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Micro and Nano Manipulations for
Biomedical Applications

For a listing of recent related Artech House titles
turn to the back of this book.

Micro and Nano Manipulations for
Biomedical Applications
Tachung C. Yih
Ilie Talpasanu
Editors
artechhouse.com

Library of Congress Cataloging-in-Publication Data
A catalog record for this book is available from the U.S. Library of Congress.
British Library Cataloguing in Publication Data
A catalogue record for this book is available from the British Library.
ISBN-13: 978-1-59693-254-8
Cover design by Igor Valdman
Chapter 3 permissions: Sections of text from pages 59–67 have been reprinted in large part
with permission from [84]. Copyright 1997 American Chemical Society. Sections of text
from pages 82–83 have been reprinted in large part with permissions from [160]. Copyright
2006 American Chemical Society. Sections of text from pages 72, 74, and 75 have been
reprinted in large part with permission from [119]. Copyright 2006 American Chemical Soci-
ety. Sections of text from pages 52–54 have been reprinted in large part with permission from
[53] Copyright 1999 WILEY-VCH verlag GmbH.
© 2008 ARTECH HOUSE, INC.
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All rights reserved. Printed and bound in the United States of America. No part of this book
may be reproduced or utilized in any form or by any means, electronic or mechanical, includ-
ing photocopying, recording, or by any information storage and retrieval system, without
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10 9 8 7 6 5 4 3 2 1

To our familes
To my wife Debbie and my daughter Jessica
—Tachung C. Yih
To my wife Lucretia-Dalia and my son Alexandru
—Ilie Talpasanu

List of Contributors
Chapter 1 Tachung C. Yih
Oakland University, Rochester, Michigan, United States
Ilie Talpasanu
Wentworth Institute of Technology, Boston, Massachusetts, United States
Chapter 2 Nicosor V. Iftmia
Physical Sciences, Inc., Andover, Massachusetts, United States
Mansoor M. Amiji
Northeastern University, Boston, Massachusetts, United States
Ileana N. Iftimia
Lahey Clinic, Burlington, Massachusetts, United States and Tufts University,
Medford, Massachusetts, United States
Chapter 3 Tianzhong Yang, Chengmin Shen, Hongjun Gao, and Congwen Xiao
Beijing National Laboratory for Condensed Matter Physics, Institute of Physics,
Chinese Academy of Sciences, Beijing, China
Anca Mocofanescu
Brigham and Women’s Hospital, Boston, Massachusetts, United States
Chapter 4 J. C. Chiao
University of Texas at Arlington, Arlington, Texas, United States
Mu Chiao
University of British Columbia, Vancouver, British Columbia, Canada
Chapter 5 J. C. Chiao
University of Texas at Arlington, Arlington, Texas, United States
Mu Chiao
University of British Columbia, Vancouver, British Columbia, Canada
Chapter 6 Leonardo Sacconi and Francesco Saverio Pavone,
LENS European Laboratory for Non Linear Spectroscopy, University of Florence,
Firenze, Italy
Alessio Masi
University of Florence, Firenze, Italy
Chapter 7 Jody Vykoukal and Dayene Mannering Vykoukal
University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States
Chapter 8 Florin Ionescu
University of Applied Sciences, Konstanz, Germany
Kostadin Kostadinov
Institute of Mechanics and Biomechanics, Bulgarian Academy of Sciences, Sofia,
Bulgaria
Ilie Talpasanu
Wentworth Institute of Technology, Boston, Massachusetts, United States
Dragos Arotaritei
University of Medicine and Pharmacy “Gr. T. Popa,” Iasi, Romania
George Constantin
Polytechnic University of Bucharest, Bucharest, Romania
Chapter 9 Weimin Tao
Brooks Automation, Inc., San Jose, California, United States
Mingjun Zhang
Agilent Technologies, Inc., Santa Clara, California, United States

Contents
Preface xiii
CHAPTER 1
Introduction 1
1.1 The Third Industrial Revolution? 1
1.1.1 The First Industrial Revolution—
Manufacturing and Transportation 1
1.1.2 The Second Industrial Revolution—
Computer and Communication 3
1.1.3
The Third Industrial Revolution—Health and Environment? 5
1.2 Microtechnologies and Nanotechnologies 6
1.2.1 Challenges and Opportunities in Nanotechnology 7
1.2.2 Micromanipulations and Nanomanipulations 9
1.3 Applications and Trends 9
1.3.1 Biomedical Science and Engineering 9
1.3.2 Health Care and Environmental Applications 10
References 10
CHAPTER 2
Nanotechnology Applications in Cancer Imaging and Therapy 13
2.1
Introduction 13
2.2 Nanotechnology Approaches for In Vivo Diagnostics 15
2.2.1
Molecular Imaging Approaches for In Vivo Diagnostics 16
2.2.2 Nanotechnology-Based Contrast Agents for In Vivo Imaging 18
2.3 Nanotechnology-Based Drug Delivery Systems for Cancer Therapy 24
2.3.1 Fundamental Requirements for Drug Delivery Systems 25
2.3.2 Cancer Therapy Approaches Using Nanotechnologies 30
2.4 Conclusions 36
References 37
CHAPTER 3
Nanoparticles for Biomedical Applications 43
3.1 Introduction 43
3.2 Synthesis of Metallic Nanoparticles 45
3.2.1
Synthesis Approaches to Noble Metal Nanoparticles 45
3.2.2 Synthesis of Magnetic Metal Nanoparticles 49
3.3 Novel Properties of Metal Nanoparticles 57
3.3.1 Unique Properties of Noble Metal Nanoparticles 57
3.3.2 Magnetic Properties of Metallic Nanoparticles 67
vii

3.4 Application of Metal Nanoparticles in Biomedicine 71
3.4.1 Biomedical Detection Using Novel Metal Nanoparticles 71
3.4.2 Drug Delivery and Biosensing with Magnetic Nanoparticles 78
3.5
Specific Properties of Quantum Dots 83
3.6 Quantum Dots as Fluorescent Biological Labels 86
3.6.1 Disadvantages of Organic Dyes, Traditional Biological Labels 86
3.6.2 Beneficial Quantum Dot Optical and Spectral Properties 87
3.7 Quantum Dots in Biomedical Applications 88
References 91
CHAPTER 4
Microactuators for In Vivo Imaging and Micromanipulators in Minimally Invasive Procedures 101
4.1 Minimally Invasive Procedure Applications 101
4.2 Endoscopic and In Vivo Imaging Applications 102
4.2.1
In Vivo Scanning Microscope 103
4.2.2 In Vivo Optical Coherent Tomography Imaging 104
4.3 Micromanipulators for Minimally Invasive Procedures 108
4.3.1 Microtools 109
4.3.2 Sensors in Micromanipulators 111
4.3.3 Navigation 112
4.5 Conclusions 113
References 114
CHAPTER 5
Microactuators 119
5.1 Introduction 119
5.2 Electrostatic Actuators 119
5.3 Thermal Actuators 122
5.4
Piezoelectric Actuators 126
5.5 Shape Memory Alloy Actuators 128
5.6 Magnetic Actuators 132
5.7 Conclusions 135
References 135
CHAPTER 6
Optical Nanomanipulation in a Living Cell 143
6.1 Two-Photon Fluorescence Microscopy 143
6.1.1 Introduction 143
6.1.2 A Brief Analytical Description 145
6.2
Second-Harmonic-Generation Microscopy 146
6.2.1 Introduction 146
6.2.2 Nonlinear Optical Processes 147
6.2.3 Single-Molecule Cross Section 148
6.2.4 Biological Membrane Imaging 149
6.3 Laser-Induced Microdissection 151
6.3.1 Summary 151
viii Contents

6.3.2 Introduction to Optical Dissection 151
6.3.3 Three-Dimensional Imaging and Optical Dissection
by Nonlinear Optical Microscopy 151
6.3.4 Physical Characterization of Nanosurgery 153
6.3.5
Mitotic Spindle Positioning 154
6.3.6 Mitotic Spindle Elongation 156
6.4 Optical Trapping 157
6.4.1 Summary 157
6.4.2 Introduction to Optical Tweezers 157
6.4.3 Optical Trapping Inside Yeast Cells 158
6.4.4 Laser-Induced Nucleus Displacement 162
6.4.5 Motion of a Displaced Interphase Nucleus Back
to the Cell Center by Microtubule Pushing 163
6.4.6 Asymmetric Cell Division as a Result of Nucleus
Displacement During Interphase 164
6.4.7 Division Plane Determination in Early Prophase 165
6.5 Optical Knockout 166
6.5.1 Introduction 166
6.5.2 One-Photon CALI 167
6.5.3 Micro-CALI 168
6.5.4 Multiphoton CALI 171
6.6 Conclusions 172
Acknowledgments 173
References 173
CHAPTER 7
Dielectrophoretic Methods for Biomedical Applications 179
7.1 Introduction 179
7.2 Theory 181
7.2.1
Dielectrophoresis 181
7.2.2 Dielectric Properties of Bioparticles and Biomolecules185
7.3 Dielectrophoretic Approaches to Bioparticle Manipulation and Characterization 191
7.3.1 Differential Manipulation of Bioparticles 191
7.3.2 Filtration and Concentration of Bioparticles 193
7.3.3 Manipulating Cells for Subsequent Analysis 195
7.3.4 Cell Patterning and Tissue Engineering 198
7.3.5 Characterizing Cell Physiology by Dielectrophoresis 200
7.4 Dielectrophoretic Approaches to Molecular Assays 202
7.4.1 Microparticle-Based Systems 202
7.4.2 Droplet-Based Systems: Digital Microfluidics 203
7.5 Conclusions and Perspectives 204
Acknowledgments 205
References 205
Contents ix

CHAPTER 8
Design, Analysis, Modeling, Simulation, and Control of Microscale and
Nanoscale Cell Manipulations 215
8.1 Introduction 215
8.1.1 Overview of Micropositioning and Nanopositioning
Systems Based on Piezoactuators 216
8.1.2
Applications of Piezoactuated Micropositioning and
Nanopositioning Systems 217
8.2 Construction of the Micro-Nano Robot as a Mechatronic System 218
8.2.1 Conceptual Design of Piezo-Actuated Microrobot
Development 218
8.2.2 Robot RoTeMiNa for Cell Micromanipulation and
Nanomanipulation 221
8.2.3 Design of the Micro Stage Robot 222
8.2.4 Design of the Nano Stage Robot 223
8.2.5 Teleoperated Control 223
8.3 Differential Kinematics of a Hybrid Robot for Cell
Micromanipulations and Nanomanipulations 225
8.3.1 Link and Joint Numbering 225
8.3.2 Oriented Graph Attached to the Mechanism 225
8.3.3 Matrix Description of Graph 226
8.3.4 Geometric Jacobean 227
8.3.5 Degrees of Freedom 232
8.3.6 Independent Equations for the Inverse Kinematics 232
8.4 Hardware and Software for the Development of Micropositioning and
Nanopositioning Systems 234
8.4.1 Guidelines for Development 234
8.4.2 Sensors for Feedback 235
8.4.3 Unified Approach for Functional Task Formulation 235
8.5 Intelligent Control of Piezoactuated Robot Using an Approximated
Hysteresis Model in Micromanipulations and Nanomanipulations 238
8.5.1 Introduction 238
8.5.2 The Mathematical Model of Hysteresis 238
8.5.3 The Neuro-Fuzzy Inverse Model 241
8.5.4 The Control System Structure 242
8.5.5 Multiobjective Optimal PI/PID Controller Design Using Genetic
Algorithms 244
8.6 Experimental Results 246
8.7 Extension of the Method and Limitations 247
8.8 Discussion and Conclusions 247
Acknowledgments 250
References 250
x Contents

CHAPTER 9
Dynamics Modeling and Analysis for Gene Manipulations 253
9.1 Introduction 253
9.1.1 Current Status 254
9.1.2
Requirements for Gene Delivery 254
9.1.3 Methods for Gene Delivery 256
9.2 Electroporation 257
9.2.1 Electrode 258
9.2.2 Electric Pulse 259
9.2.3 Tissue Damage 260
9.2.4 Gene Expression Efficiency 260
9.2.5 Dynamics Modeling 261
9.3 Hydroporation 261
9.4 Sonoporation 262
9.4.1 Impact of Ultrasound Frequency 263
9.4.2 Impact of Ultrasound Intensity 263
9.4.3 Impact of Ultrasound Exposure Time 264
9.4.4 Cell Damage with Sonoporation 264
9.4.5 Dynamic Modeling 264
9.5 Microneedle and Microinjection 266
9.5.1 Microneedle 266
9.5.2 Microinjection 266
9.6 Optoinjection and Optoporation 267
9.7 Magnetofection 268
9.8 Gene Gun 269
9.8.1 Introduction 269
9.8.2 Dynamic Modeling 272
9.9 Summary and Comparison of the Physical Methods 275
9.10 Summary and Future Challenges 275
References 277
About the Authors 281
Index 287
Contents xi

Preface
In the next decade, nanotechnology is expected to have a large impact on diseases
such as cancer, neurodegeneration, and diabetes and those of the blood, lungs, car-
diovascular system, and skeleton. The design of novel nanoparticles, capable of
sensing and drug delivery, will increase. Chapter 2 presents an overview of technol-
ogies for cancer diagnosis and therapy. The fabrication and properties of metallic
nanoparticles, as well as quantum dots, nanocrystals based on semiconductors, also
are presented in this chapter.
Chapter 3 outlines the fundamentals of MEMS/NEMS actuator design and fab-
rication. A description of thermal, magnetic, electrostatic, piezoelectric micro-
actuators is detailed in Chapter 4, which also focuses on the micromanipulators for
minimally invasive procedures. Chapter 5 discusses end effector tools and electron
microscopy for biomedical nanomanipulations.
Cell viability and behavior following nanoparticle injection are observed
through imaging techniques. Chapter 6 presents current applications and trends in
imaging manipulations such as 3-D imaging and optical dissection by nonlinear
optical microscopy, laser-induced microdissection, nanosurgery characterization,
optical trapping, and optical tweezers.
Chapter 7 discusses dielectrophoretic methods for cell and biomolecule manip-
ulations. The design, modeling, and control of a piezo-actuated robotic system with
microaccuracy and nanoaccuracy, as required in cell manipulations, are presented
in Chapter 8. The main physical methods of gene manipulations and delivery such
as electroporation, hydroporation, sonoporation, microneedle and microinjection,
optoinjection and optoporation, magnetofection, and the gene gun method are
described in Chapter 9. The performances and limitations of the gene delivery meth-
ods are also analyzed in this chapter.
The book covers basic principles and applications of micromanipulation and
nanomanipulation for biomedicine and will provide the reader with the facts he or
she needs to know about the manipulation, control, modeling, and simulation at the
microscale and nanoscale levels. Microprecision and nanoprecision robotic systems
are used by manufacturers or researchers involved in biorobotics projects, and by
practitioners involved in applications from the emerging area of micro and nano bio
devices. Presenting the most recent applications of biomanipulation of cells and
genes, the book is suitable also for undergraduate and graduate students in
biomedical applications.
We wish to thank Dr. Frank Caserta from the Wentworth Institute of Technol-
ogy for his suggestions during the preparation of the manuscript. We would like to
thank the reviewers, Dr. Jane Wang from the Northwestern University and Dr. Pak
Kin Wong from the Systematic Bioengineering Lab at the University of Arizona, for
xiii

their critical reviews. We also would like to thank Wayne Yuhasz, Barbara
Lovenvirth, Judi Stone, and Rebecca Allendorf from Artech House for their efforts
during the preparation of this book.
xiv Preface

