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A fluorescence microscopy study of quantum dots as fluorescent probes for brain tumor diagnosis

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A fluorescence microscopy study of quantum dots as fluorescent probes for brain tumor diagnosis

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Content A FLUORESCENCE MICROSCOPY STUDY OF QUANTUM DOTS
AS FLUORESCENT PROBES FOR BRAIN TUMOR DIAGNOSIS
by
Jingjing Wang
A Thesis Presented to the
FACULTY OF THE VITERBI SCHOOL OF ENGINEERING
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOMEDICAL ENGINEERING)
December 2005
Copyright 2005 Jingjing Wang
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UMI Number: 1435094
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Dedication
This work is dedicated to my beloved parents for their everlasting love and support.
ii
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Acknowledgements
I would like to thank my advisor and thesis committee chairman, Professor Laura
Marcu, for her invaluable attention, guidance, and support throughout my graduate
studies. I would also like to extend my great appreciation to the other thesis
committee members, Professor Gundersen and Professor David D'Argenio. In
addition, I would like to thank Dr. Vernier, and Yinghua Sun for their particular
influence on my way of thinking. And finally, I would like to pay my sincere thanks
to my colleagues for their generous help throughout my graduate career.
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Table of Contents
Dedication ii
Acknowledgements iii
List of Figures vi
Abbreviations vii
Abstract viii
Chapter 1 1
1.1 Molecular imaging and diagnostics using nanoprobes 1
1.2 Quantum dots — imaging probes 2
1.3 Quantum dots in cancer imaging 4
1.4 Goal 5
Chapter 2 6
2.1 Properties and Application of Quantum dots 6
2.2 Imaging o f High Grade Glioma with Quantum Dots 17
Chapter 3 24
3.1 Cell Culture 24
3.2 Fluorescence Microscopy 24
3.3 Imaging Glioma Cells and Frozen Tissue with Quantum dots 25
3.4 Preparation of Anti-EGFR Conjugated Quantum dots 25
3.5 Immunolabeling of Cells with Quantum Dots Conjugates 26
3.6 Preparation and Immunolabeling of Frozen Tissue Sections with Quantum dots Conjugates 26
Chapter 4 27
4.1 Immunolabeling of Tumor Cells with Anti-EGFR Conjugated Quantum Dots 27
4.2 Immunolabeling o f Frozen Tumor Tissues with Anti-EGFR Conjugated Quantum Dots 33
4.3 Discussion 34
iv
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4.4 Conclusions
Bibliography
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List of Figures
Figure 1 Typical absorbance and emission spectra of QDs streptavidin conjugate. 8
Figure 2 Schematic of the structure of a Qdot™ Streptavidin conjugate from Quantum Dots
Corporation. 15
Figure 3 Strategy for anti-EGFR conjugated streptavidin coated QDs selectively binding to EGFR. 25
Figure 4 Targeting QDs to EGFRs in SKMG-3 cells. 28
Figure 5 Targeting QDs to EGFRs in human GBM cell lines, SKMG-3 and U87. 29
Figure 6 Images of control cell line. 30
Figure 7 Images of SKMG-3 cells. 31
Figure 8 Multi-panel z-stack images of SKMG-3 cells at 0.5 pm interval. 32
Figure 9 Tracking QDs in SKMG-3 cells at 24 and 48 hours. 33
Figure 10 Representative images of GBM frozen tissue slices. 39
Figure 11 Images of GBM frozen tissue slices exposed to QD strepavidin conjugates. 40
vi
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Abbreviations
QDs - quantum dots
EG FR - epidermal growth factor receptor
GBM - glioblastoma multiforme
BBB - brain blood barrier
TOPO - trioctyphosphine oxide
PEG - polyethylene glycol
CNS - central nervous system
M RI - magnetic resonance imaging
CT - computed tomography
FBS - fetal bovine serum
RPM I - Roswell Park Memorial Institute
DMEM - Dulbecco’s Modified Eagle Medium
PBS - phosphate buffered saline
BSA - bovine Serum Albumin
IDG - indocyanine green
GFAP - Glial Fibrillary Acidic Protein
LAC - lactate
vii
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Abstract
In vivo fluorescent spectroscopy and imaging using endogenous and exogenous
sources of contrast can provide new approaches for enhanced demarcation of brain
tumor margins and infiltrations. Quantum dots (QDs) represent excellent contrast
agents for biomedical imaging due to their broader excitation spectrum, narrower
emission spectra, and higher sensitivity and stability. The epidermal growth factor
receptors (EGFRs) are overexpressed in high-grade glioma patients and thus a
potential target for brain tumor diagnosis. In this study, we conducted fluorescence
microscopy studies of the up-take mechanism of the anti-EGFR conjugated QDs by
human U87 and SKMG-3 glioblastoma cells. Our preliminary results show that QDs
can enter into glioma cells through anti-EGFR mediated endocytosis, indicating that
these nano-size particles can tag brain tumor cells. In addition, the success of
labeling frozen brain tumor tissue specimens suggests the possibility for enhanced
optical demarcation of brain tumors with QDs in vivo.
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Chapter 1
Introduction
1.1 Molecular imaging and diagnostics using nanoprobes
The nanostructures and nanoprobes generated through using nanotechnology
approaches have recently attracted widespread interest in biology and medicine. The
integration of nanotechnology with current biotechnology has led to many scientific
and technological advances in medical diagnostics, therapeutics and treatment over
the past several years (Freitas, 2005). Among all major tools in biology and medical
research, fluorescent microscopy plays an important role in observing labeled target
molecules or cells, indicating physiological change, and monitoring intra- and extra
cellular events. However, conventional organic dyes are limited by serious photo
bleaching, broad emission, low quantum efficiency, and wide and asymmetric
emission spectrum. Molecular imaging and diagnostics based on fluorescent
spectroscopy would benefit from recent breakthrough in nanoparticles synthesis,
modifications and funtionlizations. For example, colloidal gold nanoparticles
recently have been used for in vitro and in vivo cancer diagnostics application due to
their ease of preparation, ready bioconjugation, and potential noncytotoxicity.
