsorbed proteins samples containing GP was not sufficient for b eing introduced into any lane during 1 -d gel electrophoresis. These results from the 1 -d gel experiment on varying protein adsorption on the surface of NPs appear to corroborate the results of half -life behavior of the NP s obtained from the blood clearance studies 5. 5 Discussions The half life behavior of GP NPs was completely different from the other two gold NPs and the QDS and QDSG NPs. This confirms the effect of pegylation reported in literature for various NPs (Chapter 1). In Chapter 3, it was re ported that pegylation of the surface of bare gold NPs resulted in a reduction of surface charge to near neutral values. This indicated that the PEG functional group was successfully attached to the surface of the gold NPs. The large half -life value of GP NPs when viewed in the context of the small half life values of G+ and G clearly indicates that the GP NPs clearance from the rat blood stream was significantly delayed. This behavior can be attributed to the fact that PEG modification of the particle su rface that have been shown to reduce the non-specific binding of particles to the blood proteins (opsonins) which is the main mechanism for the partitioning of NPs out of the blood (Moghimi & Hunter 2001, Moghimi & Szebeni 2003, Owens & Peppas 2006) This imparts stealth properties to the GP NPs that help them to evade blood clearance for a much longer time, whereas, the G+ and G NPs are removed from the blood possibly due to the combined processes of rapid opsonization followed by phagocytosis.
149 It was also found that the half life values of QDSG and QDS NPs in the rat blood are similar. This implies that the gold speckles added to the surface of the QDS NPs do not significantly change the biological response of the se particle s in blood. As observed in Chapter 3, the addition of gold speckles did not significantly change the surface charge of the QDS NPs as compared to QDSG NPs Thus, even though the bare gold NPs (G ) were rapidly cleared from the rat blood stream, this same response could not be observed in the case of gold speckled surface of the QDSG NPs. Therefore, the blood clearance behavior of QDSG NPs was more comparable to the QDS NPs than that of gold NPs. The composition of the QDSG NPs consisted of 2 7 .4 % cadmium sulphide, 16.8 % zinc sulphide, 4.39% gold and the remaining being silica Therefore, it was not a surpris e that the e ffect of the quantum dot core was more significant than gold in determining the particokinetic behavior of QDSG NPs in rat blood. It wa s interesting to observe that even though G+ and G NPs have a large positive and a large negative surface charge values, respectively, they both were cleared very rapidly from the blood stream. One of the trends that can be observed from the half life values of all the five NPs is that the parti cles with a large surface charge (either positive or negative) are removed rapidly from the blood stream when compared to the particles with surface charge values close to zero (GP, QDSG and QDS). This clearly confirms the hypothesis of this research effort, that the particle surface can be altered to tailor engineered NPs for specific bioimaging applications. The half life results obtained from the animal study corroborat ed the findings o f the protein adsorption study using 1 d gel electrophoresis. The am ount and molecular weights of the different proteins that adsorbed on the surface of NPs could be visi ually observed from the gel in Figure 5 .13 It was noticed that GP could not be load ed into the lanes of the gel due to insufficient quantity of adsorbed proteins in the samples In the animal study, it wa s noticed that
150 the half life value of GP was very high due to its delayed blood clearance probably due to the inability of the opsonin proteins to adsorb and initiate the process of phagocytosis. The adso rbed proteins for G+ and G NPs were observed to be very high (Figure 5 .13 ), which corroborated the animal study data showing an extremely fast clearance from rat blood. Finally, QDS and QDSG had an intermediate amount of adsorbed proteins, and a blood cle arance rate that was higher than G+ and G but far lesser than GP. One of the major conclusions from this chapter is that protein adsorption appears to play a crucial role in regulating the blood clearance behavior of the NPs. The physicochemical paramete rs of NPs ( particle size, shape and surface charge and chemistry ) have a secondary and indirect effect on the biological activity. The primary effect can be attributed to the adsorbed proteins that contribute towards the blood partitioning behavior Regard ing the indirect effect of the physicochemical parameters, general ly it is known that opsonization of NP s with hydrophobic groups on the surface occur more quickly than NP s with hydrophilic surface groups like PEG due to enhanced adsorbability of blood se rum proteins on the surfaces (Carstensen et al. 1992, Muller et al. 1992, Norman et al. 1992) The actual role of the adsorbed prote ins in governing the activity can be explained only after identification followed by monitoring any conformational or chemical changes in the protein structure This would involve sophisticated proteomic research and detailed analysis which is included as a future recommended work.
151 Table 5 1 A ctual dose of nanoparticles administered to each rat
152 Table 5 2 Amount (mass) of blood collected from rats dosed with nanoparticles at pre determined time -points (0, 5, 15, 30, 60, 180, 360 and 1440 minutes)
153 Ta b le 5 2 Continued.
154 Table 5 2 Continued.
155 Table 5 3 Observations from d ispersion s tudy
156 Table 5 4 Details of n anoparticles
157 Table 5 5 Nanoparticle deposition on polyurethane (PU) cannulae tube
158 Table 5 6 Nanoparticle deposition on p olyethylene (PE) cannulae tube
159 Table 5 7 Quantitative data of analytical detection of blood samples collected from rats
160 Table 5 8 Preparation of n anoparticle s tock s olution
161 Figure 5 1 Effect of increasing the aqua regia concentration in dig ested samples with no nanoparticles in acid matrix (marked in b lue square s) and in blood matrix ( marked in red diamonds ).
162 Figure 5 2 Effect of increasing the aqua regia concentration with gold nanoparticles in digested samples ( marked in blue di amonds) in acid matrix and ( marked in red squares ) in blood matrix
163 Figure 5 3 Effect of increasing aqua regia concentration on the background signal (blue) and completion of nanoparticle digestion (red)
164 Figure 5 4 Standard curve for cadmi um generated using spiked samples of QDS nanoparticles Figure 5 5 Particle concentration of QDS nanoparticles in blood versus time of draw; inset shows data plotted up to 60 minutes to magnify the datapoints near the axes
165 Figure 5 6 Standard curve for cadmium generated using spiked samples of QDSG nanoparticles Figure 5 7 Particle concentration of QDSG nanoparticles in blood versus time of draw; inset shows data plotted up to 60 minutes to magnify the datapoints near the axes
166 Figure 5 8 Standa rd curve of gold generated using spiked samples of G+ nanoparticles Figure 5 9 Particle concentration of G+ nanoparticles in blood versus time of draw; inset shows data plotted up to 60 minutes to magnify the datapoints near the axes
167 Figure 5 1 0 St andard curve for gold generated using spiked samples of GP nanoparticles Figure 5 11. Particle concentration of GP nanoparticles in blood versus time of draw; inset shows data plotted up to 60 minutes to magnify the datapoints near the axes
168 1 10 100 1000 QDSG QDS G+ GGP Half-life Values [minutes] < 5 min?? < 5 min?? 14 + 6.5 12.5 + 9.7 648 + 15.5 Figure 5 1 2 Histogram showing the half -life values of QDS G, QDS, G+ G and G P NPs
169 Figure 5 1 3 Results from the one -d imensional gel electrophoresis experiment on differential protein binding on the nanoparticles The samples in the lanes are as follows (a) l adder (with protein molecular weight bands (b) QDS (c) G+ (d) G (e) QDSG NPs Molecular Weight of Protein (kDa) 250 150 100 75 50 37 25 20 15 10 (a) ( b ) ( c ) ( d ) ( e )
170 CHAPTER 6 CONCLUSIONS AND SUGGESTIONS FOR FUTURE WORK 6 .1 Conclusions Nanoparticle (NP) -based contrast agents have initiated a whole new class of robust nano sized (between 1 and 100 nm) particulate materials that have been shown to have immense potential for bioimaging applications. Some of the major advantages of the NP based contrast agents being improved sensitivity, enhanced brightness and photostability with a small siz e (approximately 2 to 3 orders of magnitude smaller than the cells), making them suitable for use as contrast agents inside the cells. However, till date there exists no clinical contrast agent with an added therapeutic functionality in the structure. Ther e is a significant clinical need for the development of NPs with in -built imaging and therapeutic functionalities, for early cancer detection and therapeutic applications. Furthermore, in order to establish a contrast agent, it is crucial to understand the various factors that influence NP uptake in vitro and biodistribution behavior in vivo. A suite of novel engineered NP systems for bioimaging and therapeutic applications were developed for this present study and the influence of certain physicochemical parameters of the NPs like particle size, material composition, surface charge and chemistry on the cellular uptake and blood clearance behavior was investigated. The different NPs developed for this research effort include: 100 nm and 190 nm fluorescent s ilica nanoparticles (FSNPs), 15 nm silica coated quantum dots (QDS) and gold spec kled silica coated quantum dots (QDSG) and 15 nm gold NPs with three different surface charge and functionalization. The particles were synthesized using reverse -microemulsion and/or sol gel techniques and characterized for particle size, shape, surface charge absorbance and fluorescence spectroscopy.