CHAPTER 1
Introduction
Tachung C. Yih and Ilie Talpasanu
1.1 The Third Industrial Revolution?
In 2000, then-President William J. Clinton launched the National Nanotechnology
Initiative (NNI) as a top science and technology priority [1]. The President’s Coun-
cil of Advisors on Science and Technology (PCAST) described NNI as an excellent
multiagency framework to ensure U.S. leadership in this emerging field that will be
essential for economic and national security leadership in the first half of the next
century [2]. The speech by President Clinton given at the California Institute of
Technology on January 21, 2000, was titled “National Nanotechnology Initiative:
Leading to the Next Industrial Revolution.” Is nanotechnology leading us into our
third Industrial Revolution? To answer this question, we must understand what
happened in previous industrial revolutions [3–12].
Humans walked through the Stone Age, Bronze Age, and Iron Age, periods cov-
ering thousands of years, with a dawdling speed of technology advancement. The
common cause of the Industrial Revolution is the advancement of new technology,
triggered by scientific innovations. The Industrial Revolution transformed countries
from agricultural to industrial, communities from rural to urban, and people from
farmers to factory workers. The significant impacts resulting from the Industrial
Revolution cover a broad range such as productivity growth, culture, and the daily
lives of the populace. The long-term effects brought about by the industrial revolu-
tions remain debatable. However, many believe that, on the whole, the benefits that
came with the Industrial Revolution outweigh the problems.
1.1.1 The First Industrial Revolution—Manufacturing and Transportation
The first Industrial Revolution occurred in Great Britain between 1750 and 1850,
followed by those of the United States and other countries in Europe during the
nineteenth century as well as those of Russia and Japan in the first half of the twenti-
eth century.
The earliest factories appeared in 1740, producing textile merchandise. Eli
Whitney, an American, changed the textile industry from using wool to using cotton
as its raw material with his invention of the cotton gin (1792). Other important
English inventions included the flying shuttle (John Kay, 1733), the hand-powered
cotton-spinning jenny (James Hargreaves, 1765) [13], the water frame spinning
1

device (Richard Arkwright, 1766), and the improvements in weaving powered by a
steam engine (Samuel Crompton, 1790). Samuel Slater, known as the father of the
American Industrial Revolution, emigrated from England to America in 1789 and
built the first successful water-powered textile mill in Pawtucket, Rhode Island, in
1793 [14, 15]. The first sewing machine was designed by Elias Howe in 1843. Sew-
ing machines were the first major consumer appliance. In 1851, Isaac Singer
invented the first practical, domestic sewing machine and widely marketed his
machines.
The early steam engines were developed in England by Thomas Savery (1698)
and Thomas Newcomen (1705) to pump water out of coal mines, but they could not
generate power. Henry Cort (1780) developed the steam-powered rolling mills that
revitalized the iron industry. James Watt (1782) developed power-generating steam
engines to drive rotary shafts in other machinery. Watt’s engines were widely used
during the period of the Industrial Revolution. Richard Trevithick (1805) built sev-
eral full-size, high-pressure steam carriages, known as locomotives, which were used
to haul coal and ore out of the mines. George Stephenson opened the first public rail-
way in the world in 1825, which was worked by a StephensonRocketlocomotive.
During the nineteenth century, steam locomotives were exported from England to
many other countries. The steam-powered locomotive and iron railway system revo-
lutionized the means of transportation in human history; people and goods could be
transported in mass quantities at lower cost, which enabled the market economy.
In the United States [16, 17], Oliver Evans pioneered and invented the high-pres-
sure steam engine. He built the first automatic mill in Delaware in 1782, and in
1789, the first U.S. patent for a steam-powered land vehicle was granted to him.
John Fitch designed and tested a steam-powered boat on the Delaware River in
1786. The first practical steamboat was built by William Symington in Scotland in
1801. Robert Fulton constructed theClermontto carry fare-paying passengers on
the Hudson River in 1807.
To construct the steam engines and machines, the growing need for machine
tools was inevitable. The oldest known machine tool was the wood-working lathe.
Jasse Ramsden, an English instrument maker, developed the first screw-cutting
lathes in 1770. Henry Maudslay, in 1800, produced the first large, high-accuracy
(1/10,000 of an inch) screw-cutting lathe. Maudslay’s assistants introduced the first
gear-cutting machine (Richard Roberts, 1818), a punching machine for making rivet
holes (Richard Roberts, 1847), a measuring machine with an accuracy of one-mil-
lionth of an inch (Joseph Whitworth, 1856; he also standardized the screw threads
in English), and the milling machine and shaper (James Nasmyth, 1854). One of the
most important inventions, which made Watt’s steam engine a practical power
source, was John Wilkinson’s cylinder boring machine (1775).
The post–Industrial Revolution (1850–1950) mainly carried on in the United
States. One of the most important landmarks in the history of engine design was the
invention of the internal combustion engine (Nikolaus A. Otto, 1876) [18]. He con-
structed the first practical gas-motor, four-stroke piston cycle internal combustion
engine, named the Otto Cycle Engine. Otto’s invention was the first practical alter-
native to the steam engine and the foundation of the modern engine. By applying
Otto’s engine, Gottlieb Daimler built a motorcycle (1885) and the first four-wheeled
automobile (1886) in the world. Karl Benz also constructed his first three-wheel
2 Introduction

automobile (1885) and four-wheel car (1891) using Otto’s engine. The two firms,
Daimler and Benz, later merged and manufactured the famous Mercedes-Benz vehi-
cle. In the United States, Henry Ford incorporated the Ford Motor Company in
1903. In 1908, he introduced the Model T car, which heralded the beginning of the
motor age. Ford also revolutionized the mass production manufacturing processes
by using a constantly moving assembly line, subdivision of labor, and coordination
of operations. Much smaller and lighter, compared to a steam engine of equal
power, diesel engines were developed after 1920.
In 1903, the Wright Brothers (Wilbur and Orville) successfully flew their first
airplane,Wright Flyer, which opened the aerial age. By 1906, they developed their
flying machine into the first practical fixed-wing aircraft in the world. Later, the
invention of jet engines (Hans von Ohain, 1939, and Frank Whittle, 1941) revolu-
tionized the airplane development from propeller to jet with much higher speed.
Other important inventions during this period included telegraph (William
Cooke and Charles Wheatstone, 1839). In the United States, the first telegraph mes-
sage was sent in 1844 using (Samuel) Morse’s code. Alexander G. Bell in 1876 was
granted a patent for his communication device to “transmit vocal or other sounds
telegraphically”; he also founded the first telephone company, Bell Telephone Com-
pany, in 1877. Radio was known as “wireless telegraphy.” Based on the theory
(James Maxwell, 1860) and discovery (Heinrich Hertz, 1886) of radio waves,
Mahlon Loomis successfully demonstrated “wireless telegraphy” in 1866 and
Guglielmo Marconi sent and received his radio signal in Italy in 1895 and success-
fully transmitted a transatlantic radiotelegraph message in 1902. In 1862, Abbe
Caselli transmitted a still image over wires using his invention—the Pantelegraph.
Eugene Goldstein described “cathode rays” (1876) as the light emitted when an
electric current was forced through a vacuum tube. Paul Nipkow sent “electric tele-
scope” images over wires using a rotating metal disk technology in 1884. Finally,
the word “television” was made known by Constantin Perskyi at the World’s Fair
in Paris in 1900. During the first Industrial Revolution, societies and countries went
through a series of major transformations: migration, urbanization, factorization,
industrialization, mass production, transportation, suburbanization, pollution, and
so forth.
1.1.2 The Second Industrial Revolution—Computer and Communication
As historian Claude Fohlen stated, “The industrial revolution is … a continuing
phenomenon which is going on in front of our eyes ….” It is a mistake to believe
that the first Industrial Revolution has ended more than 150 years ago. If we stretch
the first Industrial Revolution into three periods—initial stage (1750–1850), expan-
sion stage (1850–1900), and transition stage (1900–1950)—instead of ending at
1850 or 1950, the innovation and momentum generated in the second half of the
first Industrial Revolution actually crossed the threshold and fueled the second
Industrial Revolution. Some historians defined the second Industrial Revolution as
the period between 1871 and 1914. However, others believed that the first Indus-
trial Revolution merged into the second Industrial Revolution around 1850. If we
consider the post–first Industrial Revolution era (1850–1950) as the initial stage
of the second Industrial Revolution, the three phases of the second Industrial
1.1 The Third Industrial Revolution? 3

Revolution are then initial stage (1850–1950), expansion stage (1950–2000), and
transition stage (2000–2050).
Technology advancement in the past century accelerated and surpassed the
achievement accumulated throughout our entire human history. New technologies
and new generation of products turned around at a speed that we have never before
experienced [19–22].
History changed when the Soviet Union in 1957 launched Sputnik I and Sputnik
II, the world’s first two satellites. It marked the beginning of the Space Age. In 1958,
the United States launched Explorer I. Satellites have significantly improved our sci-
entific advancement in many different areas and have impacted our daily lives in
such areas as communication and entertainment.
Computers, especially personal computers, have revolutionized our daily lives
in many respects. Computer hardware and software advanced expeditiously and
sparked many other new scientific findings, state-of-the-art technologies, and prod-
ucts. Some of the historic milestones are summarized in the following: Konrad Zuse
developed the first freely programmable computer, the Z1, in 1936. The transistor,
invented by John Bardeen, Walter Brattain, and William Shockley (Bell Telephone
Laboratories) in 1947, miniaturized the electronic circuits and made the develop-
ment of personal computers possible. UNIVAC introduced the first commercial
computer in 1951, followed by IBM’s 701 EDPM Computer in 1953. In the follow-
ing year, IBM marketed the first high-level programming language, FORTRAN.
The computer mouse and graphical user interface were invented by Douglas
Engelbart in 1964. The original Internet, ARPANET, was introduced in 1969. The
inventions of integrated circuits (Texas Instruments, 1958) and microprocessors
(Intel 4004, 1971) enabled the development of consumer computers (Altair and
IBM, 1974/75; Apple and Commodore, 1976–1977) as well as personal home com-
puters (IBM, 1981; Apple, 1983). The Microsoft disk operating system (MS-DOS)
was first introduced in 1981, followed by Windows in 1985.
One of the major technological advancements in the second Industrial Revolu-
tion worthy of special attention is communication. Several years after the construc-
tion of ARPANET, what is now known as the Internet was formed. Internet and
satellites undeniably revolutionized the way we communicate with each other on a
global scale. In recent years, new generations of cellular phones and wireless com-
munication devices have advanced rapidly.
One of the main causes for the Industrial Revolution is the need for power/
energy. New sources of power/energy were developed to satisfy the production
needs and then further fueled the momentum of the Industrial Revolution. It took
humans a very long time to evolve from using only human power to utilizing animal
power, water power, steam power, and finally electrical power (Zenobe-Theophile
Gramme created a reliable electric generator in 1870). Michael Faraday demon-
strated how electricity could be mechanically generated in 1831. Thomas Edison
established the very first commercial electric power generating plant (1882) in New
York City. Thomas Edison and Joseph Swan invented the first incandescent filament
lamp in the early 1880s. By 1906, the tungsten filament lamp was introduced, which
had three times more illumination per watt than Edison’s carbon lamp. Later, the
nitrogen-filled lamp was developed. Other household appliances such as the vacuum
cleaner, electric stove, dishwasher, and laundry machine were invented during the
4 Introduction

early 1890s. These household appliances and machines in factories stimulated the
consumption of electricity.
One technological breakthrough during this period that cannot be overlooked
was the exploration of nuclear power and nuclear energy. However, due to the
safety and environmental concerns, highly sophisticated technologies must be
developed to resolve these two issues before nuclear power can be used extensively.
Major impacts on society during the second Industrial Revolution included
further improvement of transportation, faster-paced suburbanization, worsened
environmental pollution, information from mass media, communization without
boundary, digitization to augment capacities and functions, miniaturization for
lighter and portable devices, and so forth.
1.1.3 The Third Industrial Revolution—Health and Environment?
In analogy to estimating the spans of the first and second Industrial Revolutions, the
third Industrial Revolution should start with its initial stage (1950–2050) and be
followed by the expansion stage (2050–2100) and transition stage (2100–2150).
The first two industrial revolutions did enhance our standard of living due to the
availability of cheaper mass-produced goods and household appliances. The indus-
trial revolutions also increased economic productivity and our life expectancies.
However, chemical pollution caused by the industrial revolutions is subsequently
jeopardizing our living environment.
Is the third Industrial Revolution actually taking place in front of our eyes? By
definition, we are now in the last 50 years of the transition stage (2000–2050) of the
second Industrial Revolution as well as in the second half of the initial stage
(1950–2050) of the third Industrial Revolution. The first and second Industrial Rev-
olutions arose from the most powerful and wealthiest countries at that time, Eng-
land and the United States, respectively. The common causes of both industrial
revolutions were technology innovation and energy exigency. To scrutinize these
factors, the United States at the present time is still known as the most powerful and
wealthiest country as well as the most creative technological leader in the world.
The United States is also earnestly researching on replacing fossil fuels with other
sources of alternative energy. Consequently, there is a great possibility that the third
Industrial Revolution was just launched.
The remaining questions are then: “Is nanotechnology the key technology that
triggers the third Industrial Revolution?” and “What areas were impacted by the
third Industrial Revolution?” The conclusions will be drawn in the future by histori-
ans, most likely 150 years from now. However, by analyzing the overall progress of
technology from 1950 to the present and, more specifically, innovations created by
nanotechnology, it is predictable that nanotechnology will play an important role as
one of the major technologies in the third Industrial Revolution. The first two indus-
trial revolutions have improved our living standards, working conditions, and
medical treatment, yielding longer human life in general. Nanotechnology, in com-
bination with biotechnology, will revolutionize the health care diagnostic and treat-
ment methods and further prolong healthier human life through artificial means
such as gene repair. Furthermore, awareness of restoring our environment for
future generations has been heightened. More efficient and greener energy sources
1.1 The Third Industrial Revolution? 5

are sought with urgency. Scientists are alerting us that we are reaching the threshold
of an irreversible effect from the polluting of our environment. It is envisioned that a
breakthrough in health and environmental concerns will be pursued in the third
Industrial Revolution.
The first Industrial Revolution was originated in Europe, and the second Indus-
trial Revolution took place in the United States. Will the third Industrial Revolution
be taken over by a country in Asia in a later stage? Besides health and the environ-
ment, what other fields will be affected by the third Industrial Revolution? These
questions can only be answered by time.
1.2 Microtechnologies and Nanotechnologies
In echo of the National Nanotechnology Initiative, President George W. Bush
announced the “American Competitiveness Initiative: Leading the World in Innova-
tion” in his State of the Union speech dated January 31, 2006. The American Com-
petitiveness Initiative (ACI) encourages American innovation and strengthens our
nation’s ability to compete in the global economy. The ACI is also committed to a
10-year increased investment in research and development, strengthening of educa-
tion, and encouraging of entrepreneurship and innovation.
Microtechnology is best known as microelectromechanical systems (MEMS).
MEMS is a multidisciplinary field that develops miniaturized functional, intelligent
devices at the microscopic level, that is, the micron (10
–6
m) scale, Figure 1.1. A basic
MEMS device includes the sensing, processing, and actuating units. The first MEMS
device, a gold resonating MOS gate structure, was developed in 1967 [23]. Nowa-
days, MEMS devices are widely used in the fields of chemical engineering,
pharmaceuticals, bioengineering, appliances, automobile industry, medical, avia-
tion, defense, and communication. When we further miniaturize from the micron
level to the nanoscale (10
–9
to 10
–7
m; i.e., 1 to 100 nm), Figure 1.1, the physical laws
convert from Newtonian physics to quantum physics. Table 1.1 describes the
governing physical laws at different scales.
6 Introduction
10 m
–7
(100n)
10 m
–1
d(deci)
10 m
–2
c(centi)
10 m
–3
m(milli)
10 m
–6
µ(micro)
10
−12
m
p(pico)
10 m
–9
n(nano)
Nanoscale
Atoms (0.1~0.5 nm) Antibodies (10 nm) Bacteria (100 nm~10 m) Insects (1 mm~1 cm)µ
X-rays (0.1~10 nm) UV (100 nm) Visible light (1 m) Infrared (10 m) Microwave (1 cm)µµ
Angstrom(Å, 0.1 nm) Viruses (10~100+ nm) Red blood cells (5~10 m) Fly (10 mm)µ
DNA (2.5 nm) Molecules (0.3~23 nm) Proteins (1~10 nm) Hair (80 m) Human egg (100 m)µµ
Figure 1.1Micron scale and nanoscale.