Bioconjugated gold nanoparticals were able to selectively and specifically label
cancer cells and tissue (Sokolov et al., 2004, El-Sayed et al., 2005). The progress of
medical imaging requires new fluorescence probes, which can provide controllable
optical properties, long observation time, overblaze autofluorescence, and indicate
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multiple targets simultaneously. Recent research on fluorescent semiconductor
nanocrystals, known as quantum dots (QDs), demonstrated that these nanoparticals
have unique optical properties. The large surface areas are available for biological
molecules binding, such as antibody, peptides, and nucleic acid. These bioconjugated
QDs have the potential to monitor the long-term intracellular processes at the single
molecule level, present the high-resolution cellular imaging, and target and detect
multiple biomarkers (Jaiswal et al., 2003, Lidke et al., 2004, Gao et al., 2004). These
inorganic fluorescent QDs overcome many limitations possessed by traditional
organic fluorescent dyes and provide an alternative for both in vitro and in vivo
study (Alivisatos, 1996).
1.2 Quantum dots - imaging probes
Quantum dots are nanoscale crystals that are made of semiconductor material and
synthesized as colloids, and they emit fluorescence when excited by various light
sources including lasers. Compared with organic fluorescent dyes, QDs have many
unique optical properties, such as narrow and tunable emission, broad excitation, and
photostability, and are hypothesized to be excellent contrast agents for biological and
biomedical assays and imaging. Moreover, unlike gold nanoparticals, QDs are
extremely small, with a diameter less than 10 nanometer, which makes them much
easier to cross biological membrane and reach the targets. However, the biological
applications of QDs were limited by their solubility and biocompatibility. Until
1998, through surface modification and bioconjugation reaction, these nanocrystals
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became water-soluble, thus they can target specific molecule through surface
attachment groups in biological environment (Chan 1998, Bruchez 1998). Over the
past few years, QDs have been optimized and tested in many biotechnological
applications, including immunolabeling of target proteins in fixed and lived cells and
tissues (Jaiswal et al., 2003, Lidke et al., 2004, Gao et al., 2004). The historical
breakthrough and current progress of QDs research will be described in Chapter 2.1.
In addition to molecular and cellular biology applications, the possibility for
immunostaining some membrane antigens with QDs suggests the potential
application of these particles in cancer diagnosis and in vivo imaging (Gao and Nie,
2004). Near-infrared QDs have the potential to provide deep photon penetration into
and out of tissue (Kim et al., 2003, Lim et al., 2003), and thus become an ideal
candidate for in vivo study. To make the potential realized, some biological
interfaces, such as antibodies and peptides that can recognize specific molecular
targets, are required. The approaches to functionalize QDs with different molecules
and ligands have been developed, which confer different functionalities to individual
QDs. These promising contrast agents finally became available to the research from
commercial sources. Quantum Dots Corporation provides large selections of QDs
with different emission spectra and surface chemical groups for various imaging
purposes. For example, streptavidin-coated QDs, used in our study, permit stable
conjugation of the QDs to ligands, antibodies or other molecules that can be
biotinylated, and are suitable for in vitro cell and tissue immunostaining.
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1.3 Quantum dots in cancer imaging
QDs have been reported to label breast cancer marker Her2 in living cells (Wu 2003).
Recent report showed that use of QD-peptide conjugates was able to target tumor
vasculature (Akerman et al., 2002). An in vivo study using type II near infrared QDs
provides real-time guidance for cancer surgery in large animals (Kim et al., 2004).
Moreover, a new class of multifunctional QDs is able to simultaneously target and
image tumor in live animals. All these findings suggest that QDs are powerful probes
for in vitro and in vivo cancer imaging and diagnostics.
Among all kinds of cancers, we have particular interests in brain tumor. Although
therapeutical approaches of brain tumors have been extensively studied, surgery is
still the most effective treatment for brain tumor (Hess, 1999). The survival rates of
patients with high-grade brain tumors are closely related to extent of tumor removal.
With the drive to develop an intraoperative surgical imaging technique for enhanced
demarcation of tumor margins and infiltrations, an in vivo fluorescent microscopy
using endogenous fluorescent contrasts has been proved to distinguish brain tumors
from normal tissues in clinical trials (Lin et al., 2001). To increase the sensitivity and
selectivity of this approach, exogenous probes are needed. QDs have distinct optical
advantages for in vivo imaging and can be easily functionalized with various ligands.
In addition, their extremely small size is favorable in crossing blood brain barrier
(BBB) compared to other nanopartical probes. However, the possibility to use these
nanoparticals to differentiate brain tumors from normal cells has never been
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explored. The possibility for using QDs in brain tumors diagnosis will be discussed
in chapter 2.2.
1.4 Goal
In this study, we investigated the use of EGFR-targeted QDs for enhanced optical
imaging of brain tumors cells and tissues. The overall objective of this research is to
assess the diagnostic value of bioconjugated QDs for real-time brain tumor
differentiation. The initial work were performed to evaluate the possibility using
anti-EGFR conjugated QDs selectively highlight brain tumor cells and frozen tissue
specimens. Those results will be described in Chapter 4.
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Chapter 2
Background
2.1 Properties and Application of Quantum dots
2.1.1 Optical properties of quantum dots
Quantum dots are nanoscale semiconductor particles made up of hundreds to
thousands of atoms. The diameter of colloidal QDs is only a few nanometers, smaller
than the bulk exciton Bohr radius, and can he easily controlled by changing the
temperature, ligands, and duration during the synthesis (Alivosado, 1996). These tiny
particles, behaving like a single gigantic atom, have distinguished characteristics,
including size- and component-tunable emission, broadband absorption spectra from
ultraviolet to near infrared, narrow and symmetric luminescence bands, long
fluorescent lifetime, high photostability, and high quantum efficiency. These
properties make these nanometer-size fluorescent probes promising alternative
contrast agents to organic fluorescent dyes in biotechnology and biomedical imaging.
In QDs, the energy levels in the valence and conduction bands are no longer a
continuum, but are discrete or quantised. Quantum confinement of both the electron
and hole in all three dimension leads to an enhancement in quantisation effects and
increase in the band gap of the material with decreasing crystal size. In other words,
as the dot gets smaller, the band gap gets bigger. Therefore, optical behaviors of QDs
are directly related to their sizes. When the dots are irradiated with light, an electron
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is promoted from the valence band to the conduction band. The energy required to
promote the electron to the uppermost level in the conduction band is about the same
for dots with different sizes, although their band gaps differ considerably. Thus,
multisized QDs can be stimulated by the same light source (Figure 1). The electron
then falls to the lowest energy level in the conduction band and emits heat. When it
goes back to the valence band, the electron emits its excess energy as a photon of
visible light, rather than heat. Because the energy of emission photons depends on
the band gaps, the larger dot emits a less energetic photon than the smaller dot. Since
the energy of a photon is inversely proportional to its wavelength, the larger dot
emits light of a longer wavelength towards red end of the visible spectmm than that
of the smaller dot, which tends to emit a photon towards the blue end of the visible
spectrum. The fluorescence from many QDs can be resolved over the same spectra
due to their narrow emission spectra (Figure 1), which means that several different
colored dots can be exited simultaneously to track different processes and detect
multiple ligands without their emissions getting into each other's way.