171 The effect of particle size, time of incubation, particle concentration and surface functionality on the cellular uptake was s tudied using the 100 and 190 nm FSNPs. The effect of two different FSNP -surface functionalizations, folate conjugation and amine modification was studied using A549 cells. Uptake studies using confocal microscopy (qualitative) and protein assay (semi quant itative) techniques showed that the cellular uptake increased with increasing particle size and concentration. There was no significant effect of increasing the incubation time on the cellular uptake. The folated FSNPs were uptaken more aggressively by the A549 cells than the aminated FSNPs. The cellular uptake for the 100 nm aminated NPs was observed to be anomalously low as compared to the other FSNPs. This anomaly was explained from results of particle size measurements of NPs dispersed in cell media. It was found that the 100 nm FSNPs agglomerated in the cell media to have a size range between 40 nm and 20 microns. Similarly, the size range of the 190 nm FSNPs in cell media was 2 to 200 microns. Increased settling and cell NP interactions due to agglomer ation in the cell media for the larger sized FSNPs, along with an increase in the possibility of phagocytosis could lead to increased uptake behavior. Cell viability studies of the FSNPs were conducted using lactate dehydrogenase (LDH) assay in A549 cells. The maximum percentage cytotoxicity value for FSNPs that were pre -conditioned with cell media prior to their incubation, was found to be less than 8% whereas for FSNPs that were not pre conditioned in cell media it was approximately 20%. The decrease i n cytotoxicity upon pre conditioning was attributed to protein adsorption on the particle surface that influences the biological activity inside the cells. In order to investigate the effect of material composition and surface functionality of the NPs on t he blood clearance behavior, rats were dosed with pre -determined amounts of QDSG, QDS and gold NPs Gold NPs with three different surface charges: bare gold G (negative),
172 amine -modified gold G+, (positive) and pegylated-gold GP (neutral) NPs were synthesi zed to study the effect of surface charge. The QDSG, QDS and bare gold NPs were used to study the effect of material composition on their partitioning in blood. The synthesis protocol for QDSG was optimized for fluorescence and absorbance properties. It was observed that an excess of gold speckles on the QDSG surface quenched the QD-core fluorescence property. The amount of gold speckles on the NP surface and the thickness of the silica shell were optimized to obtain good fluorescent and absorbance properties. Bioimaging capability of QDSG was demonstrated in A549 cells and daphnia The fluorescence from the QD core was utilized for imaging and hyperthermic property of gold speckles on QDSG was demonstrated using A549 cells. The biodistribution studies of gold and quantum dot based NPs were conducted in rat blood. An acid digestion protocol using an optimized amount of aqua regia (5%) and nitric acid (2%) was established to ensure complete particle digestion with minimum background from the matrix. A one -compartmental model was generated using equations from first -order kinetics and was employed to calculate the particle concentration for dosing the rats. Stock solutions of NPs were prepared at 1 m g/ mL and dispersed in 0.45% bacteriostatic solution for dosing the rats for the blood clearance study. The rats were intravenous injected with NPs through the surgically attached cannulae tubes and samples of blood were collected at various time points. The blood samples were acid digested and analyzed for gold and cadmium using Inductively Coupled Plasma -Mass Spectroscopy. A particle concentration time profile graph was generated from the analytical data. This graph along with the one -compartmental model was used to calculate the half life values of the NPs in rat blood. The half -life values for the various NPs were determined to be: 12.59.7, 146.5 and 64015.5 minutes for QDS, QDSG and GP NPs were respectively.
173 The G+ and G NPs were cleared very fast and no significant amount of gold could be detected in the blood samples collected at the five minute time point. It was concluded that the half -life values for G+ and G NPs in rat blood w ere less than five minutes. QDSG and QDS NPs with relatively small surface charges of 6.3 2.9 mV and 4.1 1.7 mV respectively were cleared slower as compared to G+ and G NPs with surface charges of 24 5.8 mV and 34.5 7.8 mV respectively The PEG groups on GP (5 1.9 mV) led to delayed clearance with a large half -life value of more than 600 minutes. In order to furthe r investigate the trends of half -life of NPs in blood, o ne -dimensional gel electrophoresis preliminary protein adsorption studies were conducted. It showed a significantly high amount of adsorbed proteins on the G+ and G NP surface, followed by moderate a mount of proteins on the QDS and QDSG NP surface, and negligible amount on the GP surface. This trend of adsorbed proteins on the NP surface correlated directly with the results from the blood clearance study and half life of NPs in rat blood It is known that when particles enter the physiological environment, they are likely to be covered by proteins that exist in the blood. Many of these proteins, also known as opsonin proteins when adsorb ed on the NP surface, can render the NP to be visible to the pha gocytic cells and trigger their phagocytosis ( process of engulfing the NP and attempt ing to destroy or remove the particle from the bloodstream ). The opsonin proteins are continuously present inside the blood and are believed to come into contact with NPs typically as a result of random Brownian motion. However, once the proteins are close enough to the NP surface, several other attractive forces including the van der Waals, electrostatic, ionic and hydrophilic/hydrophobic interactions are involved for opso nin protein binding on the NP surface (Moghimi & Szebeni 2003, Owens & Peppas 2006) The adsorbed proteins play a crucial role in phagocytosis since
174 the phagocytes do not recognize the NPs or any other foreign material, without the presence of the opsonin proteins attached to the NP surfa ce. The last step in the clearance process, following ingestion of the NPs into the phagocytes is the secretion of various enzymes and oxidative reactive chemical like nitric oxide, hydrogen peroxide and superoxides to break down the phagocytosed NPs. Non -biodegradeable material s cannot be removed by this process and depending on their relative size and molecular weights shall be removed either by the renal system or uptaken and stored in one of the organs of the reticuloendothelial system. The tissue up take of the NPs can be investigated through biodistribution studies in relevant tissues inside living systems. 6. 2 Suggestions for Future Work The biodistribution of QDSG, QDS and gold NPs in rats sh ould be carried out by quantifying the particle content in liver, spleen, lung and kidney. The effect of material composition and surface functionalization on tissue uptake and determination of half life values for the NPs in various tissues would enable the development of a computational model similar to a phy siologically based pharmacokinetic (PBPK) model. This would help establish the complete particokinetic behavior of the NPs in the animal environment. One of the major conclusions of this research effort is that the adsorped proteins on the NP surface can i nfluence the biological activity in the physiological environment. For the current research work, a preliminary protein adsorption study using one -dimensional gel electrophoresis was conducted in this regard. More detailed proteomic studies will help to be tter understand the exact mechanism of interaction between the cells and NPs This understanding will help to tailor the NPs to mitigate the toxicological effects of NPs and help in targeted bioimaging and therap eutic applications