Nanotechnology requires the manipulation of matter at the atomic and molecu-
lar levels. The domination of quantum physics at the nanoscale adds to the degree of
difficulty in nanomanipulation and nanofabrication.
1.2.1 Challenges and Opportunities in Nanotechnology
The emerging fields of nanotechnology are leading to unprecedented understanding
of and control over the fundamental building blocks of all physical objects.
Nanotechnology will bring about the ability to precisely move matter at the molecu-
lar or atomic level. Similar to traditional technology fields, nanotechnology is
divided into two major areas: fundamental nanoscience and applied nano-
engineering. The ultimate goal of nanoengineering is to create functional materials,
systems, and devices through the control of matter, at nanoscale, by utilizing the
finding of novel properties and phenomena in nanoscience. The systems developed
by applying nanotechnology are referred to as nanoelectromechanical systems
(NEMS).
Nobel laureate and quantum theorist Richard Feynman first introduced the
concept of nanotechnology in his famous talk (1959) and paper “There’s Plenty of
Room at the Bottom” [24]. The termnanotechnologywas first used by Taniguchi
[25] to describe ultrafine machining of matter. Drexler realized the concept of
Feynman and achieved a major breakthrough in nanotechnology in 1992 [26]. He
introduced two principal approaches to manufacturing on the molecular scale: (1)
the “bottom-up” approach, self-assembly of machines from basic chemical building
blocks, and (2) the “top-down” approach, assembly by manipulating components
with much larger devices, such as manipulators on the micron scale. In 2000, IBM
constructed the first quantum computer in the world, which stores and processes
information at the atomic level.
Major research activities in nanotechnology are being conducted in many dif-
ferent application areas throughout the world today. It will be one of the most
important driving forces for industries in the next 5 to 10 years. It totally revolu-
tionizes the traditional engineering technology concepts and is likely to be an imper-
ative component to impact the future development in the areas of materials and
manufacturing, nanoelectronics and computer technology, medicine and health,
aeronautics and space exploration, environment and energy, biotechnology and
agriculture, national security, science and education, global trade and competitive-
ness, and other government applications [27–34].
As mentioned earlier, one of the main challenges in nanotechnology is that the
classical Newtonian laws cannot capture the physics at the nanoscale due to the fail-
ure of the continuum hypothesis at such a small scale. As the components of
1.2 Microtechnologies and Nanotechnologies 7
Table 1.1Physical Laws Governing at Different Scales
Scale Size Physical Laws
Macro > 1µm Classical Newtonian physics
Micro ~1 µm Classical Newtonian physics
Meso 10 nm–1 µm From Newtonian to quantum physics
Nano <10 nm Quantum physics

engineering systems become smaller, their dynamic and material properties will
change correspondingly. Newtonian physics is “deterministic” (i.e., predictable),
whereas at the quantum scale, it is not deterministic. Quantum physics is “statisti-
cal” in nature; it deals with questions of “probability.” Nanotechnology is governed
by the laws of quantum physics, which are well known to be counterintuitive. A
“quantum” can be considered as (1) a “particle” with point mass if its position is
measured, (2) a “wave” if its momentum is measured, or (3) a “clock” (“atomic
clock”) if its energy is measured.
The immediate needs in nanotechnology are to understand the behaviors of par-
ticles at the molecular level in order to develop enabled nanotechnologies. Accurate
and predictive models and simulations at the nanoscale are critical to advancement
in all fields of nanotechnology. Figure 1.2 shows the major research and develop-
ment directions, critical research and development needs, and the short-range (5–10
years), midrange (7–15 years), and long-range (15 years and more) achievable goals
in nanoscience and nanoengineering. It is essential that nanoscientists lay the foun-
dation for nanotechnology through new findings in nanoscience prior to developing
nanoengineering systems and devices by nanoengineers.
8 Introduction
Nanotech Areas
Major R&D
Directions
Critical
R&D
Needs
Achievable
Goals
Long range
Mid-range
Short range
Figure 1.2Major research and development directions and needs in nanotechnology.

1.2.2 Micromanipulations and Nanomanipulations
The process of moving, controlling, altering, and fabricating objects at the micron
or atomic/molecular scale is known as micromanipulation or nanomanipulation. At
the micron scale, the effect of adhesive forces is considerably higher than that of
gravity. Therefore, environmental factors (e.g., relative humidity and temperature)
and material properties (e.g., roughness, affinity for water, and so forth) will greatly
affect the reliability and precision of micromanipulation. Microfabricated mechani-
cal grippers were developed to complete pick-and-place nanomanipulation tasks
[35]. Other types of micromanipulators and microtools include microneedles,
microinjectors, microtweezers, microtubes, micropipettes, microcantilevers, and so
forth. An optically guided microsphere was achieved by using the femtosecond laser
for optical micromanipulation [36]. Laser micromanipulation uses optical forces to
trap, move, and rotate microscopic particles by a focused laser beam or beams.
It transfers momentum from the light to the particle. An electromagnetic
micromanipulator was also developed, which integrates microcoils and soft ferro-
magnetic microtips for localized positioning of microscopic objects in fluids.
Micromanipulation is also widely applied in the medical field, where it is defined
[37] as the manipulation of minute instruments and needles under a microscope to
perform delicate procedures such as microsurgery. Other applications and applica-
tion fields include microdissection, microinjection, imaging, neurology, fertility,
and so forth [38–42].
The atomic force microscope (AFM), scanning tunneling microscope (STM),
and scanning electron microscope (SEM) enable scientists to view and manipulate
nanoscale particles, atoms, and small molecules. Ernst Ruska, Gerd Binning, and
Heinrich Rohrer were granted the 1986 Nobel Prize for Physics for inventing the
electron and scanning tunnel microscopes.
AFM can be used to manipulate nanotubes and nanowires to fabricate electrical
circuits and measure and characterize their mechanical properties [43]. Quantum
corals can be produced by manipulating individual atoms using STM. SEM and
TEM (transmission electron microscope) are used for monitoring three-dimensional
nanomanipulation. Three-dimensional SEM and TEM nanomanipulation tools
have been developed by Zyvex, Nanofactory, and other research groups. Pacific
Nanotechnology offers different types of nanoprobes. A variety of nanopositioning
systems with subnanometer precision have been manufactured by Mad City Labs
Inc. Optical forces are also used in nanomanipulation [44]. Radiation pressure,
derived from the linear and angular momentum of light, can be exerted by a laser
beam to perform optical trapping. Nanomanipulation [45–48] will lead to a revolu-
tion in manufacturing technology—molecular manufacturing—a new technology
that may be an integral part of the third Industrial Revolution.
1.3 Applications and Trends
1.3.1 Biomedical Science and Engineering
New directions in research such as biosensing, bioimaging, bioinformatics, and tis-
sue engineering emerge from the multidisciplinary cooperation between medical
doctors, biologists, chemists, physicists, pharmacologists, and specialists from
1.3 Applications and Trends 9

traditional fields such as mechanical engineering, materials science, information
technology, and electrical engineering. The biomedical research provides new
insights and new therapies and solutions for medical problems. As a result, medical
advancements lead to better health care delivery. From basic research to manufac-
turing of a health care product, there is laboratory testing and preclinical develop-
ment, which are conducted in order to achieve validation in accordance with
regulatory rules.
1.3.2 Health Care and Environmental Applications
Recently, the biological, medical, and agricultural sciences have been expanding at
an unprecedented rate. The techniques developed for medical testing also find appli-
cations in environmental monitoring and security. Using nanoparticles, anthrax
detection will be as easy as the self-pregnancy test. The nanoimaging technologies
lead to single-cell or molecule detection in a biological environment.
Nanotechnology-based products such as window layer coatings protecting against
UVB radiation, kitchen or bathroom tiles coated with antimicrobial nanoparticles,
stain resistant clothing, and nanopore-structured zeolites for water softening will
improve the protection and sanitation in our everyday lives [49–52].
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10 Introduction

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1.3 Applications and Trends 11

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12 Introduction

CHAPTER 2
Nanotechnology Applications in Cancer
Imaging and Therapy
Nicusor V. Iftimia, Mansoor M. Amiji, and Ileana N. Iftimia
2.1 Introduction
The diagnosis of cancer poses the challenge of accurately identifying the anatomical
site of origin of the malignancy and the type of cells involved. Cancer can arise in
any organ or tissue in the body except fingernails, hair, and teeth. Early cancer diag-
nosis significantly increases the chances of successful treatment. Some early signs of
cancer include lumps, sores that fail to heal, abnormal bleeding, persistent indiges-
tion, and chronic hoarseness [1]. Early detection is particularly relevant for cancers
of the breast, cervix, mouth, larynx, colon and rectum, and skin. Various technolo-
gies and protocols are used for early cancer diagnosis. For breast cancer, for
instance, personal examination by palpation and X-ray mammography have con-
tributed significantly to the overall well-being of the patient [2]. For cervical cancer
the Pap smear tests are largely used as a first line of diagnosis [3].
Imaging plays a fundamental role in early cancer diagnosis and prognosis,
mainly through the detection of anatomically defined abnormalities. However, it is
universally accepted that early detection of cancer is essential even before anatomic
anomalies are visible. Unfortunately, since most of the imaging modalities do not
provide the necessary resolution and contrast to visualize early signs of cancer,
imaging alone is not very effective. The identification of molecular signatures of
cancer is the only way to allow the imaging modalities in distinguishing between
normal tissue and one that is cancerous.
A major challenge in cancer diagnosis is to be able to determine the exact rela-
tionship between cancer biomarkers and the clinical pathology. Currently, there is a
large discrepancy between reliable clinical biomarkers and disease progression. For
instance, nearly 20% of patients with mildly elevated prostate specific antigen
(PSA) levels have prostate cancer and about one-third of prostate cancer patients
have normal PSA levels [4].
Recent developments in areas of genomics and proteomics have made possible
the identification of cancer-specific molecular markers [5]. Molecular approaches
and nanotechnologies promise to advance diagnostic cancer imaging and ultimately
to affect the quality of cancer patient care [6–8]. The application of these new
molecular and nanotechnology approaches in medicine, callednanomedicine, has a
13

potential to impact on the prevention, early and reliable diagnosis, and treatment of
cancer diseases [9]. Synthetic nanostructures, such as nanoparticles and
nanodevices, being of the same size as biological entities, can readily interact with
biomolecules both on the cell surface and within the cell. Nanomedical develop-
ments range from nanoparticles used for diagnosis and/or therapy to integrated
medical nanosystems, which in the future may perform complex repair actions at the
cellular level inside the body [10].
Besides enhanced sensitivity and specificity imaging, the goal of using
nanotechnologies in cancer management is to create highly sensitive, highly reliable
detection agents that can also deliver and monitor therapy. This is the “find, fight,
and follow” concept of early diagnosis, therapy, and therapy control, also known as
theragnostic[11]. This term was recently proposed to describe the use of molecular
imaging in prescribing the distribution of radiation doses in four dimensions (three
spatial dimensions and time). The tissue of interest is initially imaged, using tar-
get-specific contrast nanostructures. Then, the targeting nanostructures, combined
with a pharmacologically active agent, can be used for therapy. Finally, using
sequential imaging, the results of this therapy can be monitored over time. Earlier
and more reliable disease detection will be achieved by using better tracers and con-
trast agents in combination with superior detection systems.
Modern medicine is based on the mid-nineteenth century discovery that the cell
is the source of health and disease. The ultimate objective of using molecular imag-
ing approaches in medicine is to identify diseases at the earliest stage possible, ide-
ally at the single-cell level. To achieve this goal, research and development activities
in nanotechnology need to be undertaken to improve the effectiveness of in vivo
diagnostics.
Nanotechnologies can bring very efficient tools for medical diagnostics, offering
enhanced sensitivity, specificity, and reliability. Combined with new developments
in microscopic and spectroscopic techniques toward ultrahigh spatial resolution,
such as optical coherence tomography, optical confocal microscopy, scanning probe
microscopy, electron microscopy, and imaging mass spectrometry, the
nanotechnology approaches will offer molecular resolution and ultrahigh sensitiv-
ity, allowing for a better understanding of the cell’s complex “machinery” in basic
research. The resulting accelerated progress could pave the way to more inventive
and powerful in vivo diagnostics tools.
As mentioned previously, besides medical diagnostics, the emergence of nano-
technology will have a significant impact on the drug delivery sector, affecting just
about every route of administration from oral to injectable. It is well known that
during their development, drugs with narrow therapeutic indexes create a major
challenge for pharmaceutical scientists. The use of nanotechnology for the delivery
of such drugs can significantly overcome this problem. Also, the biodistribution and
pharmacokinetic parameters of the drug encapsulated in various nanocarriers can
be significantly improved compared to free drug.
Nanotechnologies can also offer the opportunity to take different measurements
in parallel or to integrate several analytical steps, from sample preparation to detec-
tion, into a single miniaturized device. Such a device could contain enough hard-
wired intelligence and robustness to be used by the patient and deliver a multitude of
data to the practitioner. Additionally, the use of nanoelectronics will improve the
14 Nanotechnology Applications in Cancer Imaging and Therapy