Besides their unique excitation and emission wavelengths, QDs can be observed and
tracked over an extended period of time with fluorescent microscopy due to their
long lifetime and photostability. The high quantum efficiency and broad excitation
permit monitoring and observing single quantum dot, which provides capability to
identify and quantify cellular and molecular interactions (Lacoste et al., 2000, Dahan
et al., 2003), such as binding and transport phenomena. The outstanding brightness,
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which can be easily detected, is particularly helpful in the biomedical research
concerning low abundance molecules.
Extinctioa-Emissiori Plot of Qdot Conjugates
1.503.000 -
1 CDD.COO ■
Odot 525 Conjugate Absorbanc&
C l dot 665 Conjugate Absorbance
Odot 535 Conjugate Absorbance
Qdot 605 Conjugate Absorbance
Qdot 655 Conjugate A bsorbance
Qdot 705 Conjugate Absorbance
Qdot 600 Conjugate Absorbance
Qdot 625
Qdot 665
Qdot 585
Qdot
Qdot 655
Qdot 705
Qdot 800
Conjugate
Conjugate
Conjugate
Conjugate
Conjugate
Conjugate
Conjugate
Emission
Emission
Emission
Emission
Emission
Emission
Emission
S C O € £ < !
W a v e le n g th (ru n)
Figure 1 Typical absorbance and emission spectra o f QDs streptavidin conjugate.
The blue lines on the absorbance represent a broad window o f absorbance that will
excite the materials more efficiently than a single wavelength excitation (Qdots
Manual).
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Taken these properties together, QDs are excellent probes for studies that require
long-term and multicolor imaging within and among cells as they grow and
differentiate. Many biological mysteries are expected to be solved by these exciting
new properties of QDs.
2.1.2 Solubility and Functionalization of quantum dots
The biomedical application of QDs has been hampered by incompatible chemical
surface with varied biological environments, lack of techniques for selectively and
specifically labeling cells and molecular targets.
Solubility
These luminance QDs are generally prepared in organic solvent, like
trioctyphosphine oxide (TOPO), to achieve high quality. The hydrophobic surface of
QDs is not suitable for biological applications. Nie and his colleagues first solved
this problem by exchanging the TOPO absorbed on QDs surface for a layer of
amphiphilic ligand, mercaptoacetic acid, which has hydrophilic carboxylic acid
group for solubility and a thiols group for binding to ZnS sehll (Chan and Nie, 1998).
However, the water solubility of these QDs capped with mercaptocarbonic acid is
limited because of unstable thiol-ZnS bonds. At the same time, Alivisatos and
colleagues reported a more complicated but extremely stable method by adding a
silica coating for creating water soluable ZnS capped CdSe QDs (Bruchez et al.,
1998). To date, various solubilization methods using different polymers coating for
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QDs have been developed to confer the required colloidal stability to QDs (Michalet
et al., 2005).
Functionalization
In addition to solubility, QDs provide large surface areas available for multiple
binding of diagnostic and therapeutic agents, such as oligonucleotides, antibodies
and peptides. In order to target specific tissues and cell types, a large number of
surface attachment groups have been explored to equip individual QDs with different
functionalities and provide great flexibility in QD surface chemistry. Some bi
functional solubilization ligands also served as functionality groups, such as
mercaptoacetic acid. There are several strategies for bioconjugating various groups
to QDs. Reactive functional groups include primary amines, carboxylic acids,
alcohols, and thiols. Depending on the available chemical groups on the surface of
molecules, functionalization can be achieved using passive adsorption, linkage via
mercapto (-SH) groups, electrostatic interaction and covalent-lingkage formation.
Mercaptoacetic acid capped QDs as described above have the carboxyl group that
also reacts to amine group and form covalent coupling to various molecules for
ultrasensitive detection (Chan and Nie, 1998). QDs with silica shell expose mercapto
groups on their surface for further functionalization reaction (Bruchez et al., 1998).
Since most proteins contain primary amine and carboxylic acid, the carbodiimide-
mediated carboxylate-amine formation is particularly favored in protein conjugation.
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By customizing different functionality ligands, we can easily incorporate multiple
functions to these nanocrystals for specific purpose. For instance, streptavidin-coated
QDs were used to detect cancer makers Her2 on the surface of human breast cancer
cell line through three-layer approach, including primary antibody and biotinylated
secondary antibody (Lidke, 2004). In addition to antibodies, QDs with specifically
designed peptides had been delivered into subcellular site in vitro (Derfus et al., a,
2004) and targeted tumor vasculature in vivo (Akerman et al., 2002). In principle, the
similar approach could be used to target and label other molecular targets both in
vitro and in vivo. Moreover, the large surface of QDs offers the possibility for
multivalent QD-target binding with increased affinity, which is not available for
traditional organic dyes. Previous studies have demonstrated that functionalized QDs
are invaluable probes for fundamental research and biotechnology, and will have
widespread applications in medical diagnostics and imaging.
QDs fo r in vivo imaging
Most of these attempts to confer solubility to QDs result in water soluble QDs in
vitro, but they still suffer from aggregation, loss of fluorescence and low quantum
efficiency in vivo, preventing them from real-time in vivo imaging. In 2002,
researchers have developed a simple solution to add two different phospholipids -
one natural, one synthetic - to a suspension of ZnS-coated CdSe dots. The
phospholipids form a micelle around each dot, with the phospholipid tails pressed
against the dot's hydrophobic surface, and the polar head-groups of the natural
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phospholipid pointing into the solvent. These lipid-coated QDs were used for real
time tracking frog embryogenesis and can be detected for over four days (Dubertret
2002). Moreover, at the same time, another group revealed that, under guidance of
peptides, polyethylene glycol (PEG) coated ZnS-capped CdSe QDs were able to
escape from the biological particulate filter, reticuloendothelial system, and target
specific tissues and cell types in vivo (Akerman 2002). In these two studies, the
ether-rich PEG tails of the synthetic phospholipids were used and had been proved to
serve to discourage contact with biological molecules and other dots, thereby
avoiding aggregation and increase circulation time. Later, the in vivo cancer
targeting and imaging study in live animals further proved that multiple PEG
molecules could improve biocompatibility and duration of circulation of targeted
QDs in live animals (Gao and Nie, 2004). In addition, the use of an ABC triblock
copolymer in Gao’s in vivo studies helps solving the problems of aggregation and
fluorescence loss.