175 Most importantly, robust characterization protocols have to be developed in order to investigate the physicochemical parameter of NPs inside the biological environment. The biological responses are related to the physicochemical parameters of the NPs as encountered by the cells i nvitro or in vivo. Nanoparticle characterization in blood still remains a challenge and a robust methodology to accurately investigate the physicochemical parameters of NPs in blood has to be developed. The state of agglomeration of the NPs in the physiological environment will provide an insight to the actual effect of particle size distribution on NP toxicity. In future, different methods for surface functionalization of QDSG NPs should be explored. Further, t he surface of the NPs could be bio conjugated w ith fol ic acid or other bio molecules to selectively target cancer cells. This concept was demonstrated using folated FSNPs to selectively target cancer cells for this research work
176 LIST OF REFERENCES Akerman ME, Chan WCW, Laakkonen P, Bhatia SN, Ruoslahti E. 2002. Nanocrystal targeting in vivo. Proceedings of the National Academy of Sciences of the United States of America 99: 1261721 Alexis F, Pridgen E, Molnar LK, Farokhzad OC. 2008. Factors affecting the clearance and biodistributi on of polymeric nanoparticles. Molecular Pharmaceutics 5: 505 15 Antony AC. 1992. The Biological Chemistry of Folate Receptors. Blood 79: 280720 Bajpai SK, Mohan YM, Bajpai M, Tankhiwale R, Thomas V. 2007. Synthesis of polymer stabilized silver and gold nanostructures. Journal Of Nanoscience And Nanotechnology 7: 29943010 Banerjee T, Mitra S, Singh AK, Sharma RK, Maitra A. 2002. Preparation, characterization and biodistribution of ultrafine chitosan nanoparticles. International Journal of Pharmaceutics 243: 93105 Barnes CA, Elsaesser A, Arkusz J, Smok A, Palus J, et al. 2008. Reproducible Comet assay of amorphous silica nanoparticles detects no genotoxicity. Nano Letters 8: 306974 Ben -Ari ET. 2003. Nanoscale quantum dots hold promise for cancer applicat ions. Journal of the National Cancer Institute 95: 5024 Bhushan B. 2007. Springer Handbook of Nanotechnology : Springer Blodgett K, Langmuir I. 1932. Accommodation coefficient of hydrogen, a sensitive detector of surface films. PHYSICAL REVIEW 40: 78 104 Blodgett K, Langmuir I, Volume: S. 1932. A film which adsorbs atomic H and does not adsorb H 2. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 54: 37812 Borm P, Klaessig FC, Landry TD, Moudgil B, Pauluhn J, et al. 2006. Research strategies for safety evaluation of nanomaterials, Part V: Role of dissolution in biological fate and effects of nanoscale particles. Toxicological Sciences 90: 2332 Bornhop DJ, Contag CH, Licha K, Murphy CJ. 2001. Advances in contrast agents, reporters, and detection. Journal of Biomedical Optics 6: 10610 Bremer C, Ntziachristos V, Weissleder R. 2003. Optical -based molecular imaging: contrast agents and potential medical applications. European Radiology 13: 23143 Brigger I, Dubernet C, Couvreur P. 2002. Nanoparticles in cancer therapy and diagnosis. Advanced Drug Delivery Reviews 54: 63151
177 Bright RM, Walter DG, Musick MD, Jackson MA, Allison KJ, Natan MJ. 1996. Chemical and electrochemical Ag deposition onto preformed Au colloid monolayers: Approaches to uniformly -sized surface featur es with Ag like optical properties. Langmuir 12: 8107 Brown DM, Stone V, Findlay P, MacNee W, Donaldson K. 2000. Increased inflammation and intracellular calcium caused by ultrafine carbon black is independent of transition metals or other soluble compone nts. Occupational and Environmental Medicine 57: 68591 Brown SC, Kamal M, Nasreen N, Baumuratov A, Sharma P, et al. 2007. Influence of shape, adhesion and simulated lung mechanics on amorphous silica nanoparticle toxicity. Advanced Powder Technology 18: 6979 Bruchez M, Moronne M, Gin P, Weiss S, Alivisatos AP. 1998. Semiconductor nanocrystals as fluorescent biological labels. Science 281: 20136 Carstensen H, Muller RH, Muller BW. 1992. Particle Size, Surface Hydrophobicity And Interaction With Serum Of Parenteral Fat Emulsions And Model Drug Carriers As Parameters Related To Res Uptake. Clinical Nutrition 11: 28997 Cauvel A, Brunel D, DiRenzo F, Garrone E, Fubini B. 1997. Hydrophobic and hydrophilic behavior of micelle templated mesoporous silica. Langm uir 13: 27738 Cedervall T, Lynch I, Lindman S, Berggard T, Thulin E, et al. 2007. Understanding the nanoparticle protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles. Proceedings of the National Academy of Sciences of the United States of America 104: 20505 Champion JA, Mitragotri S. 2006. Role of target geometry in phagocytosis. Proceedings of the National Academy of Sciences of the United States of America 103: 49304 Chan WCW, Nie SM. 1998. Quantum dot b ioconjugates for ultrasensitive nonisotopic detection. Science 281: 20168 Chithrani BD, Chan WCW. 2007. Elucidating the mechanism of cellular uptake and removal of protein -coated gold nanoparticles of different sizes and shapes. Nano Letters 7: 154250 Ch ithrani BD, Ghazani AA, Chan WCW. 2006. Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells. Nano Letters 6: 6628 Cho SJ, Maysinger D, Jain M, Roder B, Hackbarth S, Winnik FM. 2007. Long-term exposure to CdTe quantum dots causes functional impairments in live cells. Langmuir 23: 197480 Cohen ML. 2001. Nanotubes, nanoscience, and nanotechnology. Materials Science & Engineering C -Biomimetic And Supramolecular Systems 15: 111 Committee. 2004. Final Report: Nanotoxicology Workshop, University of Florida, Gainesville, FL
178 Copland JA, Eghtedari M, Popov VL, Kotov N, Mamedova N, et al. 2004. Bioconjugated gold nanoparticles as a molecular based contrast agent: Implications for imaging of deep tumors using optoacoustic tomog raphy. Molecular Imaging and Biology 6: 3419 Craighead JE. 1988. Diseases Associated with Exposure to Silica and Nonfibrous Silicate Minerals. Archives of Pathology & Laboratory Medicine 112: 673720 Craighead JE. 1992. Do silica and asbestos cause lung c ancer? Arch Pathol Lab Med 116: 1620 Cullen RT, Tran CL, Buchanan D, Davis JMC, Searl A, et al. 2000. Inhalation of poorly soluble particles. I. Differences in inflammatory response and clearance during exposure. Inhalation Toxicology 12: 1089111 Dabbou si BO, RodriguezViejo J, Mikulec FV, Heine JR, Mattoussi H, et al. 1997. (CdSe)ZnS core -shell quantum dots: Synthesis and characterization of a size series of highly luminescent nanocrystallites. Journal of Physical Chemistry B 101: 946375 Das M, Patil S Bhargava N, Kang JF, Riedel LM, et al. 2007. Auto-catalytic ceria nanoparticles offer neuroprotection to adult rat spinal cord neurons. Biomaterials 28: 191825 De M, Ghosh PS, Rotello VM. 2008. Applications of Nanoparticles in Biology. Advanced Materia ls 20: 422541 Decker SP. 2006. Current and emerging applications involving nanoscale fillers and minerals. In Functional Fillers And Nanoscale Minerals: New Markets/New Horizons ed. JJ Kellar: Society of Mining Engineers Del Campo A, Sen T, Lellouche JP, Bruce IJ. 2005. Multifunctional magnetite and silica magnetite nanoparticles: Synthesis, surface activation and applications in life sciences. Journal of Magnetism and Magnetic Materials 293: 3340 Derfus AM, Chan WCW, Bhatia SN. 2004a. Intracellular deli very of quantum dots for live cell labeling and organelle tracking. Advanced Materials 16: 961+ Derfus AM, Chan WCW, Bhatia SN. 2004b. Probing the cytotoxicity of semiconductor quantum dots. Nano Letters 4: 118 Derjaguin BV, Abrikosova, II, Lifshitz EM. 1992. Direct Measurement Of Molecular Attraction Between Solids Separated By A Narrow Gap (Reprinted From Quart Rev, Vol 10, Pg 295329, 1956). Progress In Surface Science 40: 83117 Diagaradjane P, Shetty A, Wang J, Schwartz J, Park HC, et al. 2008. Modulation of in vivo tumor radiation response via vascular -focused hyperthermia characterizing gold nanoshells as integrated anti hypoxic and localized vascular disrupting agents. International Journal of Radiation Oncology Biology Physics 72: S64S