sensitivity of sensors based on already established methods. Nanotechnology will
also have a great impact on the methodologies available for both disease and drug
discovery and consequently will influence the scope and throughput of
pharmaceutical developments.
2.2 Nanotechnology Approaches for In Vivo Diagnostics
The application of nanotechnology to in vivo diagnostic imaging technology is pav-
ing the way for the future of health care. Using these diagnostics, doctors may one
day be able to tailor individual therapies to the very molecules that distinguish a
patient’s cancer from other cancer types. The goal of in vivo nano-based diagnostics
research is to create highly sensitive, highly reliable detection agents that can target
abnormal tissue. The use of nano-object labels, such as quantum dots, nanorods,
and nanowires, permits for highly multiplex tagging of unknown molecules in a
sample and their subsequent tag-by-tag recognition. The recognition can be carried
out using nuclear, optical, or electrochemical means. It is anticipated that these
novel nanotechnology-based assays will exhibit superior sensitivity and specificity
and the ability to measure multiple signatures (from both genome and proteome)
simultaneously. These newly developed sensors, in conjunction with integrated
sample preparation, can facilitate migration of clinical lab tests to the physician’s
office, near the patient, or other point of care, where rapid access to diagnostic
information could lead to more effective and timely medical decisions.
The nanoagents can be used in combination with various imaging modalities to
increase the diagnostic yield by substantially enhancing the imaging contrast and/or
highlighting the presence of a suspicious area. The same agents can transport vari-
ous drugs, thus also contributing to the therapeutic process. With this strategy, the
tissue of interest can initially be imaged, using target-specific contrast nano-
structures. Then, combined with a pharmacologically active agent, the same target-
ing strategy can be used for applying therapy. Finally, monitoring of treatment
effects is possible by sequential imaging.
Originally, imaging techniques were able to detect changes in the appearance of
tissues only when the symptoms were relatively advanced. Later, contrast agents
were introduced to more easily identify and map the locus of disease. Today,
through the application of nanotechnology, both imaging tools and marker/con-
trast agents are being radically refined toward the end goals of detecting disease as
early as possible, eventually at the level of a single cell, and monitoring the effective-
ness of therapy. The convergence of nanotechnology and medical imaging opens the
doors to an innovation in molecular imaging (also called nanoimaging) in the fore-
seeable future, leading to the detection of a single molecule or a single cell in a com-
plex biological environment. Nanoengineered technologies and molecular targeting
strategies provide an exciting nexus for further development in detection,
monitoring, and treatment of various diseases.
Nanotechnology facilitates further refinement of diagnostic techniques, leading
to high throughput screening (to test one sample for numerous diseases, or screen
large numbers of samples for one disease) and ultimately point-of-care diagnostics.
These technological advancements will pave the way toward major advances in the
2.2 Nanotechnology Approaches for In Vivo Diagnostics 15

manner drugs can be prescribed in the future, by enabling the goal of personalized
medicine that is tailored to individual needs. It is interesting to note that many new
in vitro techniques developed for medical testing are also finding important diverse
applications, such as in environmental monitoring and security.
2.2.1 Molecular Imaging Approaches for In Vivo Diagnostics
Molecular imaging, defined as the in vivo characterization and measurement of bio-
logical processes at the cellular and molecular levels, is an endeavor to image the
molecular makeup of the macrofeatures currently visualized using “classical” diag-
nostic imaging modalities [6]. Molecular, functional, and metabolic imaging tech-
niques currently permit the physician or scientist to visualize important
disease-causing physiologic, cellular, and molecular processes in living tissue [6–11].
With the postgenomic advances in proteomic research, there is an opportunity to
particularly image altered gene products, molecular pathways, and tumor-specific
receptors. Molecular imaging can also make available important information on
how a tumor progresses and responds to certain drug therapies [12, 13]. For exam-
ple, molecular imaging and image-guided therapy are now indispensable tools for
monitoring disease and in developing almost all the applications of in vivo
nanomedicine.
While molecular imaging with exogenous agents may be universally attractive
for most conventional imaging modalities such as magnetic resonance imaging
(MRI), nuclear imaging (such as gamma imaging), positron emission tomography
(PET), and X-ray computed tomography (CT), molecular imaging requires suffi-
cient signal arising fromminutequantities of targeting agent. MRI and X-ray imag-
ing techniques typically require millimolar amounts of an exogenous contrast agent,
but recent advances in MRI have demonstrated that potential contrast can exist at
concentrations as low as micromolar amounts. However, the amounts of exogenous
contrast agent required in MRI and X-ray imaging are still substantial for sufficient
signals for human imaging. Hence, the challenge of molecular diagnostic imaging
lies with nuclear and optical techniques, which enable imaging using exogenous
contrast agents at levels that are orders of magnitude smaller (nanomolar).
To smooth the progress of molecular imaging technology for cancer diagnosis
and treatment, there have to be linked developments in image enhancement agents,
probes, and ligands that selectively and specifically recognize the differences
between tumor and normal cells [14, 15]. For efficient cancer diagnosis, the current
methods of imaging, including nuclear, magnetic resonance, and X-ray CT, and var-
ious optical methods are continuously being improved in order to enhance the image
resolution and show molecular differences. For MRI, for instance, magnetic
nanoparticles based on iron oxide chemistry have been developed and
functionalized with various ligands such as TAT peptide [16], transferrin [13], and
folate [17] for detection of tumor cells. Functionalized magnetic nanoparticles pro-
vide a distinctive platform for molecular imaging because they can be detected by
conventional MRI methods, can be “tailored” for specific signal recognition, and
can be targeted to the disease site based on the enhanced permeability and retention
(EPR) effect of the tumor vasculature [18]. The principal effect is the decrease of
bothT
1
andT
2
*
relaxation times. For instance, uptake by tumor cells is less efficient
16 Nanotechnology Applications in Cancer Imaging and Therapy

than by healthy cells, because the former do not have effective reticuloendothelial
systems, and hence they can be differentiated in MRI scans.
Molecular imaging covers a large variety of noninvasive techniques used for
detection and visualization of biological entities in living organisms, down to the
cellular level [7, 19]. Targeted molecular imaging is important for a wide range of
diagnostic purposes, such as the identification of suspicious lesions or inflamma-
tions, the visualization of vascular structures around lesions, or the determination
of specific disease states. It is also important for research on controlled drug release,
in assessing the distribution of a drug, and for the early detection of unexpected and
potentially dangerous drug accumulations. The ability to trace the distribution of a
drug leads to the possibility of activating it only where needed, thus reducing the
potential for toxicity [20]. Molecular imaging opens up the possibility of observing
molecular and cellular events and monitoring them simultaneously by means of dif-
ferent contrast agents. It can also potentially be combined with molecular therapy in
an integrated technological platform [21]. Recent advances in molecular biology,
biochemistry, and pharmacology have increased the sensitivity and versatility of
molecular imaging techniques.
There are two distinct categories of molecular imaging. The first one uses an
exogenous probe (e.g., a radiopharmaceutical) to detect and visualize molecular
targets in a biological sample [22, 23]. To detect these molecules, a detector (e.g.,
gamma camera) is usually employed. The probes can be ions, molecular aggregates,
or nanoparticles. The latter can be adapted for improved signal strength and speci-
ficity. The second category of molecular imaging uses contrast agents (labeled
analogs of biological entities such as proteins), which localize with certain specific-
ity into subregions of an organism, tissue, or cell [24, 25]. The localization process
is not yet completely understood.
Recently, nanoparticles were used as contrast agents for such techniques. There
are currently a wide variety of nanoparticles, both organic and inorganic, capable of
targeting cells or extracellular entities as diagnostic agents. Nanoimaging is a new
concept that has much in common with the field of molecular imaging. Both catego-
ries use standard imaging techniques such as single-photon emission computed
tomography (SPECT) and PET and new optical imaging techniques such as fluores-
cence imaging, diffuse fluorescence tomography (DFT), confocal microscopy (CM),
and optical coherence tomography (OCT).
MRI and CT have recently become part of molecular imaging by using specific
contrast agents [13, 26–28]. Unlike classical anatomic imaging, molecular imaging
may not provide a complete picture of an organism. For diagnostic purposes, a com-
bination of anatomic and molecular imaging is best, as demonstrated by the recent
development of PET/CT and SPECT scanners [28].
Optical imaging modalities can also take advantage of enhanced contrast pro-
vided by various molecular markers. For instance, the introduction of refractive
index changes increases the intensity of the backscattered light. Functionalized
nanostructures can be used to induce such changes. Optical imaging has started to
gain more terrain in medical imaging. It is based on detecting the transmission of
light (photons) through biological tissue. Photon transport in biological tissue is
severely affected by the processes of absorption and scattering. Absorption occurs
when the energy associated with the photon frequency matches an energy transition
2.2 Nanotechnology Approaches for In Vivo Diagnostics 17

state within the tissue, causing the photon energy to be absorbed by the tissue and
thus reducing the total photon energy. Scattering occurs when light is incident upon
the atoms of the tissue. This causes the atom’s electrons to accelerate and radiate in
various directions and causes light to deviate from its original path. Absorption and
scattering are wavelength and penetration-depth dependent. Absorption is large in
the ultraviolet (UV), near visible, and infrared (IR) but low in the red and near-infra-
red (NIR) between 650 and 1,000 nm [29]. For animal and human in vivo and in
vitro molecular imaging, the optical imaging technologies of diffuse optical tomog-
raphy (DOT), NIR fluorescence imaging, confocal microscopy, optical coherence
tomography, fluorescence protein imaging, and bioluminescence imaging (BLI) are
emerging as powerful tools for measuring dynamic metabolic processes and probing
protease, protein, and enzymatic activity [30–40].
Fluorescence labeling of molecular markers has made possible the analysis of
protein-protein interaction, high-resolution microscopy of single molecules, cells,
and tissues, and the imaging of small animals without using ionizing radiations. The
spatial resolution is inversely proportional to the depth of imaging [36–38]. For this
reason, fluorescent imaging is limited to visualization of subcutaneous lesions or
thin (small) animals. Fluorescence tomography can be applied to small animals
using either reflectance or transmittance mode. Rotating the detector around the
animal makes possible a three-dimensional reconstruction of the fluorescence
source location. This technique is less hazardous than SPECT or PET since the
probes used are not radioactive.
Optical imaging plays a key role in the rapidly developing field of molecular
imaging. The spectroscopic nature and high-resolution imaging capabilities of light
provide a means for probing biological morphology and function at the cellular and
molecular levels. In the past few years, novel molecular imaging probes have been
employed as contrast agents for optical imaging [41–47]. Classes of probes used for
optical imaging include those that alter the local optical scattering or absorption
properties of the tissue [47], those that modulate these local optical properties in a
predictable manner [45], and those that are detected utilizing spectroscopic OCT
principles [47]. By functionalizing and targeting probes to specific cellular and
molecular sites, the detection and spatial localization of the probes through imaging
techniques add molecular specificity. Therefore, the potential of molecular optical
imaging becomes great, providing morphological, spatial, and functional
information at the molecular level.
2.2.2 Nanotechnology-Based Contrast Agents for In Vivo Imaging
A wide range of nanoparticles and molecule-type targeting and contrast enhance-
ment agents are currently used in medical imaging [5, 6, 9, 10]. These include iron
oxide–based nanoparticles, gold nanoparticles, quantum dots, dendrimers,
liposomes, and so forth.
The iron oxide nanoparticles consist of Fe
2
O
3
or Fe
3
O
4
colloids. Iron oxide
colloids (~1 to 20 nm) are usually prepared by coprecipitation of ferrous and ferric
salts in water in the presence of citric acid and sodium hydroxide. The iron oxide
colloids are washed to remove excess sodium hydroxide and then oxidized toγ-Fe
2
O
by suspension in nitric acid and subsequent stirring at moderate temperatures. The
18 Nanotechnology Applications in Cancer Imaging and Therapy

iron oxide colloids are usually embedded in poly(ethylene oxide)-modified
poly(epsilon-caprolactone) (PEO-PCL) nanoparticles a few hundred nanometers in
size, which can be used as carrier vehicles for cytotoxic drugs (e.g., paclitaxel). Scan-
ning electron microscopy (SEM) images of iron oxide colloids entrapped in 200-nm
PEO-PCL nanoparticles fabricated in Mansoor Amiji’s lab at Northeastern Univer-
sity are shown in Figure 2.1.
The iron oxide nanoparticles for medical applications (i.e., drug delivery) need
to be highly engineered in order to maximize their systemic circulation and tumor
cell targeting. They are fabricated to be 200–400 nm in size, which will allow for
better exploitation of the enhanced permeability and retention effect [18], and their
surface is usually modified with poly(ethylene oxide) for long circulating properties
upon systemic administration. These nanoparticles can be loaded with drugs and
conjugated with a monoclonal antibody (mAb) for preferential tumor targeting.
They can bind to drugs, proteins, enzymes, antibodies, or nucleotides and can be
2.2 Nanotechnology Approaches for In Vivo Diagnostics 19
(c)
(b)(a)
Figure 2.1(a) SEM image of the iron oxide PCL-coated nanoparticles (mean size ~ 200 nm). (b)
Transmission electron microscopy image of Fe
2
O
3
colloids in PCL nanoparticles. The arrows indicate
Fe
2
O
3
particle clusters. (c) SEM image of the Fe
2
O
3
colloids used for PCL iron oxide nanoparticle fab-
rication (mean size ~ 20 nm).

directed to an organ, tissue, or tumor using an external magnetic field or can be
heated in alternating magnetic fields for use in hyperthermia.
Superparamagnetic iron oxide (SPIO) nanoparticles with appropriate surface
chemistry are being widely used experimentally for numerous in vivo applications
such as magnetic resonance imaging contrast enhancement, tissue repair,
immunoassay, detoxification of biological fluids, hyperthermia, drug delivery, cell
separation, and so forth.
Currently, paramagnetic gadolinium ion complexes and SPIO nanoparticles
(e.g., Feridex) are the most common contrast agents used. Recent studies show posi-
tive results for the use of iron oxide nanoparticles in lymphangiography [48], for
liposomes with integrin antibodies and gadolinium contrast agents in angiogenesis
[20], and for nanoparticle emulsions with gadolinium contrast agents in MRI con-
trast enhancement [49].
SPIO nanocrystals are used as well, on large scales as MR contrast imaging
agents. They have a concentration-dependent influence on the relaxation times (T
1
andT
2
), enhancing the contrast of labeled domains [50–52]. They can serve as nega-
tive contrast agents (i.e., appearing dark in MR images). The SPIO agents consist of
an inorganic core of iron oxide 4 to 5 nm in diameter (e.g., magnetite Fe
3
O
4
), coated
with a polymer [50]. SPIO surfaces can be functionalized with small molecules ame-
nable to the bioconjunction of proteins and antibodies for targeted imaging [17].
SPIO particle colloids have been used to monitor gene expression and to detect dif-
ferent pathologies such as liver cancer [52], atherosclerosis [53], brain inflammation
[54], and so forth. An example of using nanoparticles for contrast and localization
enhancement in MRI is the effort to quantify angiogenesis [55, 56]. Efforts have
been directed toward the neovascularα
v
β
3
integrin receptor, which is expressed on
activated endothelial cells but not on mature quiescent cells.
Nanoparticles based on gold chemistry have attracted significant research and
practical attention recently. They are versatile agents with a variety of biomedical
applications including use in highly sensitive diagnostic assays, thermal ablation,
and radiotherapy enhancement, as well as drug and gene delivery. They offer addi-
tional advantages such as being nontoxic, being easily manufactured using mild
aqueous chemistry into a variety of particle sizes ranging from 2 to 100 nm in diame-
ter, allowing surface modification using thiol chemistry, and having high electron
density of the metal, allowing for visualization of the nanoparticles by electron
microscopy and X-ray analytical techniques. The most important advantage of gold
nanoparticles is that they can be functionally “tailored” by surface modification
using thiol chemistry to produce a variety of probes for desired applications. Trans-
mission electron microscopy (TEM) images of 20- to 30-nm gold nanoparticles
fabricated in Amiji’s lab at Northeastern University are shown in Figure 2.2.
For biomedical applications, surface functionalization of gold nanoparticles is
essential in order to target them to specific disease areas and allow them to selec-
tively interact with cells or biomolecules. Surface conjugation of antibodies and
other targeting moieties is usually achieved by adsorption of the ligand to the gold
surface. Surface adsorption, however, can denature the proteins or, in some cases,
limit the interactions of the ligand with the target on the cell surface due to steric hin-
drance. Additionally, for systemic applications, long-circulating nanoparticles are
desired for passive targeting to tumors and inflammatory sites. Poly(ethylene glycol)
20 Nanotechnology Applications in Cancer Imaging and Therapy