Past in vivo imaging fluorescence studies through targeted QDs used bioconjugated
QDs with emission in visible light range. Despite the great progress in QDs synthesis
and modifications, high-quality targeted QDs with infrared or near infrared
emissions, which are highly desired for in vivo imaging, have never been reported.
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2.1.3 Intracellular delivery of quantum dots
A major challenge in use of QDs for intracellular tracking is the deliver of QDs to
the cytoplasm and organelles. Some nonspecific biochemical and physical
approaches have been developed to transfer QDs into cells in vitro. Electroporation
and liposome complexes are efficient non-specific schemes to deliver QDs to the
cytoplasm of cells, but most QDs formed large aggregates (Derfus et al. a, 2004).
Microinjection is technically difficult to manipulate when working on large
quantities of cells (Dubertret et al., 2002). Currently, receptor-mediated endocytosis
and peptide-guided transport are still most common methods in biomedical research.
These two approaches provide specificity and can be used for in vivo imaging (Gao
et al., 2004, Dubertret et al., 2002). However, QDs entering cells through
endocytosis remain sequestered in endocytic vesicles, preventing the labeling of
other intracellular structures in vitro.
2.1.4 Cytotoxicity of quantum dots
Most reports in living cells and animals did not find obvious effects of QDs on cell
viability, function, and other physical conditions. These results suggest that, with
appropriate shell protection, it’s possible to use QDs in medical procedures without
major effects on the biological systems. In vivo QDs studies in live animals indicate
that QD-tagged cancer cells led to usual tumor growth in animal models with no
detectable affect on cell viability and growth (Voura et al., 2004, Gao et al., 2004).
However, recent work indicates that CeSe QDs are acutely toxic to cells when
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exposed to ultraviolet light (Derfus et al., b, 2004). The UV light excitation probably
cause free cadmium ion release. In addition, the study of embryonic development
using lipid coated QDs revealed that the dot dosage might be important in toxicity
(Dubertret et al., 2002). With high quantity of QDs, abnormalities were seen in the
later stages of the developing embryo. Although the doses of QDs used for in vivo
imaging are below the known toxicity levels for Cadmium, the accumulative effects
are not clear. Therefore, it is important to thoroughly investigate the potential
toxicity of QDs before any clinical application.
2.1.5 Commercialized quantum dots
Quantum Dots Corporation offers QDs with various surface chemical modifications
and functionalities for biotechnological application. Streptavidin-coated QDs are
used in combination with biotinylated anti-EGFR antibody in our study (Figure 2).
These CdSe-ZnS core-shell QDs are coated with a polymer shell, which has been
directly coupled to streptavidin, which allows the material to be conjugated to
biological molecules and to retain their optical properties (Qdot Manual). The
streptavidin on the surface permits the binding of biotinylated primary or secondary
antibody, which allows QDs labeling of most types of targets. These QDs are
designed for in vitro imaging.
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* ►
:,,Core
!.* - Shel!
Polymer Coating
Streptavidin
Figure 2 Schematic of the structure of a Qdot™ Streptavidin conjugate from
Quantum Dots Corporation, consisting of CdSe core and ZnS shell, are capped with
polymer and streptavidin (from Qdots manual).
2.1.6 Applications of quantum dots in biomedical imaging
Cancer diagnostics require high sensitivity, efficiency and resolution and potentially
high tumor vs. normal tissue specificity. The brightness, photostability, extraordinary
sensitivity, and functionality of QDs make them ideal probes for in vivo cancer
targeting and imaging. Only a very small number of QDs are necessary to produce a
detectable fluorescent signal. Quantitative measurements showed that about as few
as 100 cancer cells could be detected with long-wavelength QDs (Gao and Nie,
2003). Their resistance to bleaching effects is particularly useful for three-
dimensional optical sectioning and long-term observation. Large absorption
coefficients of QDs give more efficient probe excitation, which allows the signal
passing through tissue absorption and scattering. In addition, it has been know that
there is low tissue scattering and absorption in the near infrared and infrared regions
(700-2000nm), which allow the greatest tissue penetration depth and optical signal.
15
10-15 nm
< -------------------------
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Therefore, fluorescence QDs with near infrared emission are extremely useful for in
vivo imaging. Theoretical model predicted that near-infrared QDs probes are able to
penetrate deep tissue with low absorbance and scatter, thus providing real-time
visual guidance to the surgeon (Lim et al., 2003). Moreover, the bioconjugated QDs
are able to specifically and effectively label molecular targets at a sub-cellular level,
providing a basis for antibody based selectively targeting of tumor cells.
The initial impact of QDs in clinical cancer surgery was visualization of sentinel
lymph nodes through injection of polydentate phosphine coated near-infrared QDs,
which allows image-guided resection of lymph nodes in pig (Kim et al., 2004). Later,
antibody-conjugated QDs were injected into mice and accumulated at prostate
tumors through enhanced permeability and retention and antibody medicated binding
to biomarkers (Gao et al., 2004). This technology allowed combined QD targeting
and imaging studies in live animals. These results suggested the possibility for
sensitive and multicolor imaging of molecular target in live animals and clinical
cancer surgery. Because of the potential cytotoxicity, QDs have never been used in
human. With the increased knowledge and rapid progress in QDs research, these
nanoparticles may be available for clinical biomedical imaging in near future.
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2.2 Imaging of High Grade Glioma with Quantum Dots
2.2.1 Brain tumor clinical representation and epidemiology
Primary central nervous system (CNS) tumors constitute a small fraction of the
overall incidence of human cancer each year, but they represent a major source of
cancer-related mortality. In 1999, about 13,100 people died with primary cancers of
CNS (Deangelis, 2001). Gliomas, arising as a result of genetic aberrations in normal
precursor glial cells (Rao and James, 2004), are the most common malignancies of
brain tumors. They are divided into two main categories: astrocytic and
oligodendroglial, both of which can be either low-grade or high-grade. They are
mainly graded based on the presence or absence of nuclear atypia, mitosis,
microvascular proliferation, and necrosis (Deangelis, 2001). As the accumulation of
genetic mutations, most low-grade astrocytomas progress to high-grade malignant
gliomas in 5 years (Deangelis, 2001). High-grade astrocytomas, such as Grade IV
astrocytomas known as Glioblastoma multiforme (GBM), grow very rapidly with
widely infiltrating into normal tissues. Tumor cells typically extend microscopically
several centimeters away from the obvious area of disease, thereby are difficult to be
completely removed during surgery. Despite aggressive treatment by surgery,
radiotherapy, and chemotherapy, there has been little progress in extending the
survival time or quality of life for brain tumor patients. The prognosis for patients
with brain cancer is dependent on the location, type, and grade of tumors. The
median survival of patients diagnosed with highly malignant GBM is less than 12
months (Deangelis, 2001).