179 Ding LH, Stilwell J, Zhang TT, Elboudwarej O, Jiang HJ, et al. 2005. Molecular characterization of the cytotoxic mechanism of multiwall carbon nanotubes and nano onions on human skin fibroblast. Nano Letters 5: 244864 Dobrovolskaia MA, Aggarwal P, Hall JB, McNeil SE. 2008. Preclinical studies to understand nanoparticle interaction with the immune system and its potential effects on nanoparticle biodistribution. Molecular Pharmaceutics 5: 48795 Donaldson K, Borm PJA. 1998. The quartz hazard: A variable entity. Ann als of Occupational Hygiene 42: 28794 Donaldson K, Stone V, Tran CL, Kreyling W, Borm PJA. 2004. Nanotoxicology. Occupational and Environmental Medicine 61: 7278 Drexler E. 1987. Engines of Creation: The Coming Era of Nanotechnology New York: Anchor Boo ks Edition Driscoll KE, Carter JM, Howard BW, Hassenbein DG, Pepelko W, et al. 1996. Pulmonary inflammatory, chemokine, and mutagenic responses in rats after subchronic inhalation of carbon black. Toxicology and Applied Pharmacology 136: 37280 Driscoll K E, Guthrie GD. 1997. Crystalline Silica and Silicosis New York: Elsevier. 37391 pp. Dubertret B, Skourides P, Norris DJ, Noireaux V, Brivanlou AH, Libchaber A. 2002. In vivo imaging of quantum dots encapsulated in phospholipid micelles. Science 298: 175962 Dutta D, Moraga D, Santra S. In Review for Publication. In vitro biocompatibility assessment of folate -conjugated silica nanoparticles. Materials Science & Engineering C Dutta D, Moudgil BM. 2007. Crystalline Silica Particles Mediated Lung Injury. Kon a Powder and Particle 25: 7687 Dutta D, Sundaram SK, Teeguarden JG, Riley BJ, Fifield LS, et al. 2007. Adsorbed proteins influence the biological activity and molecular targeting of nanomaterials. Toxicological Sciences 100: 30315 Eichelbaum M, Schmidt BE, Ibrahim H, Rademann K. 2007. Three photon -induced luminescence of gold nanoparticles embedded in and located on the surface of glassy nanolayers. Nanotechnology 18 Eijkel JCT, van den Berg A. 2006. The promise of nanotechnology for separation devices from a top down approach to nature -inspired separation devices. Electrophoresis 27: 67785 El Sayed IH, Huang XH, El -Sayed MA. 2005. Surface plasmon resonance scattering and absorption of anti -EGFR antibody conjugated gold nanoparticles in cancer diagnost ics: Applications in oral cancer. Nano Letters 5: 82934
180 Elghanian R, Storhoff JJ, Mucic RC, Letsinger RL, Mirkin CA. 1997. Selective colorimetric detection of polynucleotides based on the distance -dependent optical properties of gold nanoparticles. Scienc e 277: 107881 Espinosa -Mansilla A, Duran -Meras I, Salinas F. 1998. Highperformance liquid chromatographic fluorometric determination of glyoxal, methylglyoxal, and diacetyl in urine by prederivatization to pteridinic rings. Analytical Biochemistry 255: 26373 Evans CO, Reddy P, Brat DJ, O'Neill EB, Craige B, et al. 2003. Differential expression of folate receptor in pituitary adenomas. Cancer Research 63: 421824 Fang XH, Liu XJ, Schuster S, Tan WH. 1999. Designing a novel molecular beacon for surface im mobilized DNA hybridization studies. Journal of the American Chemical Society 121: 29212 Ferin J, Oberdorster G, Penney DP, Soderholm SC, Gelein R, Piper HC. 1990. Increased Pulmonary Toxicity of Ultrafine Particles.1. Particle Clearance, Translocation, M orphology. Journal of Aerosol Science 21: 3814 Ferrari M. 2005. Cancer nanotechnology: Opportunities and challenges. Nature Reviews Cancer 5: 16171 Feynman R. 1959. Plenty of Room at the Bottom. ed. APS meeting. Caltech Finney LA, O'Halloran TV. 2003. T ransition metal speciation in the cell: Insights from the chemistry of metal ion receptors. Science 300: 9316 Franklin WA, Waintrub M, Edwards D, Christensen K, Prendegrast P, et al. 1994. New Anti Lung -Cancer Antibody Cluster 12 Reacts With Human Folate Receptors Present On Adenocarcinoma. International Journal Of Cancer : 89 95 Frost RL, Cejka J, Weier ML, Martens W. 2006. Molecular structure of the uranyl silicates a Raman spectroscopic study. Journal of Raman Spectroscopy 37: 53851 Fubini B. 1997. S urface reactivity in the pathogenic response to particulates. Environmental Health Perspectives 105: 101320 Gao XH, Cui YY, Levenson RM, Chung LWK, Nie SM. 2004. In vivo cancer targeting and imaging with semiconductor quantum dots. Nature Biotechnology 22: 96976 Gao XH, Nie SM. 2003. Molecular profiling of single cells and tissue specimens with quantum dots. Trends in Biotechnology 21: 3713 Gao XH, Yang LL, Petros JA, Marshal FF, Simons JW, Nie SM. 2005. In vivo molecular and cellular imaging with quantu m dots. Current Opinion in Biotechnology 16: 6372
181 Geoghegan WD, Ackerman GA. 1976. Electron-Microscopic Study Of Concanavalin-A (Con A) And Wheat Germ Agglutinin (Wga) Binding On Mouse Peritoneal Cells Using Colloidal Gold As An Indirect Marker. Journal O f The Reticuloendothelial Society 20: A1 -A Geoghegan WD, Scillian JJ, Ackerman GA. 1978. Detection Of Human Lymphocytes -B By Both Light And Electron -Microscopy Utilizing Colloidal Gold Labeled Anti Immunoglobulin. Immunological Communications 7: 1 12 Godt J, Scheidig F, Grosse -Siestrup C, Esche V, Brandenburg P, et al. 2006. The toxicity of cadmium and resulting hazards for human health. J Occup Med Toxicol 1: 22 Goodsell DS. 2004. Nanobiotechnology: Lessons from Nature : Wiley Liss Govorov AO, Richardson H H. 2007. Generating heat with metal nanoparticles. Nano Today 2: 303 8 Govorov AO, Zhang W, Skeini T, Richardson H, Lee J, Kotov NA. 2006. Gold nanoparticle ensembles as heaters and actuators: melting and collective plasmon resonances. Nanoscale Research L etters 1: 8490 Green M, Howman E. 2005. Semiconductor quantum dots and free radical induced DNA nicking. Chemical Communications : 1213 Grodzinski P, Silver M, Molnar LK. 2006. Nanotechnology for cancer diagnostics: promises and challenges. Expert Review Of Molecular Diagnostics 6: 30718 Grzelczak M, PerezJuste J, Mulvaney P, Liz -Marzan LM. 2008. Shape control in gold nanoparticle synthesis. Chemical Society Reviews 37: 178391 Guo JX, Zhang X, Li QN, Li WX. 2007. Biodistribution of functionalized multiw all carbon nanotubes in mice. Nuclear Medicine and Biology 34: 57983 Guo SJ, Wang EK. 2007. Synthesis and electrochemical applications of gold nanoparticles. Analytica Chimica Acta 598: 18192 Hamoir J, Nemmar A, Halloy D, Wirth D, Vincke G, et al. 2003. Effect of polystyrene particles on lung microvascular permeability in isolated perfused rabbit lungs: role of size and surface properties. Toxicology and Applied Pharmacology 190: 27885 Hardman R. 2006. A toxicologic review of quantum dots: Toxicity depe nds on physicochemical and environmental factors. Environmental Health Perspectives 114: 16572 Harper S, Usenko C, Hutchison JE, Maddux BLS, Tanguay RL. 2008. In vivo biodistribution and toxicity depends on nanomaterial composition, size, surface function alisation and route of exposure. Journal Of Experimental Nanoscience 3: 195206 Haugland RP. 2003. Handbook of fluorescent probes and research products Eugene, OR