(PEG) modification of nanoparticles affords a long circulating property by evading
macrophage-mediated uptake and removal from the systemic circulation. Surface
modification of gold nanoparticles through the PEG spacer would, therefore, allow
the modified nanoparticles to remain in the systemic circulation for the prolonged
period and provide flexibility to the attached ligand for efficient interaction with its
target.
In Figure 2.3 are shown coumarin-PEGthiol functionalized gold nanoparticles
binding to breast cancer cells MDA-MB-231. The coumarin-PEGthiol function-
alized gold nanoparticles were internalized by nonspecific endocytosis within the
first few minutes. Within 30 minutes, a large fraction of the administered dose was
found to be in the early endosomes. As time progressed, the nanoparticles traversed
through the cytosol and reached the perinuclear region within 1 hour of incubation.
The hetero-bifunctional PEG derivative with a molecular weight of 1,500 daltons
was sufficient to allow flexibility in the attached fluorescent ligand but did not pre-
vent cellular entry of the nanoparticles. In addition, none of the gold nanoparticles
was observed inside the nucleus even after 24 hours of incubation.
Particle-based technologies have also generated much interest for use in enhanc-
ing contrast for optical imaging and microscopy due to their optical capabilities and
ease of adding surface modifications. Optical technologies combined with molecu-
lar contrast agents show a real promise for high-resolution noninvasive functional
imaging of tissue with improved sensitivity, specificity, and cost effectiveness rela-
tive to current approaches. Numerous studies have shown the viability of scatter-
ing-based optical approaches, including spectroscopy, confocal microscopy, and
optical coherence tomography [43, 44, 47]. For example, Lee et al. [57] have devel-
oped oil-filled encapsulating protein microspheres that can incorporate various par-
ticles such as gold and carbon to alter backscattering optical signatures for OCT.
These microspheres were recently used as a contrast agent in a mouse study [57].
Work by Sokolov et al. [58] demonstrated that the detection of precancerous cells
using confocal reflectance imaging can be enhanced with gold nanoparticles
bioconjugated with molecule-specific markers bound to their targets.
Various nanoparticles conjugated with monoclonal antibodies have been used
by several investigators as contrast agents for CM [41] and OCT [42, 43]. Probes
designed for efficient light scattering are sensed either directly by detecting their
2.2 Nanotechnology Approaches for In Vivo Diagnostics 21
(a) (b)
0.7 2 4 3010 100 50000
0
50
100
SDP weight result
Weight %
1000
Size (nm)
Figure 2.2Gold nanoparticles used in medical imaging. (a) Coulter particle size analysis and (b)
transmission electron microscopy image of gold nanoparticles.

scattered light or indirectly through their attenuation of the incident light [44]. Ste-
ven Boppart from the University of Illinois at Urbana has pioneered work as well to
enhance the OCT imaging contrast by using both iron oxide and gold nanoparticles
[43]. An example of OCT contrast enhancement using magnetic nanoparticles is
shown in Figure 2.4.
Ligand-targeted acoustic nanoparticles are also being used as contrast agents in
ultrasound (US) imaging [59]. The contrast enhancement technique has allowed for
the imaging of specific cell-surface receptors and has thus brought this technique
into the molecular imaging field. The resolution of US imaging is approximately 1
mm, but the sensitivity is poor, so this technique is used mainly for morphological
characterization.
Recently, rare Earth–doped nanocrystals and quantum dot nanocrystals
(Qdots) were also used as contrast enhancement agents [60, 61]. Depending on their
coating and their physical and chemical properties, they can target a specific tissue
or cell and can be made to fluoresce for imaging purposes. Qdots are nanocrystals (1
to 10 nm in diameter) made of inorganic semiconductor materials in which absorp-
tion of a photon with the energy of a few electron volts can result in the creation of a
bound electron-hole pair, called anexciton. The electron and hole recombine, emit-
ting a photon with lower energy than that of the one used for excitation. Qdots have
size-dependent optical properties. For example, the shift of absorption and emission
spectra is a function of particle diameter. Using appropriate material composition
22 Nanotechnology Applications in Cancer Imaging and Therapy
(a) (b)
(c)
Figure 2.3Cellular uptake and distribution of coumarin-poly(ethylene glycol)-thiol functionalized
gold nanoparticles in MDA-MB-231 human breast adenocarcinoma cells. (a) Differential interfer-
ence contrast, (b) epifluorescence, and (c) merged images.

and size of Qdots, the visible, NIR, and even far IR spectra can be covered. This
property is very useful, especially if one wants to cover the NIR spectrum, for which
only a few good dyes exist. Qdots have a broad excitation spectrum. A combination
of nanocrystals can be excited with a certain laser wavelength and detected concur-
rently in different color channels, so the multiplexing capabilities are greater than
those of conventional materials. This property facilitates the use of Qdots in com-
plex surroundings such as living cells. They are brighter than conventional dyes,
offering a more intense fluorescent light emission, which means that even a small
number of Qdots is enough to produce an adequate signal. They also are more
photostable, so the acquisition time can be longer.
Qdots are expected to be particularly useful for imaging in living tissues, where
signals can be obscured by scattering. The Qdots have already demonstrated their
potential application in medical imaging, such as for lymph node surgery and track-
ing metastatic tumors [47, 48]. Qdots have a large surface area (tens to hundreds of
nm
2
). Chemical groups with different functionalities can be attached to the surface
of a Qdot, producing a multimodality probe. The biodistribution of Qdots in a
2.2 Nanotechnology Approaches for In Vivo Diagnostics 23
CombinedMM-OCTStructural
No magnetic agents Injected magnetic agents
Magnetomotive OCT signal (dB)Structural OCT signal (dB) −300 20 0200 mµ
Figure 2.4Contrast enhancement in OCT using magnetic nanoparticles. Images were acquired
from in vitro chicken breast tissue. Structural and magnetomotive OCT images were acquired in tan-
dem, without and with the topical administration ofiron oxide nanoparticles. Note the large dynamic
range and excellent background signal rejection of this technique [26]. (Courtesy of Professor S.
Boppart, the Biophotonics Imaging Laboratory,University of Illinois at Urbana-Champaign.)

living organism can be studied using different techniques, as micro-PET, two-pho-
ton light microscopy, electron microscopy, and so forth. Micro-PET scanning, for
example, has already been used for small animal studies. The animals were injected
with PEGylated Qdots having DOTA (a chelator for heavy metals, such as
64
Cu
radioactive isotope used for PET) attached to their surfaces. Toxicological studies
are being undertaken to precisely study their impact on humans, animals, and the
environment. New developments are focusing on the nanoparticle coating, to
improve its targeting efficiency and biocompatibility.
Dendrimers form another class of contrast enhancement agents. They are poly-
meric macromolecules with multiple branches originating from a central core. Their
size can be tuned to a desired value (usually a few nanometers) by controlling the
polymer growth. The dendrimers are very attracting for bioimaging because of the
cavities in the central core and because of their multiple branches, which are open to
bioconjunction and functionalization. Contrast agents used for PET, MRI, and fluo-
rescence techniques have already been conjugated to dendrimers and used for
bioimaging [62, 63].
Liposomes are used as contrast imaging agents as well. They are closed bilayer
vesicles that form upon hydration of dry phospholipids. They can be created either
as large multilamellar liposomes (up to 1µm onionlike structures) or unilamellar
vesicles (approximately hundreds of nanometers in size). These liposomes can act as
contrast agent carriers. Recently, MRI tumor contrast was enhanced using magne-
tite-loaded liposomes conjugated with targeting antibodies [64].
The contrast agents used to generate a measurable signal should interfere as lit-
tle as possible with the physiology. The signal increase is usually obtained by
increasing the contrast agent concentration, and this can result in cytotoxic effects.
For example, the radioactive labels used for PET imaging can damage the DNA,
while heavy metals such as cadmium present in Qdots can induce cell apoptosis.
Unfortunately, these effects are difficult to characterize and quantify. The contrast
agent concentration should be high enough to have a good signal-to-noise ratio and
low enough to reduce the cytotoxic effects. Another important parameter is the
duration of the exposure. The exogenous probes can be either rapidly eliminated by
excretion or retained for several days, resulting in long-term toxic effects.
Cytotoxicity is a major problem and may be a limitation to the use of nanoparticles.
The studies in this direction will be critical for future implementation of the
nanoimaging techniques in human medicine. The National Cancer Institute has
established the Nanotechnology Characterization Laboratory (http://ncl.cancer.
gov) to perform preclinical efficacy and safety testing of nanoparticles and to facili-
tate regulatory review of nanotechnologies intended for cancer diagnosis, imaging,
and therapy.
2.3 Nanotechnology-Based Drug Delivery Systems for Cancer Therapy
Although significant advances have occurred in our understanding of tumor origin,
growth, and metastasis and many different types of pharmacological agents have
been developed over the years to treat tumors, the problem of optimum delivery
remains a formidable challenge. It is further compounded by newer anticancer
24 Nanotechnology Applications in Cancer Imaging and Therapy

drugs, developed through advances in molecular and cellular biology, which are
either very hydrophobic or hydrophilic macromolecules (proteins and DNA) with
very poor solubility and diffusional properties, respectively, when administered into
the systemic circulation. The method by which a drug is delivered can have a signifi-
cant effect on its efficacy. Some drugs have an optimum concentration range within
which maximum benefit is derived, and concentrations above or below this range
can be toxic or produce no therapeutic benefit at all. On the other hand, the very
slow progress in the efficacy of the treatment of severe diseases has suggested a
growing need for a multidisciplinary approach to the delivery of therapeutics to tar-
gets in tissues. From this, new ideas on controlling the pharmacokinetics, pharm-
acodynamics, nonspecific toxicity, immunogenicity, biorecognition, and efficacy of
drugs were generated. These new strategies, often calleddrug delivery systems
(DDSs), are based on interdisciplinary approaches that combine polymer science,
pharmaceutics, bioconjugate chemistry, and molecular biology.
The emergence of nanotechnology is likely to have a significant impact on the
drug delivery sector, affecting just about every route of administration from oral to
injectable. NanoMarkets expects the dosing benefits of nanoenabled drug delivery
systems to be extended to compounds used in treating both infectious disease and
cancer and has identified six types of drug delivery systems in which nano-
technology is likely to have a significant impact. For injectable drugs, nano-
technology is already generating new dosage forms that are easier to administer, are
more pleasant for the patient to receive, and confer a competitive advantage in the
marketplace. Nanotechnology is also opening up new opportunities in implantable
delivery systems, which are often preferable to the use of injectable drugs, because
the latter frequently display first-order kinetics (the blood concentration goes up
rapidly but drops exponentially over time). This rapid rise may cause difficulties
with toxicity, and drug efficacy can diminish as the drug concentration falls below
the targeted range. In contrast, implantable time release systems may help minimize
peak plasma levels and reduce the risk of adverse reactions, allowing for more pre-
dictable and extended duration of action, reducing the frequency of redosing, and
improving patient acceptance and compliance. Nanoimplants will also be used in
the not-too-distant future for treating cancer. Among the first nanoscale devices to
show promise in anticancer therapeutics and drug delivery are structures called
nanoshells, which NanoMarkets believes may afford a degree of control never
before seen in implantable drug delivery products.
2.3.1 Fundamental Requirements for Drug Delivery Systems
To minimize drug degradation and loss, to prevent harmful side effects, and to
increase drug bioavailability and the fraction of the drug accumulated in the
required zone, various drug delivery and drug targeting systems are currently under
development [65–68]. A large number of pharmaceutical carriers are used for effi-
cient drug delivery. Among these, colloidal drug carrier systems such as micellar
solutions, vesicle and liquid crystal dispersions, as well as nanoparticle dispersions
consisting of small particles of 10- to 400-nm diameter show great promise [69–72].
When developing these carrier formulations, the goal is to obtain systems with opti-
mized drug loading and release properties, long shelf life, and low toxicity. The
2.3 Nanotechnology-Based Drug Delivery Systems for Cancer Therapy 25

incorporated drug participates in the microstructure of the system and can influence
the drug delivery efficiency due to molecular interactions, especially if the drug
possesses amphiphilic and/or mesogenic properties.
Controlled drug release and subsequent biodegradation are also very important
for developing successful formulations. Two major mechanisms can be distin-
guished for addressing the desired sites for drug release: passive and active targeting.
For passive targeting, the natural defense mechanisms can be utilized, in which
phagocytic cells remove foreign particles from the body. Macrophages are among
the most important components of the immune defense system and play a major role
in clearance of particulate contrast media such as ultrasmall paramagnetic iron
oxide particles (USPIO).
An example of passive targeting is the preferential accumulation of
chemotherapeutic agents in solid tumors as a result of the enhanced vascular perme-
ability effect of tumor tissues compared with healthy tissue [18]. For the cancer
therapy strategy to be effective, the systemically administered agent must be able to
reach the tumor’s mass in sufficient concentration, traverse through the tumor’s
microcirculation, diffuse into the interstitium in sufficient concentrations, and
remain at the site for the duration to induce tumoricidal effect. Blood vessels in most
solid tumors possess unique characteristics that are not usually observed in normal
blood vessels. Examples of such characteristics are extensive angiogenesis and high
vascular density extensive extravasation (vascular permeability), induced by various
vascular mediators such as bradykinin or nitric oxide. Tumor vascular permeability
is important in delivery of macromolecular anticancer agents. This makes it possible
to do selective targeting of drugs for tumors. Such selective targeting is not possible
with low-molecular-weight substances. Small molecules, as are many of the drugs
being used today for chemotherapy, do not discriminate tumor tissue from normal
tissue; they reach most normal tissues and organs as well as tumor tissues by free dif-
fusion-dependent equilibrium. For example, common drugs such as antibiotics
spread throughout the body within a few minutes after subcutaneous injection. Due
to the porosity of the tumor vasculature and the lack of lymphatic drainage,
blood-borne macromolecules and nanoparticles are preferentially distributed in the
tumor due to the EPR effect. It has been found that the effective pore size of most
peripheral human tumors ranges from 200 to 600 nm in diameter, with a mean of
about 400 nm. The EPR effect allows for passive targeting to tumors based on the
cutoff size of the leaky vasculature.
Systemic circulation of the drug carrier is very important as well. For example,
polymeric nanoparticles administered into the systemic circulation will be essen-
tially removed within an hour of administration by the macrophages of the
reticuloendothelial systems. To prolong the circulation of nanoparticles by evading
the macrophages, the surface of the nanoparticle is modified with water-soluble
polymers [73]. Poly(ethylene glycol) or poly(ethylene oxide) are very popular for
surface modification of nanoparticulate drug delivery systems since they have a long
history of safe use in biological and pharmaceutical products [74, 75]. Sur-
face-bound PEG chains extend into the aqueous physiological environment and
repel proteins, decrease antibody formation, and increase the circulation of the for-
mulation in the plasma for an extended period of time by the steric repulsion
mechanism.
26 Nanotechnology Applications in Cancer Imaging and Therapy