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2.2.2 Glioma diagnosis and treatment
Currently, the cranial magnetic resonance imaging (MRI) is the most accurate test to
diagnose a brain tumor. Surgery is the first treatment option for brain tumors. The
feasibility of surgery is usually determined by MRI or computed tomography (CT)
scanning. Radiation therapy, often used as an adjuvant to surgery to irradiate tumor
cells invading normal tissues, is the most effective non-surgical therapy and
significantly prolongs survival. However, the response of astrocytomas to radiation
is relatively low due to their highly resistance to radiotherapy. The adverse reactions
are very common and can lead to neurological deterioration, memory loss, and
impaired intellectual function. The use of chemotherapy in addition to radiotherapy
consistently increases the proportion of long-term survivors (DeAngelis et al., 1998),
but its effects are limited by the ability of the drugs to cross the BBB. The toxicity is
also a major concern for this treatment. Thus, resection is still the initial and major
intervention in malignant gliomas treatment. The clinical prognostic outcomes are
closely related to the extent and accuracy of surgical resection (Hess, 1999, Fadul et
al., 1988). Unlike other tumors in most parts of body and low-grade gliomas, high-
grade gliomas, such as GBM, are of infiltrative nature. Thus, surgery to remove brain
tumors located near vital brain centers may be very risky because of the chances of
damaging neurological functions. Every effort should be made to maximize tumor
removal without sacrificing brain functions. However, the difficulty of visually
differentiating malignant tissue from normal brain during surgery often results in a
subtotal resection and recurrence of tumors or neurological morbidity due to the
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resection of normal tissue. Therefore, the primary challenge in brain tumor treatment
is to develop strategies for effective removal of tumor cells without damaging the
healthy brain tissue.
Currently, differentiation of brain tumors from normal tissues primarily depends on
the neurosurgeons visual examination, biopsy and image guided navigation
techniques, including preoperative (CT), MRI, and intraoperative ultrasound.
However, these techniques are restricted by the sensitivity and post imaging brain
shifts due to retraction and cerebrospinal fluid drainage (Hill et al., 1998, Dorward et
al., 1998). CT and MRI images are unable to define some infiltrating margins. Intra
operative MRI makes "real-time" three-dimensional imaging possible, but requires
highly specialized and expensive systems. Hence, there is requirement on high
sensitivity, high resolution and real-time imaging guidance tools to help surgeon on
thorough tumor resections.
Fluorescent spectroscopy has attracted great interests in cancer diagnostics. The
occurrence of histological and biochemical alterations induced by pathological
processes can change intrinsic autofluorescence properties of biological tissues,
which can be measured using fluorescence spectroscopy. Recently studies
demonstrated that brain tumor tissues have distinguished autofluorescence emission
spectra and longer lifetime (Chung et al., 1997, Lin et al., 2001, Croce et al., 2003).
Besides detecting emission wavelength, the advanced time-resolved laser-induced
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fluorescence spectroscopy is able to measure the fluorescence lifetime of
biomolecules in vivo and offer a greater degree of specificity (Marcu et al., 2004).
The ability and spectral ranges related to the diagnosis of brain tumors using time-
resolved fluorescence spectroscopy are under investigation by Marcu and Black’s
groups in Cedars-Sinai hospital. Based on these findings, using endogenous sources
of contrasts, fluorescent spectroscopy and imaging approach offer a potential method
for intraoperative brain tumor demarcation and diagnosis, with high specificity,
sensitivity, resolution, and relatively low cost. The high clarity images provided by
this new fluorescence technology are expected to facilitate real time differentiation
of aggressive infiltrating tumor tissue with poorly defined borders, which is
extremely difficult for current imaging techniques.
However, all of these studies were focused on natural biological fluorophores present
in the tissues. The possibility of using exogenous fluorescent probes to highlight
tumor tissue and facilitate autofluorescence-based spectroscopy in intraoprative brain
tumor delineation has never been explored. The implication of fluorescent probes
with moiety recognizing cancer markers, such as bioconjugated QDs, with current
spectroscopy is expected to provide higher sensitivity and specificity. Considered
their optical advantages and functionality described in Chapter 2, QDs are promising
contrast agents to selectively highlight brain tumor cells for real-time fluorescent
spectroscopy. With the extraordinary brightness and high quantum efficiency, it’s
possible to differentiate infiltrations of high-grade gliomas from surrounding brain
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tissues with specific designed QDs in vivo. Therefore, QDs may facilitate brain
tumor demarcation using laser-induced fluorescence spectroscopy and provide
higher specificity, sensitivity and resolution.
2.2.3 Overexpression of EGFRs in gliomas
As with other cancers, gliomas occur as a result of gene aberrations in normal cells.
The epidermal growth factor receptors (EGFRs) signaling pathway appears to play
an important role in the development of gliomas. EGFRs are implicated in the
development and progression of a number of human solid tumors. About 40% GBM
are associated with a high ratio of overexpression coupled with various mutations of
the EGFRs (Rao and James, 2004), which are currently of intense interest in the
treatment of GBM.
EGFRs are transmembrane glycoprotein that is composed of an extracellular ligand-
binding domain and an intracellular tyrosine kinase domain. As a readily accessible
receptor on the cell membrane, EGFRs have been used as a docking site for
delivering cytotoxic agent, such as tyrosine kinase inhibitors and toxin-conjugated
TGF-cc (Phillips et al., 1994, Lawrence and Niu, 1998), in EGFR-based therapeutic
research. The anti-EGFR monoclonal antibodies, which inhibit binding of natural
EGFR ligands, have been shown in non-clinical studies to bind specifically with and
exhibit high affinity to EGFR, providing an approach for both drug delivery and
molecular targeting (Sampson et al., 2000). In principle, similar approach can be
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applied to the detection of tumor cells with bioconjugated QDs. Stimulation of
EGFRs results in activation of series downstream kinases, which promote cell
proliferation, resistance to apoptosis, tumor invasion, and tumor angiogenesis (Rao
and James, 2004). The EGFRs expression is relatively low in low-grade glioma and
absent in normal brain tissues, suggesting that over expressed EGFRs contribute to
the malignant phenotype of human glioblastomas, and thus a promising target for
brain tumor diagnosis and treatment. Moreover, EGFR overexpression in GBM is
correlated with poor prognosis results among glioblastoma patients and confers
radioresistance to the tumor cells (Barker et al., 2001). Inhibition of EGFR function
will be critical to improve tumor response to radiotherapy for EGFR overexpression
patients. In total, determining EGFR expression level in tumor cells can provide
potentially useful clinical information to the physician for identifying a subgroup of
glioma patients and designing the specific therapeutic strategies for patients.