182 He XX, Duan JH, Wang KM, Tan WH, Lin X, He CM. 2004. A novel fluorescent label based on orga nic dye -doped silica nanoparticles for HepG liver cancer cell recognition. Journal of Nanoscience and Nanotechnology 4: 5859 He XX, Nie HL, Wang KM, Tan WH, Wu X, Zhang PF. 2008. In Vivo Study of Biodistribution and Urinary Excretion of Surface -Modified S ilica Nanoparticles. Analytical Chemistry 80: 9597603 He XX, Wang KM, Tan WH, Li J, Yang XH, et al. 2002. Photostable luminescent nanoparticles as biological label for cell recognition of system lupus erythematosus patients. Journal of Nanoscience and Na notechnology 2: 31720 Hett A. 2004. Nanotechnology. Small matter, many unknowns. Swiss Reinsurance Company, Zurich Hiremath SRR, Hota A. 1999. Nanoparticles as drug delivery systems. Indian Journal of Pharmaceutical Sciences 61: 6975 Hirsch LR, Stafford RJ, Bankson JA, Sershen SR, Rivera B, et al. 2003. Nanoshell -mediated near -infrared thermal therapy of tumors under magnetic resonance guidance. Proceedings of the National Academy of Sciences of the United States of America 100: 1354954 Holm BA, Bergey EJ, De T, Rodman DJ, Kapoor R, et al. 2002. Nanotechnology in biomedical applications. Molecular Crystals and Liquid Crystals 374: 58998 Hood JD, Bednarski M, Frausto R, Guccione S, Reisfeld RA, et al. 2002. Tumor regression by targeted gene delivery t o the neovasculature. Science 296: 24047 Hoshino A, Fujioka K, Oku T, Suga M, Sasaki YF, et al. 2004a. Physicochemical properties and cellular toxicity of nanocrystal quantum dots depend on their surface modification. Nano Letters 4: 21639 Hoshino A, Ha naki K, Suzuki K, Yamamoto K. 2004b. Applications of T -lymphoma labeled with fluorescent quantum dots to cell tracing markers in mouse body. Biochemical and Biophysical Research Communications 314: 4653 Hu M, Wang X, Hartland GV, Salgueirino -Maceira V, Li z -Marzan LM. 2003. Heat dissipation in gold -silica core -shell nanoparticles. Chemical Physics Letters 372: 76772 Huang XH, El Sayed IH, Qian W, El -Sayed MA. 2006. Cancer cell imaging and photothermal therapy in the near infrared region by using gold nanor ods. Journal of the American Chemical Society 128: 211520 Huang XL, Zhang B, Ren L, Ye SF, Sun LP, et al. 2008. In vivo toxic studies and biodistribution of near infrared sensitive Au -Au2S nanoparticles as potential drug delivery carriers. Journal o f Mat erials Science -Materials In Medicine 19: 25818
18 3 Huff TB, Hansen MN, Zhao Y, Cheng JX, Wei A. 2007a. Controlling the cellular uptake of gold nanorods. Langmuir 23: 15969 Huff TB, Tong L, Zhao Y, Hansen MN, Cheng JX, Wei A. 2007b. Hyperthermic effects of go ld nanorods on tumor cells. Nanomedicine 2: 12532 Huo Q, Worden JG. 2007. Monofunctional gold nanoparticles: synthesis and applications. Journal Of Nanoparticle Research 9: 101325 IARC M. 1997. IARC Monograph on the Evaluation of the Carcinogenic Risk of Chemicals to Humans: Silica, some silicates, coal dust and paraaramid fibrils Geneva: IARC Press Ivanitsky GR, Medvinsky AB, Deev AA, Tsyganov MA. 1998. From Maxwell's Demon to the self -organization of mass transfer processes in living systems. Uspekhi Fizicheskikh Nauk 168: 122133 Jain TK, Reddy MK, Morales MA, Leslie -Pelecky DL, Labhasetwar V. 2008. Biodistribution, clearance, and biocompatibility of iron oxide magnetic nanoparticles in rats. Molecular Pharmaceutics 5: 31627 Jakobs S, Subramaniam V, Schonle A, Jovin TM, Hell SW. 2000. EGFP and DsRed expressing cultures of Escherichia coli imaged by confocal, two-photon and fluorescence lifetime microscopy. FEBS Letters 479: 1315 Jiang W, Papa E, Fischer H, Mardyani S, Chan WCW. 2004a. Semiconductor q uantum dots as contrast agents for whole animal imaging. Trends in Biotechnology 22: 6079 Jiang ZL, Pan HC, Yuan WE. 2004b. Change color effect and spectral properties of gold nanoparticle -cationic surfactants system. Chemical Research In Chinese Universi ties 20: 5238 Juang JY, Bogy DB. 2005. Nanotechnology advances and applications in information storage. Microsystem Technologies -Micro -And Nanosystems -Information Storage And Processing Systems 11: 9507 Karakoti AS, Hench LL, Seal S. 2006. The potential toxicity of nanomaterials The role of surfaces. Jom 58: 7782 Khan MK, Nigavekar SS, Minc LD, Kariapper MST, Nair BM, et al. 2005. In vivo biodistribution of dendrimers and dendrimer nanocomposites Implications for cancer imaging and therapy. Technology In Cancer Research & Treatment 4: 60313 Kim DK, Jo YS, Dobson J, Haj AE, Muhammed M. 2006. Nanomaterials for the controlled release of anti -cancer agent: Nanomaterials for Cancer Therapy : Wiley -VCH Kirchner C, Liedl T, Kudera S, Pellegrino T, Javier AM et al. 2005. Cytotoxicity of colloidal CdSe and CdSe/ZnS nanoparticles. Nano Letters 5: 331 8
184 Klostranec JM, Chan WCW. 2006. Quantum dots in biological and biomedical research: Recent progress and present challenges. Advanced Materials 18: 195364 Kobayashi H, Hama Y, Koyama Y, Choyke PL, Asanuma D, et al. 2008. In vivo real time monitoring of cancer cell viability using a molecularly targeted pH activatable fluorescent imaging probe. Proceedings of the American Association for Cancer Research Annual Me eting 49: 1077 Khler M, Fritzsche W. 2007. Nanotechnology: An Introduction to Nanostructuring Techniques Federal Republic of Germany: Wiley -VCH Langmuir I, Blodgett K. 1935. Regarding some new methods for the investigation of monomolecular layers. Kolloid -Zeitschrift 73: 25763 Leamon CP, Low PS. 1991. Delivery Of Macromolecules Into Living Cells A Method That Exploits Folate Receptor Endocytosis. Proceedings Of The National Academy Of Sciences Of The United States Of America 88: 55726 Leatherdale CA, Woo W -K, Mikulec FV, Bawendi MG. On the absorption cross section of CdSe nanocrystal quantum dots. Journal of Physical Chemistry B 106: 761922 Lee HA, Imran M, Monteiro -Riviere NA, Colvin VL, Yu WW, Rivlere JE. 2007. Biodistribution of quantum dot nanopa rticles in perfused skin: Evidence of coating dependency and periodicity in arterial extraction. Nano Letters 7: 286570 Levine EM, Robbins EB. 1970. Differential Temperature Sensitivity Of Normal And Cancer Cells In Culture. Journal Of Cellular Physiology 76: 373& Levy L, Sahoo Y, Kim KS, Bergey EJ, Prasad PN. 2002. Nanochemistry: Synthesis and characterization of multifunctional nanoclinics for biological applications. Chemistry of Materials 14: 371521 Lewen N, Mathew S, Schenkenberger M, Raglione T. 2004. A rapid ICP MS screen for heavy metals in pharmaceutical compounds. Journal of Pharmaceutical and Biomedical Analysis 35: 739 Li SD, Huang L. 2008. Pharmacokinetics and biodistribution of nanoparticles. Molecular Pharmaceutics 5: 496504 Licha K, Olbri ch C. 2005. Optical imaging in drug discovery and diagnostic applications. Advanced Drug Delivery Reviews 57: 1087108 Licha K, Riefke B, Ebert B, Grotzinger C. 2002. Cyanine dyes as contrast agents in biomedical optical imaging. Academic Radiology 9: S320S2 Limbach LK, Li YC, Grass RN, Brunner TJ, Hintermann MA, et al. 2005. Oxide nanoparticle uptake in human lung fibroblasts: Effects of particle size, agglomeration, and diffusion at low concentrations. Environmental Science & Technology 39: 93706