A project was carried out in Amiji’s laboratory at Northeastern University to
study the intracellular distribution of poly(ethylene oxide)-modified poly(epsi-
lon-caprolactone) nanoparticles in MCF-7 human breast cancer. As shown in Fig-
ure 2.5, high binding was observed.
Another study was carried out in the same laboratory to determine the
biodistribution profile of tamoxifen when administered intravenously (IV) as a sim-
ple solution and when encapsulated in 200-nm polymeric nanoparticles, with or
without surface stabilizing agents. FemaleNu/Nu(athymic) mice bearing human
breast carcinoma were used in this study. The results of in vivo biodistribution stud-
ies are summarized in Figure 2.6. The tumor concentrations of different standard
(plain injection, unmodified PCL nanoparticles) and test (PCL nanoparticles sur-
face-modified with Pluronic F-68 and Pluronic F-108) formulations of tamoxifen
are shown. At the 1-hour time point, a higher tumor concentration was reached
with Pluronic-modified nanoparticles, the maximum being with F-108. However,
at 6-hour postinjection, the amount retained within the tumor was maximal for
nanoparticles having Pluronic F-68 as the adsorbed layer, though no significant
difference was seen among the test formulations.
Dealing with cancer outside of surgery is akin to waging guerrilla warfare on an
unknown enemy, sometimes destroying the whole village to attack a few or, in other
instances, being unable to locate the enemy who are well hidden from the incoming
2.3 Nanotechnology-Based Drug Delivery Systems for Cancer Therapy 27
(a)
(b) (c)
Figure 2.5(a) Scanning electron micrograph of poly(ethylene oxide)-modified
poly(ε-caprolactone) nanoparticles and the intracellular distribution of the nanoparticles in MCF-7
human breast cancer cells after 2 hours of incubation as observed with (b) differential interference
contrast and (c) fluorescence confocal microscopy where the rhodamine-containing nanoparticles
are localized in the endosomal compartments.

missiles. To wage an effective war against cancer, we must have the ability to selec-
tively attack the cancer cells while saving the normal tissue from excessive burdens
of drug toxicity. This is calledactive targetingof cancer. However, because many
anticancer drugs are designed to simply kill cancer cells, often in a semispecific fash-
ion, the distribution of anticancer drugs in healthy organs or tissues is especially
undesirable due to the potential for severe side effects. Consequently, systemic appli-
cation of these drugs often causes severe side effects in other tissues (e.g., bone mar-
row suppression, cardiomyopathy, or neurotoxicity), which greatly limits the
maximal allowable dose of the drug. In addition, rapid elimination and widespread
distribution into nontargeted organs and tissues requires the administration of a
drug in large quantities, which is often not economical and sometimes complicated
due to nonspecific toxicity. This vicious cycle of large doses and the concurrent tox-
icity present major limitations to current cancer therapy. In many instances, it has
been observed that the patient succumbs to the ill effects of the drug toxicity far
earlier than the tumor burden.
Active targeting is based on the use ofligandsand can be defined as the
ligand-directed, site-specific accumulation of contrast and/or therapeutic agents.
Ligands are molecules that bind to a protein, such as cell surface receptors. A wide
variety of ligands can be used to target cellular biomarkers, including antibodies,
peptides, polysaccharides, aptamers, and drugs. The ligands are attached to the con-
trast agent, either covalently (direct conjugation) or noncovalently (indirect conju-
gation). The circulating half-life of such agents should be in the range of hours, to
permit sufficient exposure and interaction with the binding sites (cellular epitopes).
Additional requirements are that they bind specifically to the epitope chosen for
visualization, and that they combine a prominent contrast-to-noise enhancement
28 Nanotechnology Applications in Cancer Imaging and Therapy
0
1 hour postinjection 6 hours postinjection
% activity recovered per gram
5
10
15
20
25
30
35
NP with PF-108
NP with PF-68
NP without Pluronic
Plain injection
Figure 2.6Tumor concentrations as a function of time of [
3
H]-tamoxifen administered as controls
and in poly(ethylene oxide)-modified PCL nanoparticles in femaleNu/Nu(athymic) mice bearing
human breast carcinoma (MDA-MB-231) xenografts. Results are expressed as mean±SD (n= 4).

with a low background signal. Finally, these agents require an acceptable toxicity
profile and should meld easily into the clinical routine. Targeted contrast should be
applicable with standard commercially available imaging modalities and should
offer the additional possibilities for delivery of therapeutics. The effort for develop-
ment of target-specific anticancer agents, such as Herceptin and Gleevec, and vac-
cines is highly encouraging. However, many of the current anticancer drugs can be
made target specific by inclusion in the delivery system, such as polymeric
nanoparticles, liposomes, and micelles. As previously shown, in order to reach the
tumor cells, systemically administered drugs have to overcome a number of trans-
port obstacles in the blood stream, which may include rapid metabolism and clear-
ance of drugs from the body, physiological barriers in transportation of the drugs
from the site of administration to the tumor cells, drug resistance, and toxicity of the
anticancer drugs to normal cells. An additional major barrier to systemic drug deliv-
ery in solid tumors has been identified as being caused by the tumor microvascula-
ture, such as highly convoluted and tortuous blood supply in the tumor periphery,
the high interstitial pressure, and absence of a functional lymphatic drainage, which
were reported to cause problems in the distribution of the drug in the solid tumor.
Once the drug reaches the tumor cells, many peptide and protein drugs as well as
antibodies exert their action extracellularly, by receptor-mediated interactions.
Many other drugs, however, have their targets inside the cell. In the latter case, low
permeability of cell membranes to macromolecules often represents an additional
obstacle for the development of peptide- and protein-based anticancer
formulations.
Multidrug resistance (MDR) of tumors is another problem when fighting
against cancer. Due primarily to lack of tumoricidal concentrations of drug reach-
ing the cell or insufficient contact time for cell cycle–specific cytotoxicity agents,
many tumor cells become resistant to one or more cytotoxic agents. Many times
MDR tumors are refractory to single- and multi-anticancer drug therapy. There are
many putative origins of MDR, but one of the most studied today is the
P-glycoprotein efflux pump. The pump serves to remove drug molecules from the
cell before they can act at their particular subcellular target inside the cell. Other
forms of MDR are associated with the nucleus, including the DNA repair system.
An example that illustrates the limitations of current approaches for cancer therapy
is that of prostate cancer. Despite improvements in both primary surgical and radia-
tion management of prostate cancer and less advanced disease, prostate cancer
remains a leading cause of cancer death in men living in the United States. The use of
androgen suppression therapy in conjunction with external beam radiation therapy
(RT) has been shown to improve survival compared to the use of external beam RT
alone for patients with locally advanced prostate cancer. This, coupled with
improved response rates reported for combined chemotherapeutic regimens in met-
astatic disease, provides the basis for the use of combined systemic and local therapy
in men with clinically localized prostate cancer who are at high risk for harboring
occult micrometastatic disease. Thus, while progress is being made, it is commonly
recognized that improvements in the accuracy of current diagnostic methods, cou-
pled with an ability to deliver localized chemotherapies, are needed in the ongoing
battle against prostate cancer. These elements underscore the need for imaging
2.3 Nanotechnology-Based Drug Delivery Systems for Cancer Therapy 29

modalities, chemotherapy, and other advanced treatments that are specific to the
prostatic microenvironment either by selective delivery or activation.
Another problem is the toxicity of the administered drugs. To minimize the side
effects of various drugs that have high toxicity, drug encapsulation is necessary [65].
In this way the drug is carried to the site of interest and then released, without affect-
ing the rest of the body. Hydrophobic drugs, such as tamoxifen and paclitaxel, can
be encapsulated with high efficiency in hydrophobic polymeric nanoparticles, such
as PEO-modified poly(epsilon-caprolactone) nanoparticles [76].
2.3.2 Cancer Therapy Approaches Using Nanotechnologies
For cancer therapy with drug-loaded nanoparticles to be effective, functionalization
is required. Various approaches have been taken by different research groups to
functionalize carrier nanovehicles and load them with cancer killing agents (i.e.,
tamoxifen). For example, Amiji’s team at Northeastern University has used
transferrin-conjugated iron oxide nanoparticles for intracellular delivery [77]. Since
many different tumor cells are known to overexpress transferrin receptors, the
functionalized nanoparticles are internalized by receptor-mediated endocytosis.
Transferrin was attached to the iron oxide nanoparticles through noncovalent or
covalent bonds. For covalent attachment, the surface of the iron oxide was treated
with aminopropyltriethoxy silane (APTES) and transferrin was linked by
carbodimide coupling. The iron oxide nanoparticles were loaded with a cytotoxic
drug (PTX) and a near-infrared-sensitive dye (Cy-5 or ICG). Dye loading allowed
for tumor location and a very efficient control of drug delivery by means of
fluorescence imaging.
Antibody attachment is another strategy. For example, in order to prepare
immunomicelles, which are new targeted carriers for poorly soluble
pharmaceuticals, Vladimir Torchilin et al. [78] have developed a procedure to chem-
ically attach mAbs to reactive groups incorporated into the corona of polymeric
micelles made of polyethylene glycol–phosphatidylethanolamine conjugates.
Immunomicelles with attached antitumor mAb 2C5 effectively recognized and
bound various cancer cells in vitro and showed an increased accumulation in experi-
mental tumors in mice when compared with nontargeted micelles. Intravenous
administration of tumor-specific 2C5 immunomicelles loaded with a sparingly solu-
ble anticancer agent, paclitaxel, into experimental mice bearing Lewis lung carci-
noma resulted in an increased accumulation of taxol in the tumor compared with
free paclitaxel or paclitaxel in nontargeted micelles and in enhanced tumor growth
inhibition.
Various strategies are adopted for therapy. They are either drug release based or
hyperthermic. These approaches can be independent or coupled together. Drug
encapsulation is used on a large scale. As shown before, PCL encapsulation is very
effective. In the presence of serum lipases, PEO-PCL nanoparticles degrade within
10 hours and release the encapsulated drug by diffusion and degradation. Both PEO
and PCL and their degradation products do not cause any toxicity in the body and
are excreted by the renal route. While various drugs are used, one of the very effi-
cient drugs is tamoxifen. For example, tamoxifen-loaded PEO-PCL nanoparticles
could efficiently deliver the drug inside the MCF-7 breast cancer cells, where the
30 Nanotechnology Applications in Cancer Imaging and Therapy

estrogen receptors are known to be localized. Paclitaxel-loaded PEO-PCL
nanoparticles have been prepared by various research groups [72, 76, 79]. They are
currently under evaluation for the efficacy of the formulation, relative to the solu-
tion form of the drug, in a variety of tumor cell lines. For example, at Northeastern
University in Amiji’s lab, plasmid DNA has been encapsulated in PEG-modified gel-
atin nanoparticles for systemic delivery to solid tumors [74, 75]. It has been
observed that the nanoparticles enter the cell through nonspecific endocytosis and
that the PEG chains protect the encapsulated DNA in the cytoplasm until the
nanoparticles reach the nuclear membrane in about 12 hours. The PEG-modified
gelatin nanoparticles can transfect tumor cells with 60% efficiency and without any
toxicity after 96 hours (Figure 2.7). Biodistribution studies in murine tumor model
show that the PEG-modified nanoparticles have an extended circulation time (t
1/2
of
25 hr) in the plasma and are preferentially targeted to the tumor.
Hypothermia alone or coupled with drug delivery is used as well. An example of
a coupled approach is that of PCL iron oxide nanoparticles loaded with a toxic
drug. PCL-coated iron oxide nanoparticles loaded with paclitaxel can be heated
with a variable magnetic field in order to melt the PCL matrix and release the toxic
drug [80]. This makes it possible to have the cytotoxic drugs released in a controlled
mode. Increase in temperature above 40°C melts the PCL matrix, thus allowing for
the payload to be released in the tumor. Heating tumors at temperatures above
42°C for 30 to 60 minutes is referred to ashyperthermia, whereasthermal ablation
2.3 Nanotechnology-Based Drug Delivery Systems for Cancer Therapy 31
(a)
(b) (c)
Figure 2.7(a) Scanning electron micrograph of poly(ethylene glycol)- modified gelatin
nanoparticles and the transfection of enhanced green fluorescent protein plasmid DNA in NIH-3T3
cells after 96 hours of incubation as observed with (b) differential interference contrast and (c) fluo-
rescence confocal microscopy.

is when the temperature is raised to above 60°C, leading to complete heat-induced
necrosis, carbonation, and destruction [80].
The use of hyperthermia as an adjunct to radiation or chemotherapy of various
types of solid tumors has been an area of active investigation for the past 20 years, in
part due to the improvements in instrumentation and the temperature-monitoring
technique, as well as an increasing understanding of the biology of hyperthermia. In
addition, tumor cells seem to be more sensitive to heat-induced damage than normal
cells. In 1996, the International Collaborative Hyperthermia Group reported on the
outcomes of a trial that focused on hyperthermia treatment of superficial breast can-
cer [81, 82]. A total of 306 patients with advanced primary or recurrent breast can-
cer were randomized to receive either radiation therapy alone or combined radiation
and hyperthermia therapy. The primary endpoint of the trial was complete local
control; 59% of those in the combined treatment group achieved complete local
response compared to 41% in the radiation therapy only group. The best results
with hyperthermia in conjunction with radiation therapy are seen in lesions measur-
ing 3 cm or less in diameter. Clinically, hyperthermia is typically administered every
72 hours (i.e., twice a week) for a total of 10 to 12 treatments.
The majority of hyperthermia studies have used ferromagnetic iron oxide–
containing liposomes or nanoparticles. However, more recently, particular atten-
tion has been given to gold nanoparticles with a cylindrical shape, as well as to
gold nanoshells. These gold nanoparticles have special properties that might be
very beneficial for both cancer detection and therapy. Their plasmon resonance in
the near-IR region makes them ideal for hyperthermic therapy, as well as for
two-photon imaging [83, 84]. Work done by O’Neal et al. [83] demonstrates the
capability of gold nanoshells to produce hyperthermia in small animals (nude
mice) when the tumor area is irradiated with a near-infrared laser source. For
example, by treating mice tumors with about 4 W/cm
2
for several minutes, a tem-
perature increase to about 50°C has been produced to induce hyperthermia. Sub-
stantial resorption of the tumor was observed after several days of treatment.
Functionalized gold nanoparticles can alsobe loaded with a cytotoxic drug (e.g.,
paclitaxel, or PTX) by thiol conjugation. Once internalized in the tumor cells,
thiol-modified PTX exerts a synergistic cytotoxic effect on tumor cells upon sus-
tained release from the nanorods due to glutathione reduction. In this way, very
effective cancer therapy is possible.
More recently, gold nanorods are being used for in vivo therapy studies. The use
of gold nanorods has a dual advantage: They can highlight the presence of the can-
cer lesion and they can also contribute to the therapeutic effect by inducing
hyperthermia. This is possible by exploiting their capability to strongly absorb
near-infrared laser radiation. It has already been proved that for particles much
smaller than the wavelength of the incident light, the electrons in the particle move
in phase. The electrons can generate a giant dipole, which gives surface polarization
charges on each side of the particle. This acts as a restoring force on the conduction
of electrons and leads to a resonance frequency in the absorption spectrum. The
resultant sharp band is termed a surface plasmon absorption band. The absorption
spectrum of gold nanorods is characterized by two maxima (see Figure 2.8). The
first one is located around 530 nm and is attributed to the transverse surface
plasmon resonance. This band has some contribution from the spherical gold
32 Nanotechnology Applications in Cancer Imaging and Therapy