2.2.4 Enhanced tumor imaging with anti-EGFR conjugated quantum dots
Molecular-specific contrast agents are expected to facilitate the detection of tumor
and its margins and improve diagnostic accuracy and contrast. EGFR overexpression
in high-grade gliomas can be a clinical biomarker for cancer diagnostics. Several
studies have demonstrated that anti-EGFR antibody conjugated gold nanoparticles or
organic fluorescent contrast agents were able to specifically and homogeneously
bind to the targeted cancer cells and distinguish abnormal from normal tissues
(Sokolov et al., 2004, Hsu et al., 2004, El-Sayed et al., 2005). None of these
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researches had ever explored the potential of using QDs as fluorescent probes. As
described in chapter 2.1, QDs with unique optical properties have many advantages
in biomedical imaging. The outstanding brightness provides possibility to highlight
glioma infiltrations, which have very low cell density. Moreover, transport of
nanoparticles across the BBB is possible by either passive diffusion or by carrier-
mediated endocytosis (Pulfer et al., 1999, Alyaudtin et al., 2001). In vivo
multiphoton microscopy demonstrated that QDs were able to highlight large blood
vessels about 800 pm below surface of wild-type mouse cortex after administration
of fluorescent QDs by tail vein injection (Michael et al., 2005). This result proved
that, with extremely small diameters, QDs were able to cross the BBB and stain
tumors for in vivo imaging. To differentiating brain tumors, antibodies recognizing
EGFR extracellular domain were linked to QDs. With anti-EGFR guidance, QDs are
expected to label brain tumor cells and frozen tissues with high specificity and
sensitivity.
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Chapter 3
Experiment Materials and Methods
3.1 Cell Culture
The U87 human glioblastoma and MCF-7 human breast cancer cell lines were kindly
provided by Dr. H Phillip Koeffler (Cedars-Sinai Medical Center, Los Angeles). The
SKMG-3 human GBM cells were supplied by Dr. Christopher Y. Thomas
(University of Virginia, Charlottesville). The U87 and MCF-7 cells were maintained
in RPMI-1640 containing 10% fetal bovine serum (FBS), 50 units/mL penicillin G,
50 pg/mL streptomycin, and 2mM L-glutamine and incubated at 37°C with 5% C02.
The SKMG-3 cells were grown in DMEM and incubated at 37°C with 5% C02.
3.2 Fluorescence Microscopy
Quantum dots stained cells were cultured on 8-well Lab-Tek cover glass chamber
(Nalge Nunc International, Naperville, IL) in PBS during observation. Images were
examined with an inverted Zeiss Axiovert 200 fluorescence microscopy equipped
with a Zeiss Axiocam MRm. The excitation filter is 425/45 nm. The emission was
collected using a 525/30 nm filter. Image acquisition, processing, and analysis were
done by using AxioVision 3.1.
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3.3 Imaging Glioma Cells and Frozen Tissue with Quantum dots
With the goal of immunolabeling glioma tissues, we develop a scheme using anti-
EGFR mediated endocytosis to deliver QDs into tumor cells. The antibodies are
attached to QDs streptavidin conjugates before mixing with cells (Figure 3). The
preparation of antibody-QD complexes is based on non-covalent binding of biotin to
streptavidin in aqueous solution, which is irreversible and extremely stable over a
wide range of temperature and pH.
Streptavidin
Cell membrane
A nti-EG FR Biotin
Figure 3 Strategy for anti-EGFR conjugated streptavidin coated QDs selectively binding
to EGFR.
3.4 Preparation of Anti-EGFR Conjugated Quantum dots
Through non-covalent binding between biotin and streptavidin, biotinylated
antibodies bind to QD streptavidin conjugates and form a stable QD-antibody
complex. In this study, biotinylated human monoclonal anti-EGFR antibody
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(Biodesign International), which is targeted against the extracellular domain of
EGFR, and QD 525 streptavidin conjugates (Quantum Dot Corporation) were diluted
to 2 ng/mL and 20 nM/mL, respectively, in PBS containing 2.5% (wt/vol) BSA and
incubated for 30 min at room temperature.
3.5 Immunolabeling of Cells with Quantum Dots Conjugates
Cultured live cells were first washed with PBS, then blocked with PBS containing
2.5% BSA for 30 min at 37°C with 5% CO2 and incubated sequentially with QD-
anti-EGFR complexes in PBS containing 2.5% BSA for 2 hours 37°C with 5% CO2.
Unbound QD-anti-EGFR complexes were removed by washing with PBS for three
times before observation.
3.6 Preparation and Immunolabeling of Frozen Tissue Sections with
Quantum dots Conjugates
The frozen tissues were prepared by Dr. Charles Yong from Cedar-Sinai Hospital.
All slides were first washed with PBS, then blocked with PBS containing 2.5% BSA
for 30 min at 37°C with 5% C 02 and incubated sequentially with QD-anti-EGFR
complexes in PBS containing 2.5% BSA for 2 hours 37°C with 5% C02. Unbound
anti-EGFR conjugated QDs were removed by washing with PBS for three times
before observation. The adjacent tissue sections were subjected to H&E staining.
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Chapter 4
Results and Discussion
4.1 Immunolabeling of Tumor Cells with Anti-EGFR Conjugated
Quantum Dots
This targeted delivery strategy was designed to investigate both binding and
internalization of QDs in brain tumor cells. In order to use QDs in cancer diagnosis,
the QDs have to be able to highlight tumor from normal tissues. To test the
specificity of anti-EGFR mediated QDs labeling, we exposed various cell line
expressing different level of EGFRs on the membrane to anti-EGFR conjugated
QDs. To detect QDs inside cells, we used fluorescence microscopy with 63x
objective to evaluate the QDs internalization and subcellular localization in live
cells. False-color fluorescence images were obtained at excitation 425/45 nm with a
525/20 nm band-pass filter.