185 Liu M X, Li HF, Luo G, Liu QF, Wang YM. 2008. Pharmacokinetics and biodistribution of surface modification polymeric nanoparticles. Archives of Pharmacal Research 31: 54754 Liu P, Zhang A, Xu Y, Xu LX. 2005. Study of nonuniform nanoparticle liposome extravasat ion in tumour. International Journal of Hyperthermia 21: 25970 Loo C, Hirsch L, Lee MH, Chang E, West J, et al. 2005a. Gold nanoshell bioconjugates for molecular imaging in living cells. Optics Letters 30: 10124 Loo C, Lin A, Hirsch L, Lee MH, Barton J, et al. 2004. Nanoshell enabled photonics based imaging and therapy of cancer. Technology in Cancer Research & Treatment 3: 3340 Loo C, Lowery A, Halas N, West J, Drezek R. 2005b. Immunotargeted nanoshells for integrated cancer imaging and therapy. Nano Letters 5: 70911 Lovern SB, Owen HA, Klaper R. 2008. Electron microscopy of gold nanoparticle intake in the gut of Daphnia magna. Nanotoxicology 2: 438 Lovric J, Bazzi HS, Cuie Y, Fortin GRA, Winnik FM, Maysinger D. 2005a. Differences in subcellular distribution and toxicity of green and red emitting CdTe quantum dots. Journal of Molecular Medicine -Jmm 83: 37785 Lovric J, Cho SJ, Winnik FM, Maysinger D. 2005b. Unmodified cadmium telluride quantum dots induce reactive oxygen species formation leading to m ultiple organelle damage and cell death. Chemistry & Biology 12: 122734 Lundqvist M, Stigler J, Elia G, Lynch I, Cedervall T, Dawson KA. 2008. Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts. Proceedings of the National Academy of Sciences of the United States of America 105: 1426570 Lynch I, Cedervall T, Lundqvist M, Cabaleiro -Lago C, Linse S, Dawson KA. 2007. The nanoparticle protein complex as a biological entity; a complex fluid s and surface science challenge for the 21st century. Advances in Colloid and Interface Science 134 35: 16774 Lynch I, Dawson KA. 2008. Protein -nanoparticle interactions. Nano Today 3: 407 Magrez A, Kasas S, Salicio V, Pasquier N, Seo JW, et al. 2006. C ellular toxicity of carbon based nanomaterials. Nano Letters 6: 11215 Mann S, Shenton W, Li M, Connolly S, Fitzmaurice D. 2000. Biologically programmed nanoparticle assembly. Advanced Materials 12: 14750 Maye MM, Zheng WX, Leibowitz FL, Ly NK, Zhong CJ. 2000. Heatinginduced evolution of thiolate -encapsulated gold nanoparticles: A strategy for size and shape manipulations. Langmuir 16: 4907
186 Maysinger D, Lovric J, Eisenberg A, Savic R. 2007. Fate of micelles and quantum dots in cells. European Journal of Pharmaceutics and Biopharmaceutics 65: 27081 Medintz IL, Uyeda HT, Goldman ER, Mattoussi H. 2005. Quantum dot bioconjugates for imaging, labelling and sensing. Nature Materials 4: 43546 Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Doose S, et al. 2005. Quantum dots for live cells, in vivo imaging, and diagnostics. Science 307: 53844 Moghimi SM. 2006. The effect of methoxyPEG chain length and molecular architecture on lymph node targeting of immunoPEG liposomes. Biomaterials 27: 13644 Moghimi SM, Hun ter AC. 2001. Capture of stealth nanoparticles by the body's defences. Critical Reviews in Therapeutic Drug Carrier Systems 18: 52750 Moghimi SM, Szebeni J. 2003. Stealth liposomes and long circulating nanoparticles: critical issues in pharmacokinetics, o psonization and protein-binding properties. Progress in Lipid Research 42: 46378 Mrksich M. 2000. A surface chemistry approach to studying cell adhesion. Chemical Society Reviews 29: 26773 Muller RH, Wallis KH, Troster SD, Kreuter J. 1992. Invitro Charac terization Of Poly (Methyl Methacrylate) Nanoparticles And Correlation To Their Invivo Fate. Journal Of Controlled Release 20: 23746 Murray CB, Norris DJ, Bawendi MG. 1993. Synthesis and Characterization of Nearly Monodisperse Cde (E = S, Se, Te) Semicond uctor Nanocrystallites. Journal of the American Chemical Society 115: 870615 Nardin C, Thoeni S, Widmer J, Winterhalter M, Meier W. 2000. Nanoreactors based on (polymerized) ABA -triblock copolymer vesicles. Chemical Communications : 14334 Nayak S, Lyon LA 2005. Soft nanotechnology with soft nanoparticles. Angewandte Chemie International Edition 44: 7686708 Nel A, Xia T, Madler L, Li N. 2006. Toxic potential of materials at the nanolevel. Science 311: 6227 Niidome T, Yamagata M, Okamoto Y, Akiyama Y, Tak ahashi H, et al. 2006. PEG -modified gold nanorods with a stealth character for in vivo applications. Journal of Controlled Release 114: 3437 Norman ME, Williams P, Illum L. 1992. Human Serum -Albumin as a Probe for Surface Conditioning (Opsonization) of B lock Copolymer -Coated Microspheres. Biomaterials 13: 8419
187 O'Neal DP, Hirsch LR, Halas NJ, Payne JD, West JL. 2004. Photo -thermal tumor ablation in mice using near infrared absorbing nanoparticles. Cancer Letters 209: 1716 Oberdorster G. 1993. Lung Dosime try Pulmonary Clearance of Inhaled Particles. Aerosol Science and Technology 18: 27989 Oberdorster G. 2000. Toxicology of ultrafine particles: in vivo studies. Philosophical Transactions of the Royal Society of London Series a-Mathematical Physical and Engineering Sciences 358: 271939 Oberdorster G, Ferin J, Finkelstein G, Wade P, Corson N. 1990. Increased Pulmonary Toxicity of Ultrafine Particles.2. Lung Lavage Studies. Journal of Aerosol Science 21: 3847 Oberdorster G, Ferin J, Gelein R, Soderholm SC Finkelstein J. 1992. Role of the Alveolar Macrophage in Lung Injury Studies with Ultrafine Particles. Environmental Health Perspectives 97: 1939 Oberdorster G, Ferin J, Lehnert BE. 1994. Correlation between Particle -Size, in -Vivo Particle Persistence, and Lung Injury. Environmental Health Perspectives 102: 1739 Oberdorster G, Finkelstein J, Ferin J, Godleski J, Chang LY, et al. 1996. Ultrafine particles as a potential environmental health hazard. Studies with model particles. Chest 109: 68S 9S Oberdo rster G, Gelein RM, Ferin J, Weiss B. 1995. Association of Particulate Air Pollution and Acute Mortality Involvement of Ultrafine Particles. Inhalation Toxicology 7: 11124 Oberdorster G, Maynard A, Donaldson K, Castranova V, Fitzpatrick J, et al. 2005a Principles for characterizing the potential human health effects from exposure to nanomaterials: elements of a screening strategy. Part Fibre Toxicol 2: 8 Oberdorster G, Oberdorster E, Oberdorster J. 2005b. Nanotoxicology: An emerging discipline evolving from studies of ultrafine particles. Environmental Health Perspectives 113: 82339 Oberdorster G, Oberdorster E, Oberdorster J. 2005c. Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect 113: 82339 Owens DE, Peppas NA. 2006. Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles. International Journal of Pharmaceutics 307: 93102 Pan XQ, Lee RJ, Ratnam M. 2004. Penetration into solid tumor tissue of fluorescent latex microsph eres: A mimic of liposome particles. Anticancer Research 24: 3005 8 Pellegrino T, Kudera S, Liedl T, Javier AM, Manna L, Parak WJ. 2005. On the development of colloidal nanoparticles towards multifunctional structures and their possible use for biological applications. Small 1: 4863
188 Pepperkok R, Squire A, Geley S, Bastiaens PI. 1999. Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy. Current Biology: CB 9: 26972 Perez Juste J, Pastoriza -Santos I, Liz -Marzan LM, Mulvaney P. 2005. Gold nanorods: Synthesis, characterization and applications. Coordination Chemistry Reviews 249: 1870901 Peters K, Unger RE, Kirkpatrick CJ, Gatti AM, Monari E. 2004. Effects of nano-scaled particles on endothel ial cell function in vitro: Studies on viability, proliferation and inflammation. Journal Of Materials Science -Materials In Medicine 15: 3215 Pissuwan D, Cortie CH, Valenzuela SM, Cortie MB. 2007a. Gold nanosphere antibody conjugates for hyperthermal ther apeutic applications. Gold Bulletin 40: 121-9 Pissuwan D, Valenzuela SM, Cortie MB. 2006. Therapeutic possibilities of plasmonically heated gold nanoparticles. Trends in Biotechnology 24: 627 Pissuwan D, Valenzuela SM, Killingsworth MC, Xu XD, Cortie MB. 2007b. Targeted destruction of murine macrophage cells with bioconjugated gold nanorods. Journal of Nanoparticle Research 9: 110924 Pissuwan D, Valenzuela SM, Miller CM, Cortie MB. 2007c. A golden bullet? Selective targeting of toxoplasma gondii tachyzoit es using anti body-functionalized gold nanorods. Nano Letters 7: 380812 Pitsillides CM, Joe EK, Wei XB, Anderson RR, Lin CP. 2003. Selective cell targeting with light absorbing microparticles and nanoparticles. Biophysical Journal 84: 402332 Poland CA, D uffin R, Kinloch I, Maynard A, Wallace WAH, et al. 2008. Carbon nanotubes introduced into the abdominal cavity of mice show asbestos like pathogenicity in a pilot study. Nature Nanotechnology 3: 4238 Powers M. 2006. Nanomedicine and nano device pipeline surges 68%. In Nanomedicine, Device and Diagnostics Report; NanoBiotech News pp. 1 69 Qhobosheane M, Santra S, Zhang P, Tan WH. 2001. Biochemically functionalized silica nanoparticles. Analyst 126: 12748 Qu L, Peng X. 2002. Control of photoluminescence properties of CdSe nanocrystals in growth. Journal of the American Chemical Society 124: 204955 Renovanz HD. 1984. Amorphous Silica Pathogenicity And Toxicity. Staub Reinhaltung Der Luft 44: 21720 Renwick LC, Brown D, Clouter A, Donaldson K. 2004. Incr eased inflammation and altered macrophage chemotactic responses caused by two ultrafine particle types. Occupational and Environmental Medicine 61: 4427