nanoparticles present in the solution, and it shifts only slightly to a shorter wave-
length with increasing nanorod aspect ratio. The other maximum appears at a lon-
ger wavelength and corresponds to the longitudinal surface plasmon (SPL)
resonance. The position of the SPL band can be changed by varying the aspect ratio
of the nanorods. As a result, this maximum can be tuned for a wavelength of inter-
est. The relation between the average aspect ratio (R) of the nanorods in solution
and the absorption maximumλ
max
of the SPL band is linear [85]. The aspect ratio of
the gold nanorods can be chosen to have the SPL band overlapping with the emis-
sion spectrum of the commercially available laser sources that can be used for the
nanorods’ heating. However, the position of the SPL band needs to be carefully
tuned in order to avoid the highly absorptive bands of some of the tissue constitu-
ents (water and blood). The SPL band also can be used for enhanced contrast imag-
ing. Boppart et al. [86] have shown for the first time OCT contrast enhancement
using gold nanoparticles.
With regard to carrier vehicle strategy for drug delivery, in many cases,
microreservoir-type (nanoparticulate) carriers may represent a valid alternative to
soluble polymeric carriers. These types of systems include liposomes, micelles, poly-
meric nanoparticles, and cell hosts. The use of such carriers allows for the achieve-
ment of a much higher active moiety/carrier material ratio compared to “direct”
molecular conjugates. They also provide a higher degree of protection against enzy-
matic degradation and other destructive factors upon parenteral administration
because the carrier wall completely isolates drug molecules from the environment.
An additional advantage of these carriers is that a single carrier is capable of deliver-
ing multiple drug species. All nanoparticles have a size that excludes the possibility
of loss by renal filtration.
Among particulate drug carriers, liposomes are the most extensively studied
and possess the most suitable characteristics for peptide (protein) encapsulation.
Liposomes are vesicles formed by concentric spherical phospholipid bilayers encap-
sulating an aqueous space. These particles are completely biocompatible, are
2.3 Nanotechnology-Based Drug Delivery Systems for Cancer Therapy 33
0
400 500 600 700 800 900 1000 1100 1200 13001400
1
2
3
4
5
l/d = 13.2
l/d=9
l/d = 4.2
l/d=1
Extinction coefficient (M*cm )
−1
610
−3
×
Wavelength (nm)
Figure 2.8Absoption spectrum of gold nanorods for different aspect ratios.

biologically inert, and cause very little toxic or antigenic reaction. Their inner aque-
ous compartment can be used for encapsulation of peptides and proteins. Many
techniques for liposome preparation require only manipulations that are compatible
with maintaining the drug (including peptide and protein) integrity. Torchilin et al.
[87] have developed long-circulating PEG-modified liposomes linked with HIV-1
TAT peptide for efficient DNA delivery in vitro and in vivo in tumor model
(Figure 2.9). However, as with other nanoparticulate delivery systems, conventional
liposomes suffer from raid elimination from the systemic circulation by the cells of
the reticuloendothelial system (RES). In order to make liposomes capable of deliver-
ing pharmaceutical agents to targets other than the RES, attempts were made to
prolong their circulation lifetime. This was achieved with the development of sur-
face-modified long-circulating liposomes grafted with a flexible hydrophilic poly-
mer, such as PEG or PEO. PEG and PEO, being the most common examples, prevent
plasma protein adsorption to the nanoparticle surface and the consecutive
recognition and uptake of liposomes by the RES.
A broad variety of examples show that, similar to macromolecules, liposomes
are capable of accumulating in tumors of various origins via the EPR effect.
Liposomal forms of at least two conventional anticancer drugs, daunorubicin and
doxorubicin, are currently used in the clinic. Liposomal doxorubicin, incorporated
into long-circulating PEG-coated liposomes (Doxil) demonstrates excellent effects
34 Nanotechnology Applications in Cancer Imaging and Therapy
(a)
(d)
(b)
(c)
Figure 2.9Intracellular trafficking of HIV-1 TAT petide–modified rhodamine-labeled liposomes
encapsulated with fluorescein-labeled dextran in BT-20 human breast cancer cells. (a) Differential
interference contrast images, and fluorescence confocal microscopy images, were obtained with (b)
a rhodamine filter, (c) a fluorescein filter, and (d) an overlay of both. After 2 hours of incubation, a
majority of the TAT peptide–modified liposomes were found in the perinuclear region of the cells.
(Courtesy of Vladimir Torchilin, Center for Pharmaceutical Biotechnology and Nanomedicine,
Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University, Boston,
Massachusetts.)

in EPR-based tumor therapy and diminishes the toxic side effects of the original
drug. Long-circulating, PEG-modified liposomes can be easily adapted for the deliv-
ery of peptide (protein)-based pharmaceuticals to the tumor [88]. There were, how-
ever, reports indicating that in some cases liposome size was too large to provide
efficient accumulation via the EPR effect, presumably due to a relatively small
vasculature cutoff size in certain tumors. In the cases of these particular tumors,
alternative delivery systems with smaller sizes, such as peptide (protein)/polymer
conjugates or drug-loaded micelles should be more efficient. Among various phar-
maceutical micelles, polymeric micelles including those prepared from amphiphilic
PEG-phospholipid conjugates are of a special interest because of their stability.
These particles are smaller than liposomes and lack the internal aqueous space. To
load micelles, peptide or protein pharmaceutical agent can be attached to the sur-
face of these particles or incorporated into them via a chemically attached hydro-
phobic group (“anchor”). It has been shown in mice that the use of micelles as a
carrier allows for the delivery of a model protein with higher efficiency compared to
PEG-liposomes into a tumor with a low vasculature cutoff size. PEG-based anti-
body-linked micelles, with low critical micelle concentrations, have been developed
for tumor-targeted drug delivery.
The use of specific “vector” molecules can further enhance tumor targeting of
peptide/protein carriers or make them EPR effect independent. The latter is espe-
cially important in the case of tumors with immature vasculature, such as tumors in
the early stages of their development, and for delocalized tumors. Vector molecules
(those having affinity toward ligands characteristic for target tissues) capable of rec-
ognizing tumors were found among antibodies, peptides, lectins, saccharides, hor-
mones, and some low molecular weight compounds. From this list, antibodies and
their fragments provide the most universal opportunity for targeting and have the
highest potential specificity. Vector molecules can be used for the targeting of
nanoreservoir delivery systems as well. PEG-modified long-circulating
doxorubicin-containing immunoliposomes targeted with anti-HER-2/neu
monoclonal antibody fragments represent a recent example of increased efficiency
of targeted delivery systems. In all studied HER-2–overexpressing models,
immunoliposomes showed potent anticancer activity superior to that of control
nontargeted liposomes. In part, this superior activity was attributed to the ability of
the immunoliposomes to deliver their loads inside the target cells via the recep-
tor-mediated endocytosis, which is obviously important if the drug’s site of action
locates inside the cell. As with nontargeted liposomes, liposomes targeted by vector
molecules can be adapted very easily for the delivery of peptide and protein
anticancer pharmaceutics.
Self-assembled nanosystems (nanoassemblies) for targeting subcellular
organelles, such as the mitochondria, are also developed. It has become increasingly
evident that mitochondrial dysfunction contributes to a variety of human disorders.
Moreover, since the mid-1990s, mitochondria, the “powerhouses” of the cell, have
also become accepted as the cell’s “arsenals,” which reflects their increasingly
acknowledged key role during apoptosis. Based on these recent developments in
mitochondrial research, increased pharmacological and pharmaceutical efforts
have led to the emergence of “mitochondrial medicine” as a whole new field of bio-
medical research. Yet the development of “mitochondrial pharmaceutics” is lagging
2.3 Nanotechnology-Based Drug Delivery Systems for Cancer Therapy 35

behind. No mitochondria-specific drug carrier system allowing the organelle-spe-
cific delivery of drugs and DNA in living mammalian cells has been made available.
The targeting of biologically active molecules to mitochondria in living cells will
open avenues for manipulating mitochondrial functions, which may result in the
selective protection, repair, or eradication of cells [89].
2.4 Conclusions
Nanotechnology has advanced greatly in recent years and antiangiogenic therapy is
becoming a promising approach for cancer treatment. Development of improved
and multifunctional nanoparticles is in progress for the enhancement of imaging,
targeting, delivery, and other processes.
This is an extremely active field where the miniaturization of complex reporting
devices adapted to in vivo imaging would be extremely beneficial. Different imaging
techniques require different reporting devices; for example, quantum dots may
“report back” by fluorescing on contact with diseased cells. It is not difficult to
make the leap from a reporter device to a device that not only indicates the locus of
the disease but also delivers a cure; for example, nanomagnetic particles “report
back” by providing increased contrast and also take part in the therapeutic process.
In addition, this concept underpins the need for research into gene and cellular ther-
apy and drug delivery. To improve reporting, research is required into the design
and composition of nanoparticles, enabling them to better target diseased cells,
including those situated behind barriers such as epithelial tissues. Further research is
required into the creation of an “all-purpose” nanoparticle that can be imaged by
the variety of existing instruments (e.g., optical, acoustic, magnetic, and the like).
Areas that can be expected to benefit most from nanotechnology within the next
10 years are cancer, diseases of the cardiovascular system, the lungs, and the blood,
neurological (especially neurodegenerative) diseases, diabetes, inflammatory/infec-
tious diseases, and orthopedic problems. Cancer is a complex disease involving a
multitude of molecular and cellular processes, arising as the result of a gradual accu-
mulation of genetic changes in specific cells. Nanotechnology-based, highly efficient
markers and precise, quantitative detection devices for early diagnosis and for ther-
apy monitoring will have a wide influence in patient management, in improving the
patient’s quality of life, and in lowering mortality rates. Devices capable of bypass-
ing biological barriers to deliver therapeutic agents with accurate timing and at
locally high concentrations directly to cancer cells will play a critical role in the
development of novel therapeutics. Applications of nanotechnology to diseases of
the cardiovascular system include the noninvasive diagnosis and targeted therapy of
atherosclerotic plaque. Devices to monitor thrombotic and hemorrhagic events can
have a high impact (e.g., in the diagnosis and treatment of stroke and embolisms).
Multifunctional devices could detect events, transmit real-time biological data
externally, and deliver anticoagulants or clotting factors while the patient seeks
further treatment.
Nanotechnology could also have a large impact in the area of blood purification/
decontamination, based on intelligent sorbents and hemocompatible and immuno-
tolerated implantable nanodevices or on novel separation techniques using, for
36 Nanotechnology Applications in Cancer Imaging and Therapy

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The Project Gutenberg eBook of Experience of
a Confederate States Prisoner

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Title: Experience of a Confederate States Prisoner
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*** START OF THE PROJECT GUTENBERG EBOOK EXPERIENCE OF
A CONFEDERATE STATES PRISONER ***

Transcriber’s Notes
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Obvious typographical errors have been silently corrected. Variations in hyphenation
and consistent non-standard spelling remain unchanged.

EXPERIENCE
OF A
Confederate States Prisoner,
BEING
AN EPHEMERIS
REGULARLY KEPT BY
An Officer of the Confederate States Army.
RICHMOND:
WEST & JOHNSTON, PUBLISHERS.
1862.
G. W. GARY, Printer .

PREFACE.
The gallant Morgan has said that our independence is an achieved
fact. “Privation and suffering have won it.” It is true that the noble
South has been deprived of many of its wonted necessaries, not to
say luxuries, by the present invasion of those disciples of Satan,
commonly called “Yankees.” Paper, among other things, is scarce in
the South, and paper may be turned into excellent account in the
composition of cartridges, while metal that might be moulded into
bullets is run into type. Yet newspapers and books are printed, and
most of them eagerly read, especially any that have the most
remote bearing upon the present contest. In these stern times of
war’s realities, plain facts challenge our attention rather than the
gaudy fiction of novels. Honey from Mount Hybla, or Nectar from
Olympus, would fail on the palate, unless relieved by homelier
viands; and it would certainly require considerable stoicism to sit
down to a tale of imaginary woes and sorrows while one great wail
is going up from our sick and wounded—an incredible amount of
apathy to sit leisurely down to such a book under the shade of a tree
while the nation is sending out a heartcry for reinforcements to our
brave legions, in order to speedily defeat the unscrupulous enemy.
This little book is intended as, and professes no more than a plain
statement of facts, so that others may learn what I have read, seen
and heard, without undergoing the pain of incarceration in the hands
of Yankees, whose tyranny increases in proportion to the power they
possess over their victims.

EXPERIENCE
OF A
CONFEDERATE STATES PRISONER.
May, 1862. A “heavy march” on the 6th and 7th instant resulted in
a Confederate victory at McDowell, Highland county, at which place a
battle was fought on the 8th. General Jackson routed and drove the
enemy, commanded by the Yankee Generals, Milroy and Schenck,
twenty-five miles into Pendleton county, and captured a large
amount of ammunition, commissary stores, arms, and many
prisoners. Our forces afterwards completely routed Banks’ column at
Winchester, and thoroughly defeated Fremont and Shields at Cross
Keyes and Port Republic. After the battle at Front Royal, I remained
at that place upon the recommendation of the regimental surgeon,
on account of having strong symptoms of the Typhoid fever, which
turned out to be the genuine disease. Dr. Brown, the resident
physician, attended me; and a member of my own company, Mr.
Oxford, nursed me faithfully from the 23d May, the day our forces
entered Front Royal, to the 30th May, the day that the Yankees
under General Shields recaptured it. The 12th Georgia regiment was
the only force left at Front Royal. The Provost Marshal, or the
Colonel commanding the 12th Georgia, gave us notice but one hour
before the Yankees were in the town that they were advancing.
When Mr. Oxford informed me of the near approach of the Yankees,
I quickly jumped out of bed, and we hastily made a retreat towards
Winchester. The salutary and kind attentions of Dr. Brown and Mr.
Oxford had much improved me in strength, but I soon discovered
that I could not keep pace with the latter in our eager efforts to
escape. We succeeded in getting about one mile and a half from the
town when the Yankee cavalry were heard closing on us so fast that
we leaped over a fence on the left of the road, thinking that we

might conceal ourselves in the high grass until the cavalry passed,
and be enabled to elude them by getting into the woods near by. In
the confusion, however, Mr. Oxford and I became separated, and by
this time the Yankee cavalry were close enough to fire twice on
myself and two others from the 33d Virginia, who attempted to
make their escape in the same direction. The cavalry soon after had
surrounded us, and we were compelled to surrender, and were
marched into town under a heavy guard. The commissioned officers
were carried before General Shields, and the non-commissioned
officers and privates to the building used by our army as a hospital,
where we had some hundred sick at the time. The commissioned
officers at first confined to any house they might select, were
afterwards paroled the town. I was taken to Mr. John B. Petty’s
house, and ordered to remain there “for the present” by one of
General Shields’ staff. About an hour after I was left at the above
named house, a Pennsylvania Major came into the room where I
was, and very abruptly asked me, “What are you doing here?” I
informed him that by order of General Shields I was to remain there
“until further orders;” he would not believe me, and placed two
sentinels in the room until he found that my statement was correct.
Captain Keogh (on General Shields’ staff) gave me the following
note, saying, when he did so, that I would not be “any further
annoyed by officers in other regiments” that had nothing to do with
my case:
“Headquarters, Shields’ Division , May 30, 1862.
“Captain W. is allowed to remain at the house of Mr. John B.
Petty (until further arrangements are made,) the said Captain W.
being a prisoner of war. By order of Major General Shields.
MILES W. KEOGH,
Ass’t Adj.”
After the lapse of two days I was allowed the limits of the town,
but being sick I did not go out of the house for five days after I was
captured, when I walked down to the barbers’ shop. While passing

the hotel I was called by a Federal officer, whose name I learned
afterwards was General Duryea, of New York. I went into his room,
around which were sitting several other Federal officers, and the
General addressed me, “What are you doing walking about the
streets? Are you not a Southern officer?” I replied “I am,” and told
him that Major Shedd, the Provost Marshal, had paroled me the
town. General Duryea then said, “I understand, sir, that when the
Rhode Island cavalry had you in their power, and could have killed
you, that as one of the cavalry dismounted to take your sword, and
was proceeding to mount again, you fired your pistol twice at the
back of his neck.” I replied such could not be true, for I had no pistol
about me when captured. General Duryea then said, “I may be
mistaken, but I wish to find out what Captain it was, and visit the
proper vengeance upon him.” The day before the Yankees entered
Front Royal, a colored man died of small pox in a small frame house
near the railroad depot, and by general consent of both citizens and
the Yankee paroled prisoners in the town, it was agreed as advisable
to burn the house and body, in order to prevent the spread of the
dangerous and contagious disease. The Yankees were told by some
traitor, or else themselves originated the lie, that we had burned up
two of the Yankee prisoners in our hands, and they swore
vengeance against us—declared that they intended to “put the town
in ashes,” and nothing but a special order of General Shields to the
contrary, and forbidding interference with any property whatever,
prevented the soldiers from giving vent to spleen engendered by a
false and malicious report. General Shields was informed by Major
Collins, (Vermont cavalry,) in my presence, that while a prisoner in
our hands he was treated most kindly, and that all reports to the
contrary had no foundation in truth; and all the other Federal
prisoners endorsed the statement of Major Collins.
June 6th. We have been told from day to day that all “General
Jackson’s men” would be paroled until exchanged, and yet at the
same time preparations are being made to take us to Washington, i.
e., about nineteen officers, and one hundred and fifty non-
commissioned officers and privates. The kindness of the people of