The fluorescence was firstly seen primarily on the cell membrane (Figure 4a) and
later in the cytoplasm (Figure 5a, 5d). The distribution of QDs depends on incubation
time and observation time. Intracellular accumulations of QDs in small vesicles were
detected in EGFR-overexpressing glioma cell line, SKMG-3, within 15 min. This
result indicates that these nano-particles did not impair the biological process of the
antiEGFR-mediated endocytosis. The intensity of QDs fluorescence inside of
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SKMG-3 cells kept increasing until reaching a plateau after 2 hours incubation. QDs
fluorescence was also clearly detected in U87 GBM cell line, which is lack of EGFR
overexpression, but much weaker compared to SKMG-3 cells. In contrast, there is no
detectable QDs in breast cancer cell line, MCF-7, which have extremely low quantity
of EGFRs on the membrane (Figure 6a), indicating the quantity of QDs entered cells
is depending on the membrane EGFR level. Cells mixed with anti-EGFR (Figure 7a)
or QD525 streptavidin conjugates (Figure 7d) have no detectable QDs fluorescence
inside of cells or on the cell membranes, suggesting that QDs attached and entered
the cells through anti-EGFR binding to EGFRs. After removing solution of anti-
EGFR conjugated QDs, the fluorescence of QDs on cell membrane disappeared in a
few minutes, suggesting this receptor-mediated delivery of QDs is a fast and
irreversible process.
Figure 4 Targeting QDs to EGFRs in SKMG-3 cells. SKMG-3 cells were incubated
with anti-EGFR conjugated QDs for 30 minutes at 37°C before imaging. The images
were taken at room temperature with a 63x water-immersion objective immediately
after removing QDs solution, (a) Fluorescence image; (b) corresponding transmitted
DIC image; (c) Merged image of a and b
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Figure 5 Targeting QDs to EGFRs in human GBM cell lines, SKMG-3 and U87.
Cells were incubated with anti-EGFR conjugated QDs for 2 hours at 37°C before
imaging. The images were taken at room temperature with a 63 x water-immersion
objective immediately after removing QDs solution, (a) Fluorescence image of
SKMG-3 cells; (b) Corresponding transmitted DIC image; (c) merged image of a and
b; (d) fluorescence image of U87 cells; (e) Corresponding transmitted DIC image, (f)
Merged image of d and e.
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Figure 6 Images of control cell line, MCF-7 taken with a 63 x water-immersion
objective. MCF-7 cells were incubated with anti-EGFR conjugated QDs for 2 hours
at 37°C before imaging. The images were taken at room temperature immediately
after removing QDs solution, (a) Fluorescence image o f MCF-7 cells; (b)
Corresponding transmitted DIC image; (c) merged image of a and b.
Upon binding of antibody on the external domain, EGFRs soon become internalized
through endocytosis. Hence, the QDs attached to the anti-EGFR through biotin and
streptavidin reaction were brought into cells together with antibody and EGFR
through receptor-mediated endocytosis. Instead of diffusing into cytoplasm, QDs
were restricted in the small vesicles, which have been reported to be early
endosomes. The QDs-containing vesicles are uniformly distributed in the cytoplasm
after 2 hours incubation with anti-EGFR conjugated QD (Figure 8). The fluorescence
of QDs didn’t decrease during the 3-dementional optical sectioning, indicating their
extraordinary anti-bleaching properties.
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Figure 7 Images o f SKMG-3 cells taken with a 63x water-immersion objective, (a)
SKMG-3 cells were exposed to anti-EGFR for 2 hours at 37°C before imaging; (b)
Corresponding transmitted DIC image; (c) merged image of a and b; (d) SKMG-3
cells were exposed to streptavidin conjugated QD525 for 2 hours at 37°C before
imaging; (e) Corresponding transmitted DIC image; (f) merged image of d and e.
QDs did not enter cells when antibody or QDs streptavidin conjugates were
presented alone.
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Figure 8 Multi-panel z-stack images at 0.5 pm interval. SKMG-3 cells were
incubated with anti-EGFR conjugated QDs for 2 hours at 37°C before imaging. The
images were taken at room temperature with a 63x water-immersion objective
immediately after removing QDs solution.
The fluorescence of QDs inside cells can last for a few minutes when exposed to
excitation light without visually detectable decrease due to QDs photo stability.
However, the fluorescence intensity of QDs decreased dramatically after 24 hours
(Figure 7), which might be due to the incompatibility of QDs surface with the
biological environment. After 24 hours, the QDs were accumulated in a small region
in the cytoplasm (Figure 9a). Compared to images of 24 hours, there were no
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significant changes after 48 hours (Figure 9d). The fluorescence of QDs inside cells
could be detected in a few cells even after 7 days.
Figure 9 Tracking QDs in SKMG-3 cells at 24 and 48 hours. SKMG-3 cells were
exposed to anti-EGFR for 2 hours at 37°C. Then, replaced the QD-anti-EGFR
complex solution with growth medium and incubated cells at 37°C. Cells were
washed and placed in PBS. Image was taken at room temperature after 24 hours and
48 hours incubation, (a) Fluorescence image after 24 hours; (b) orresponding
transmitted DIC image; (c) merged image of a and b; (d) fluorescence image after 48
hours; (e) Corresponding transmitted DIC image; (f) merged image of a and b.
4.2 Immunolabeling of Frozen Tumor Tissues with Anti-EGFR
Conjugated Quantum Dots
Similar approach was applied to frozen tumor tissue specimens from three GBM
patients. The tumor tissue specimens were confirmed with H& E staining (Figure 10
a). GBM samples were characterized by darkly stained polymorphic nuclei with
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atypia. Fluorescence microscopy with 40x objective and 63x objective was used to
evaluate the QDs staining in frozen tissues. False-color fluorescence images were
obtained at excitation 425/45 nm with a 525/20 nm band-pass filter. All samples
from three high-grade glioma patients were immunostained with anti-EGFR
conjugated QDs. Fluorescence images shows that anti-EGFR conjugated QDs
attached to the frozen tissue slices (Figure 10 b). In contrast, no fluorescence can be
detected in slides incubated with streptavidin conjugated QDs (Figure 11 a),
indicating that QDs attached to tissue sections through anti-EGFR. The fluorescence
in GBM frozen tissue slices loaded with anti-EGFR conjugated QDs could be
detected within 15 minutes. Compared to traditional immunohistochemistry assay for
EGFR, QDs with extraordinary brightness can greatly reduce the time needed to
assess biopsy tissue by eliminating most of steps for signal amplification.