189 Renwick LC, Donaldson K, Clouter A. 2001. Impairment of alveolar macrophage phagocytosis by ultrafine particles. Toxicology and Applied Pharmacology 172: 11927 Rhyner MN, Smith AM, Gao XH, Mao H, Yang LL, Nie SM. 2006. Quantum dots and multifunctional nanoparticles: new contrast agents for tumor imaging. Nanomedicine 1: 20917 Richardson HH, Hickman ZN, G ovorov AO, Thomas AC, Zhang W, Kordesch ME. 2006. Thermooptical properties of gold nanoparticles embedded in ice: Characterization of heat generation and melting. Nano Letters 6: 7838 Richardson HH, Thomas AC, Carlson MT, Kordesch ME, Govorov AO. 2007. Th ermo -optical responses of nanoparticles: Melting of ice and nanocalorimetry approach. Journal of Electronic Materials 36: 158793 Romig AD. 2004. Nanotechnology: Scientific challenges and societal benefits and risks. Metallurgical And Materials Transactions A Physical Metallurgy And Materials Science 35A: 36418 Roser M, Fischer D, Kissel T. 1998. Surface -modified biodegradable albumin nano and microspheres. II: effect of surface charges on in vitro phagocytosis and biodistribution in rats. European Journal Of Pharmaceutics And Biopharmaceutics 46: 25563 Rosi NL, Mirkin CA. 2005. Nanostructures in biodiagnostics. Chemical Reviews 105: 154762 Ross JF, Chaudhuri PK, Ratnam M. 1994. Differential Regulation Of Folate Receptor Isoforms In Normal And Malignant Tissues In -Vivo And In Established Cell Lines Physiological And Clinical Implications. Cancer 73: 243243 Ryman -Rasmussen JP, Riviere JE, Monteiro -Riviere NA. 2006. Penetration of intact skin by quantum dots with diverse physicochemical properties. Toxic ological Sciences 91: 15965 Safarik I, Safarikova M. 2002. Magnetic nanoparticles and biosciences. Monatshefte Fur Chemie 133: 73759 Sahoo SK, Labhasetwar V. 2003. Nanotech approaches to delivery and imaging drug. Drug Discovery Today 8: 111220 Salata O V. 2005. Tools of nanotechnology: Electrospray. Current Nanoscience 1: 2533 Santra S, Bagwe RP, Dutta D, Stanley JT, Walter GA, et al. 2005a. Synthesis and characterization of fluorescent, radio -opaque, and paramagnetic silica nanoparticles for multimoda l bioimaging applications. Advanced Materials 17: 21659 Santra S, Dutta D. 2007. Nanoparticles for Optical Imaging of Cancer. In Nanotechnologies for the Life Sciences ed. C Kumar: Wiley -VCH Verlag GmbH & Co.
190 Santra S, Dutta D, Walter GA, Moudgil BM. 20 05b. Fluorescent nanoparticle probes for cancer imaging. Technology in Cancer Research & Treatment 4: 593602 Santra S, Liesenfeld B, Bertolino C, Dutta D, Cao ZH, et al. 2006. Fluorescence lifetime measurements to determine the core -shell nanostructure of FITC doped silica nanoparticles: An optical approach to evaluate nanoparticle photostability. Journal of Luminescence 117: 7582 Santra S, Liesenfeld B, Dutta D, Chatel D, Batich CD, et al. 2005c. Folate conjugated fluorescent silica nanoparticles for labeling neoplastic cells. Journal of Nanoscience and Nanotechnology 5: 899904 Santra S, Wang KM, Tapec R, Tan WH. 2001a. Development of novel dye doped silica nanopartic les for biomarker application. Journal of Biomedical Optics 6: 1606 Santra S, Xu JS, Wang KM, Tan WH. 2004a. Luminescent nanoparticle probes for bioimaging. Journal of Nanoscience and Nanotechnology 4: 5909 Santra S, Yang H, Dutta D, Stanley JT, Holloway PH, et al. 2004b. TAT conjugated, FITC doped silica nanoparticles for bioimaging applications. Chemical Communications : 28101 Santra S, Yang H, Stanley JT, Holloway PH, Moudgil BM, et al. 2005d. Rapid and effective labeling of brain tissue using TAT -co njugated CdS: Mn/ZnS quantum dots. Chemical Communications : 31446 Santra S, Yang HS, Holloway PH, Stanley JT, Mericle RA. 2005e. Synthesis of water dispersible fluorescent, radio -opaque, and paramagnetic CdS: Mn/ZnS quantum dots: A multifunctional probe f or bioimaging. Journal of the American Chemical Society 127: 16567 Santra S, Zhang P, Wang KM, Tapec R, Tan WH. 2001b. Conjugation of biomolecules with luminophore -doped silica nanoparticles for photostable biomarkers. Analytical Chemistry 73: 498893 Sau TK, Pal A, Jana NR, Wang ZL, Pal T. 2001. Size controlled synthesis of gold nanoparticles using photochemically prepared seed particles. Journal of Nanoparticle Research 3: 25761 Schellenberger EA, Reynolds F, Weissleder R, Josephson L. 2004. Surface fun ctionalized nanoparticle library yields probes for apoptotic cells. Chembiochem 5: 2759 Schins RPF, Duffin R, Hohr D, Knaapen AM, Shi TM, et al. 2002. Surface modification of quartz inhibits toxicity, particle uptake, and oxidative DNA damage in human lu ng epithelial cells. Chemical Research in Toxicology 15: 116673 Schubert D, Dargusch R, Raitano J, Chan SW. 2006. Cerium and yttrium oxide nanoparticles are neuroprotective. Biochemical and Biophysical Research Communications 342: 8691
191 Semmler -Behnke M, Kreyling WG, Lipka J, Fertsch S, Wenk A, et al. 2008. Biodistribution of 1.4 and 18 nm Gold Particles in Rats. Small 4: 210811 Sharma P, Brown SC, Walter G, Santra S, Scott E, et al. 2007. Gd nanoparticulates: from magnetic resonance imaging to neutron capture therapy. Advanced Powder Technology 18: 66398 Shiohara A, Hoshino A, Hanaki K, Suzuki K, Yamamoto K. 2004. On the cytotoxicity caused by quantum dots. Microbiology and Immunology 48: 66975 Shvedova AA, Castranova V, Kisin ER, Schwegler Berry D, Murray AR, et al. 2003a. Exposure to carbon nanotube material: Assessment of nanotube cytotoxicity using human keratinocyte cells. Journal of Toxicology and Environmental Health-Part A 66: 190926 Shvedova AA, Kisin ER, Murray AR, Kommineni C, Gunther MR, et al. 2003b. Inflammatory response and free radical formation in skin of B63CF1 mice with diminished levels of glutathione after phenol exposure. Toxicological Sciences 72: 378Silva GA. 2004. Introduction to nanotechnology and its applications to medi cine. Surgical Neurology 61: 21620 Smith AM, Duan HW, Rhyner MN, Ruan G, Nie SM. 2006. A systematic examination of surface coatings on the optical and chemical properties of semiconductor quantum dots. Physical Chemistry Chemical Physics 8: 3895903 Smith PW. 2002. Fluorescence emissionbased detection and diagnosis of malignancy. Journal of Cellular Biochemistry : 54 9 Sokolov K, Aaron J, Hsu B, Nida D, Gillenwater A, et al. 2003a. Optical systems for In vivo molecular imaging of cancer. Technology in Cancer Research & Treatment 2: 491504 Sokolov K, Follen M, Aaron J, Pavlova I, Malpica A, et al. 2003b. Real -time vital optical imaging of precancer using anti -epidermal growth factor receptor antibodies conjugated to gold nanoparticles. Cancer Research 63: 19992004 Sonavane G, Tomoda K, Makino K. 2008. Biodistribution of colloidal gold nanoparticles after intravenous administration: Effect of particle size. Colloids And Surfaces B -Biointerfaces 66: 27480 Sonvico F, Dubernet C, Colombo P, Couvreur P. 2005. Metallic colloid nanotechnology, applications in diagnosis and therapeutics. Current Pharmaceutical Design 11: 2091105 Stober W, Fink A, Bohn E. 1968. Controlled Growth of Monodisperse Silica Spheres in Micron Size Range. Journal of Colloid and Interface Science 26: 62& Stone V, Tuinman M, Vamvakopoulos JE, Shaw J, Brown D, et al. 2000. Increased calcium influx in a monocytic cell line on exposure to ultrafine carbon black. European Respiratory Journal 15: 297303