Front Royal, and especially the ladies to the Confederate prisoners,
deserves the highest praise. Devoted to our cause, they omit no
opportunity to show their regard for those who are endeavoring to
rescue them from the obnoxious presence and depredations of the
Yankees. They keep aloof from the Yankees as much as possible,
and are always on the alert to do something for the relief of our sick
and wounded.
June 7th. Among the Yankees I made the acquaintance of
Adjutant Griffin, 5th New York cavalry, who treated me kindly, as
also Captain Abraham Moore, Captain Isaac S. Tichenor, and Major
Shedd, 105th New York regiment, and Lieutenant H. Hobert Mason,
of General McDowell’s staff. Met with the celebrated Miss “Bell Boyd”
to-day. Miss B. is a sprightly, intelligent lady, au fait in all the
movements of our army, and moderately good looking. Her general
information, and nonchalant mode of fluent conversation, renders
her tout ensemble quite interesting. It is said she has obtained
valuable information from Yankee officers in regard to their
movements, and conveyed the same to our army. A great many
soldiers talk to me every day, and they all so far have expressed
themselves tired of the war, but say that it will soon be ended,
inasmuch as they have General Jackson “in a trap,” out of which he
cannot escape. They say “Stonewall” is our greatest General—
incomparably so—that he is cunning and strategic, but that it is not
within the range of human possibility for him “to elude us this time;”
that they would like to capture him, but under no consideration
would they kill either him or Ashby if they knew it.
June 8th. They say we are to be sent to Washington city on to-
morrow, but we have been told so many things that have failed to
come to pass, that we are too reluctant to believe any more reports.
Nous verron, to-morrow. Mr. and Mrs. Petty have been untiring in
their attentions to the sick and wounded prisoners here. They will
never be forgotten by those who have been the recipients of their
kindness, especially those who had the fortune to be under their
roof. Mr. P. has been made to pay the Yankees a heavy penalty on

account of being “Secesh;” they have stolen three of his most
valuable negroes, any number of horses, cattle, &c., besides laying
waste his two farms. One of his negro men left him one day, and the
next time he saw him the negro was dressed in the cavalry uniform,
with a sabre hanging to his side, and passed his master with silent
contempt on the street. The negro was now a member of the
“Michigan cavalry,” a company notorious for its success in robbery
and plunder of every description. This same negro visited Mr. Petty’s
house afterwards in company with three Yankee officers, and
demanded of Mrs. Petty (Mr. P. was absent) the key to the wine
room; Mrs. P. told them that she had only a few bottles of wine,
which she kept for medicinal purposes, and requested them not to
disturb it, but the negro persisted with threats in having it, and told
Mrs. P. “she lied” in saying she only had a few bottles. Having
obtained all the wine in the house, by frightening this excellent lady
they drank it in her presence, when they smashed the bottles on the
floor, exclaiming, “the damned Secesh don’t deserve to have
anything.”
Monday, June 9th. To-day the prisoners were put on the cars to be
taken to Washington city. A lady gave one of the prisoners a boquet
with a small Confederate flag attached, which, as he was about to
get into the cars, was noticed by General Duryea, of New York, and
as soon as the latter saw it he quickly severed the flag from the
boquet, and with an air of contempt and triumph tore it into
fragments, at the same time trampling each fragment under his feet.
The people of Front Royal manifest the greatest interest in the
Confederate prisoners. They carry provisions to them daily at the
hospital, while those prisoners who are paroled are invited to their
houses. It would seem that interest would sometimes prompt them
to court Yankee favor, but they spurn it, and remain loyal and true in
their deportment at the sacrifice of thousands of dollars worth of
property, for Yankee regiments camp on the wheat fields, and steal
the horses and negroes, and kill the hogs, and commit every sort of
depredation upon the property of those who are known to be
Secessionists. The ladies avoid the Yankees whenever they can, and

when thrown into their presence, treat them with that reserve with
which they might be expected to treat those whom they regard as
the deadly enemies of their dearest friends and interest, but whose
presence they cannot avoid. The people seemed sad when the
prisoners left Front Royal; the ladies filled their haversacks with
refreshments, and loaded the cars with flowers.
June 10th. We arrived at Alexandria at 2 o’clock this morning—
saw the depot which was burned by the bold General Geary, when
he imagined that he saw 50,000 rebels advancing on him, when, in
fact, the rebels were no where near him. The 104th New York
regiment in their fright burned up everything they had. A fellow
prisoner informs me that he was lately a prisoner in the hands of
Geary, who had him hand-cuffed, and kept him without food for four
days, and that he led his command to believe, by repeated
assurances, that Richmond was in possession of the Federal army. At
daybreak this morning a crowd assembled around the cars, and
many were eager to talk with us, but were not permitted to do so.
Nor were our friends allowed to give us anything to eat, although
they had provided various refreshments, and although the Yankees
had furnished us nothing to eat since yesterday morning, or it may
be said with nothing at all, for what we eat then was given by the
people at Front Royal. At 7 o’clock in the morning the crowd became
very great, and the guards were increased in proportion. The ladies
could not be prevented from kissing their hands to the prisoners. A
young man attempted to throw an orange in the cars for a lady, who
requested him to do so, but he was contemptuously thrust aside,
and had to leave in “double quick” time. Our friends had provided for
us coffee, bread and butter, ham, eggs, cakes, pies, candies in
variety, and tobacco and cigars in profusion, but like the thirsty
Tantalus, and the water we were almost in reach, without being able
to enjoy them. Boquets were thrown in showers into the cars, while
there was the greatest demand for our buttons. Some cut all the
buttons off their coats, and then could not gratify all who requested
to be given “one.” This scene, and the sympathy manifested for our
cause by so many Alexandrians, made us feel happy, while at the

same time we were sad in knowing that they were then writhing
under the heel of Lincoln despotism. The Yankee soldiers seemed to
envy the attentions sought to be lavished upon the prisoners by the
people of Alexandria; some cursed us, some shook the United States
flag in our faces, &c. One fellow remarked, “If the 11th
Massachusetts was in those cars, you would not get to Washington
city.” Others vented their spleen by insulting remarks to the ladies.
We arrived at Washington at 12, M., having started from Alexandria
in a steamboat about 11. We were then marched in two ranks (with
a strong guard of infantry on either side and rear, and a display of
cavalry in front) to the “old capitol military prison.” We were very
wet when we arrived at the latter place, on account of the rain
which commenced before we left the steamboat, but were
compelled to stand out in the yard from 12, M., to 5, P.M., when we
were assigned our quarters. The room in which seven officers and
myself were confined was about twelve feet square. My prison
companions are Captain Samuel M. Sommers, quartermaster,
Lieutenants Chas. E. Bott and John F. Everly, 33d Virginia regiment,
and Lieutenant James K. Decrow, Newton T. Johnston, James M.
Brown, and Edward Waterman, of the 12th Georgia regiment. Roll
was called to-night, and our names, rank, regiment, company letter,
and State, taken in full. Our door is locked all the time, except when
officers come in, or when we are allowed to go into the yard an half
hour for exercise.
June 11th. The superintendent of this prison is William P. Wood,
and the officers in command Captain Benjamin Higgins, and
Lieutenants J. Miller and —— Holmes. Mr. Wood is an infidel, who so
far from blushing to proclaim it, takes frequent occasion to do so.
When endeavoring to enforce his doctrines, he addresses his
opponent as “You mullet-headed Christian,” and speaks in the
greatest derision of our Saviour, while he denies the existence of a
God, or hell. He is a sharp-featured, serpentine-looking specimen of
humanity, medium height, and by trade a cabinet maker, before his
black republican proclivities secured him his present position. Mr.
Wood, a prisoner, soon finds out to be the most important among

“the powers that be” connected with the prison, and all “privileges”
must be reached through him. He professes to be a great Southern
man, and sometimes demonstrates this by knocking down a
contraband, who does not wait upon him in accordance with his
fastidious notions.
It is cloudy, and my close confinement, together with the continual
sight of dark blue uniforms makes me feel as gloomy as the sky is in
appearance. I would that I could be with our army in the “Old
Dominion.” From my prison window I see an old United States
soldier cultivating flowers in a row of flower pots. One knows him to
be a soldier by his regular walk, and the style of his grey moustache,
not to speak of his uniform. Indeed one might have guessed as
much from the care he takes of his little garden, for there are two
things I have noticed especially, loved by old soldiers, viz: flowers
and children. They have so long been obliged to look upon the earth
as a field of battle, and so long cut off from the peaceful pleasures
of a quiet lot, that they seem to begin life at an age when others
end it.
June 12th. Have been here a day and a half and two nights, and
can form some idea of the way things are managed at this prison.
Roll is called night and morning, and as to fare, we are allowed a tin
cup of what is called coffee, but which is really mock-coffee, a slice
of bread six inches long, five inches wide, and a quarter of an inch
thick, and a piece of beef or fat bacon twice a day—forming a
repast, the sight of which is almost enough to cause any respectable
stomach to revolt, so unclean seems both it and its surroundings. A
lady came into our room to-day leaning on the arm of Dr. Stewart,
the prison surgeon. As the Doctor ushered her in, he remarked,
“This is the room in which Mrs. Rose O. N. Greenhow was confined.”
Lieutenant D., of the 12th Georgia regiment, was lying on a blanket
in one corner of the room, and the lady seemed to recognise him,
and asked “What’s your name?” “Are you from Georgia?” Being
answered promptly by the Lieutenant, and in the affirmative as to
the latter question, the surgeon observed, “You have a remarkable

recollection of faces,” and they left the room, which was then quickly
locked. It is supposed that she is the correspondent of some
Northern journal. No doubt she will say that we live in a palace, and
have hotel fare, thus emulating the editor of the “Evening Star,” who
a short time ago informed its readers that we “fared equal to any
hotel in the city.” If a sentinel is caught in conversation with a
prisoner, the punishment is two weeks in chains. The prisoners are
allowed an half hour in the yard after each meal. After dinner to-day,
the surgeon, Dr. Stewart, a coarse, vulgar mean Yankee came
among us in the yard, and had the audacity to say, “All who desire to
take the oath of allegiance to the United States Government, and
thereby obtain their liberty step this way.” A deserter and two men of
Northern birth obeyed the call. I am informed by prisoners, who
have been here sometime, that the greatest effort is constantly
made to induce prisoners to take the infamous “oath of allegiance.”
At roll call to-night I was informed that “several friends” called to see
me. I was not told who the friends are, and I infer that they do not
intend to tell me, or allow me to see them at all.
Friday, June 13th. Among the prisoners confined here, is Charles
C. Randolph, Esq., a venerable looking old gentleman, seventy-five
years of age, from Fauquier county, Virginia. He served in the war of
1812 as Captain, under General Parks’ command, and received his
commission through the influence of the celebrated “Harry Lightfoot
Lee,” of the revolution. He says that he went to Richmond about the
first of April last, and when he returned to his home he found that
the Yankees had devastated everything about his valuable premises.
They stole his horses, sheep and cattle, and destroyed his crops, and
took everything of value he had from a library worth $5,000, to his
bed, and even his wife’s likeness, and the family bible, besides
breaking all the hinges of the doors, and committing waste and
robbery generally. He, himself, was arrested as soon as he arrived
home, and brought here, for what he knows not, unless it be for
implied sympathy for the cause of the soil of his birth and the people
of his blood. There was a prisoner here named Wharton, a
Californian. He was a Lieutenant in the United States Navy at the

beginning of the war, when he resigned, and started for the South
via Washington city, but was arrested on his arrival here and brought
to this prison. A short time since he cursed one of the sentinels for
insulting language used towards him, when the sentinel called for
the “corporal of the guard,” who being equally insolent, was in turn
treated in the same way by Lieutenant Wharton. The “officer of the
guard” was then called, who proving equally offensive in language to
Lieutenant W., the latter cursed him in the heat of anger, whereupon
said Lieutenant Wharton was shot, and soon afterwards died of his
wounds. A respectable gentleman, Mr. Stewart of Maryland, who was
incarcerated here, was promised by the guard to be allowed to
escape, on condition of the payment of $50; but although the
sentinel pocketed the money, when Mr. Stewart was effecting his
escape the sentinel shot him, and this sentinel was immediately
promoted from a private to a sergeant.
Saturday, June 14th, 1862. It is reported this morning that Colonel
Ashby is killed, and General “Stonewall” Jackson a prisoner, and the
Yankees profess to place great reliance upon the report. From
Northern sources, I learn that when the war-tax was being collected
in Southern Illinois, it required three regiments to accomplish the
task. It seems plain that Southern Illinois would like to break the
chains that now bind her. In the beginning of the war the people of
that section were told by Yankees that wished to raise regiments of
soldiers to fight us, that the Mississippi would be blocked against
them, when the very first act of the Confederate Congress insured
the free navigation of the Mississippi river.
The Yankees say that by the first of July their public debt will be
650 million dollars! It is now 1,500 millions!! They have 65,000 sick
from their own account. Who will pay their pensions?
This is a struggle on the side of the Yankees for supremacy, and
on our side for independence. It is urged that the Northern States
are a great deal stronger than the Southern States, and therefore
must win in this contest. England was a great deal stronger than
Scotland, but when it was the object of England to establish by force

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Micro And Nano Manipulations For Biomedical Applications 1st Edition Tachung C Yih Tachung Yih

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