192 Stroh M, Zimmer JP, Duda DG, Levchenko TS, Cohen KS, et al. 2005. Quantum dots spectrally distinguish multiple species within the tumor milieu in vivo. Nature Medicine 11: 67882 Stsiapura V, Sukhanova A, Artemyev M, Pluot M, Cohen JHM, et al. 2004. Functionalized nanocrystal tagged fluoresce nt polymer beads: synthesis, physicochemical characterization, and immunolabeling application. Analytical Biochemistry 334: 25765 Tan WH, Fang XH, Li J, Liu XJ. 2000. Molecular beacons: A novel DNA probe for nucleic acid and protein studies. Chemistry -a E uropean Journal 6: 110711 Taniguchi N. 1974. "On the Basic Concept of 'NanoTechnology'". Proceedings of International Commission on Physics Education Tanojo H, Junginger HE, Bodde HE. 1997. Influence of pH on the intensity and stability of the fluorescen ce of p aminobenzoic acid in aqueous solutions. European Journal of Pharmaceutical Sciences 5: 315 Tarnuzzer RW, Colon J, Patil S, Seal S. 2005. Vacancy engineered ceria nanostructures for protection from radiation induced cellular damage. Nano Letters 5: 25737 Teeguarden J. 2008. Protocol Review Form Institutional Animal Care and Use Committee. ed. Battelle. Richland, WA Teeguarden JG, Hinderliter PM, Orr G, Thrall BD, Pounds JG. 2007. Particokinetics in vitro: Dosimetry considerations for in vitro nanop article toxicity assessments. Toxicological Sciences 95: 30012 Tomazic Jezic VJ, Merritt K, Umbreit TH. 2001. Significance of the type and the size of biomaterial particles on phagocytosis and tissue distribution. Journal of Biomedical Materials Research 55: 5239 Tong L, Zhao Y, Huff TB, Hansen MN, Wei A, Cheng JX. 2007. Gold nanorods mediate tumor cell death by compromising membrane integrity. Advanced Materials 19: 3136+ Tran CL, Buchanan D, Cullen RT, Searl A, Jones AD, Donaldson K. 2000a. Inhalation of poorly soluble particles. II. Influence of particle surface area on inflammation and clearance. Inhalation Toxicology 12: 111326 Tran CL, Buchanan D, Miller BG, Jones AD, Donaldson K. 2000b. Mathematical modeling to predict the responses to poorly solu ble particles in rat lungs. Inhalation Toxicology 12: 4039 Wang HF, Wang J, Deng XY, Sun HF, Shi ZJ, et al. 2004. Biodistribution of carbon single -wall carbon nanotubes in mice. Journal of Nanoscience and Nanotechnology 4: 101924 Wang S, Low PS. 1998. F olate -mediated targeting of antineoplastic drags, imaging agents, and nucleic acids to cancer cells. Journal of Controlled Release 53: 3948
193 Wang X, Yang LL, Chen Z, Shin DM. 2008. Application of nanotechnology in cancer therapy and imaging. Ca -a Cancer Jo urnal for Clinicians 58: 97110 Wang Z, Levy R, Fernig DG, Brust M. 2005. The peptide route to multifunctional gold nanoparticles. Bioconjugate Chemistry 16: 497500 Warheit DB, Webb TR, Reed KL, Sayes CM, Colvin VL. 2005. Impact of titania and ouartz nanoparticulate exposures on respiratory health: Role of particle size. Abstracts of Papers of the American Chemical Society 229: U911-U Wasdo SC, Barber DS, Denslow ND, Powers KW, Palazuelos M, et al. 2008. Differential binding of serum proteins to nanoparti cles. International Journal of Nanotechnology 5: 92115 Weissleder R. 2002. Scaling down imaging: Molecular mapping of cancer in mice. Nature Reviews Cancer 2: 118 Weissleder R, Tung CH, Mahmood U, Bogdanov A. 1999. In vivo imaging of tumors with protease activated near infrared fluorescent probes. Nature Biotechnology 17: 3758 Wilcox D, Dove B, McDavid D, Greer d. 1995. UTHSCSA. Wu XY, Liu HJ, Liu JQ, Haley KN, Treadway JA, et al. 2003. Immunofluorescent labeling of cancer marker Her2 and other cellula r targets with semiconductor quantum dots. Nature Biotechnology 21: 416 Xia T, Kovochich M, Brant J, Hotze M, Sempf J, et al. 2006. Comparison of the abilities of ambient and manufactured nanoparticles to induce cellular toxicity according to an oxidativ e stress paradigm. Nano Letters 6: 1794807 Xia T, Kovochich M, Nel AE. 2007. Impairment of mitochondrial function by particulate matter (PM) and their toxic components: implications for PM induced cardiovascular and lung disease. Frontiers in Bioscience 1 2: 123846 Yamamoto N. 2004. Cellular dynamics visualized in live cells in vitro and in vivo by differential dual -color nuclear -cytoplasmic fluorescent -protein expression. Cancer Res. 64: 4251 Yang H, Holloway PH. 2004. Efficient and photostable ZnS Passiv ated CdS: Mn luminescent nanocrystals. Advanced Functional Materials 14: 1526 Yang HS, Holloway PH, Cunningham G, Schanze KS. 2004a. CdS: Mn nanocrystals passivated by ZnS: Synthesis and luminescent properties. Journal of Chemical Physics 121: 1023340 Ya ng HS, Holloway PH, Santra S. 2004b. Water -soluble silica -overcoated CdS: Mn/ZnS semiconductor quantum dots. Journal of Chemical Physics 121: 74216 Yang ST, Guo W, Lin Y, Deng XY, Wang HF, et al. 2007. Biodistribution of pristine single walled carbon nan otubes in vivo. Journal of Physical Chemistry C 111: 177614
194 Yin H, Too HP, Chow GM. 2005. The effects of particle size and surface coating on the cytotoxicity of nickel ferrite. Biomaterials 26: 581826 Zhang W, Qiao X, Chen J. 2007. Synthesis of silver n anoparticles Effects of concerned parameters in water/oil microemulsion. Materials Science And Engineering B -Solid State Materials For Advanced Technology 142: 115 Zhao ZJ, Wakita T, Yasui K. 2003a. Inoculation of plasmids encoding Japanese encephalitis virus PrM -E proteins with colloidal gold elicits a protective immune response in BALB/c mice. Journal Of Virology 77: 424860 Zhao ZJ, Wakita T, Yasui K. 2003b. Inoculation of plasmids encoding Japanese encephalitis virus PrM -E proteins with colloidal gol d elicits a protective immune response in BALB/c mice (vol 77, pg 4248, 2003). Journal Of Virology 77: 9107Zhu DH, Lennon SP, Peters MH, Finney WC, Singh M. 2006. Brownian diffusion and surface kinetics of liposome and viral particle uptake by human lung cancer cells in -vitro. Annals Of Biomedical Engineering 34: 157386 Zsigmondy R. 1916. Some remarkable reactions of colloidal gold. Zeitschrift Fur Elektrochemie Und Angewandte Physikalische Chemie 22: 1024
195 BIOGRAPHICAL SKETCH Debamitra Dutta was bor n and brought up in Calcutta, India She completed her Bachelor of Engineering in Metallurgical Engineering from Bengal Engineering College, Shibpore, India in 1998 and worked as a Product Development Engineer and Export Coordinator in the R&D Division at Indian Aluminum Company Limited, Belur, India for two years. Subsequently, in 2001, she came to the United States to pursue higher ed ucation and received her Master of Science degree in M aterials E ngineering at Washington State University in 2003. She joi ned the Particle Engineering Research Center at the University of Florida as a Research Scholar in 2004 to work on projects related to the use of multi -functional nanoparticles for biological imaging. In January 2005, she joined the PhD program at the University of Florida in the Department of Materials Science and Engineering and conducted her research to develop nanoparticles for bioimaging and to study their biological responses in vitro and in vivo for her dissertation. While obtaining her PhD, she spen t 4 months during the summer of 2006 and 8 months in 2007 2008 as a Visiting Scientist at the Pacific Northwest National Laboratory (PNNL), Richland, WA working with scientists in the Fundamental and Computational Sciences Directorate (FCSD) at the labor atory. She obtained her PhD in May 2009 and joined Intel Corporation as a R&D Engineer at Portland.