4.3 Discussion
In vitro experiment with cell cultures demonstrated that the QDs with anti-EGFR on
the surface target the EGFR on the cell membrane. The SKMG-3 human glioma cell
line, which has been known to have the highest EGFR level on the membrane in
vitro, took up the largest quantity of QD-antibody complex and showed the highest
fluorescence intensity of QDs inside cells among three cell lines (Figure 5, 6) in this
study. The U87 glioma cells overexpressing EGFR also took up QDs and present
much greater fluorescence intensity (Figure 5) than the MCF-7 cells lacking EGFR
expression (Figure 4A). These results suggest that non-specific QDs labeling is
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unlikely to occur in normal tissue or tumors that lack of EGFR expression. The
intensity of QDs is related to the EGFR expression level. We are expected to achieve
specificity for brain tumors and get information for EGFR expression by using this
approach. In addition, immunostaining GBM frozen tissues specimens with anti-
EGFR conjugated QDs suggests that bioconjugated QDs as fluorescent biological
labels could serve as a complimentary exogenous contrast agent and facilitate real
time demarcation of brain tumors with increased speed, specificity and sensitivity.
Currently, the ability to discriminate between neoplastic and normal brain tissue
using optical spectroscopy depends on spectral and time-domain measurement of
autofluorescence emission (Poon et al., 1992, Chung et al., 1997, Lin et al., 2000,
Corce et al., 2003, Marcu et al., 2004). Investigations were conducted on both in
vitro and in vivo conditions and demonstrated higher sensitivity and specificity.
However, none of these studies had ever explored the possibility to use exogenous
targeted fluorescent probes for a real-time diagnostic application in neurosurgery to
enhance tumors and their margins. Unlike results based on autofluorescence, the
measurements from cancer-marker-targeted fluorescence probes are unlikely to
change significantly under different oxidation states, thereby providing more
consistent and accurate diagnostic results for in vitro and in vivo studies. Initial
attempt to use exogenous near-infrared contrast-enhancing dye, indocyanine green
(IDG), for in vivo enhanced optical imaging of human gliomas demonstrated the
potential to differentiate between normal brain and tumor tissue at the cortical
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surface and the depths of the resection margins by measuring optical signals from
IDG instead of autofluorescence (Haglund et al., 1996). But using conventional
organic fluoreophore suffers from low quantum yield, photo-bleaching effect and
difficulties in controlling excitation and emission wavelengths. To solve these
problems, we used targeted inorganice fluorescent QDs to selectively stained brain
tumor cells and tissues. Our results suggest that QDs can recognize hiomakers on
tumor cells through binding of antibody antigen, thereby providing higher specificity
and accuracy. In addition, with high quantum efficiency and brightness, QDs are
expected to be able to highlight tumor in filtrations, which have relatively low
quantity of tumor cells.
With the unique characteristics and optical advantages, bioconjugated QDs have
ability to detect various biomarkers and localize potential tumors and tumor margins.
QD probes can be delivered to tumors by passive and active targeting mechanisms
(Gao et al., 2004). To achieve high specificity and efficiency, we used QDs
conjugated with antibodies that recognize readily accessible membrane receptor,
EGFR, in our study. QDs were proved to actively attach to cells and tissues with
EGFR overexpression. Besides anti-EGFRs, other antibodies against well-known
brain tumor biomarkers, such as glial fibrillary acidic protein (GFAP) and lactate
(LAC), could be linked to QDs through similar streptavidin-biotin interaction for
diagnostics. Considering QDs broad excitation and narrow emission, it’s possible to
perform multicolor and multitarget imaging for cancer diagnostics. In addition, the
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large surface of QDs allows the attachment of a large number of functional groups
for diagnostic and therapeutic purposes. With additional conjugation step, in addition
to work as fluorescent probes, QDs could serve as drug carriers and bring therapeutic
agents to brain tumors.
The QDs streptavidin conjugates used in this study were not optimized for in vivo
imaging. Our in vitro study showed that the fluorescence of these QDs inside cells
was significantly reduced after 24 hours. With respect to in vivo use of QDs as
biological labels for tumor targeting and imaging, question concerning surface
modifications must be addressed. To push our study into animal model, QDs
designed to provide adequate circulating lifetime, minimal nonspecific deposition
and a sufficient fluorescence time are required. Studies focused on long-term in vivo
imaging revealed that the QDs coating played an important roles in reducing
accumulation in the liver and the bone marrow (Akerman et al., 2002, Gao et al.,
2004, Ballou et al., 2004). The PEG molecules prevent non-specific phagocytosis
and significantly improve the biocompatibility and circulation time (Akerman et al.,
2002, Gao et al., 2004). ABC triblock copolymers help preventing the particle
aggregation and fluorescence loss (Gao et al., 2004). In addition to surface chemistry,
the selection of QD wavelength is also important for in vivo imaging. Theoretical
model predicted deep tissue imaging require near infrared QDs to avoid tissue
absorbance and scatter (Lim et al., 2003). A non-targeted type II near-infrared QDs
with emission at 850 nm has been injected into mice and pig and successfully aided
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image-guided resection of nearby lymph node (Kim et al., 2004). However, the
techniques for the synthesis, coating, and bioconjugation of near-infrared QDs are
not well developed. Targeted QDs with near-infrared emission are needed for in vivo
enhanced optical imaging of brain tumor.
Previous studies demonstrated that QDs with a stable polymer coating didn’t affect
cell division and other physiological functions (Akerman et al., 2002, Gao et al.,
2004). Our in intro results were consistent with these reports. QDs used in this study
did not interfere receptor mediated endocytosis, cell viability and growth. There was
no morphological change among cells stained with QDs. However, the metabolism
and clearance of QDs in vivo are not clear. Although QDs are similar in size range to
many common biomolecules, it’s difficult to clear them from the circulation through
retinal filtration. This problem has yet to be addressed carefully before clinical
applications.
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a
Figure 10 Representative images of GBM frozen tissue slices, (a) GBM H&E stating;
(b) Slices were incubated with antiEGFR conjugated QDs for 2 hours; (c)
Corresponding transmitted DIC image.
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Figure 11 Images of GBM frozen tissue slices exposed to QD strepavidin conjugates.
(a) Fluorescent image; (e) Corresponding transmitted DIC image.
Bioconjugated QDs used in our study were achieved by mixing commercial QDs
with biotinylated anti-EGFRs. Missing of purification step resulted in free anti-
EGFRs and QD streptavidin conjugates in staining solutions. BSA was used to
eliminate non-specific binding between strepavidin and other molecules in cell
cultures and tissues. Images of cells loaded with QDs only (Figure 7d) showed no
detectable fluorescence, which excludes the possibility that QDs stained cells
through streptavidin and endogenous biotin non-specific interactions. However, it’s
not feasible to use blocking buffer for in vivo real time imaging. Purified
bioconjugated QDs are highly desired to increase the specificity and sensitivity for in
vivo studies. Another limitation in this study is that all the frozen tissue specimens
were from three GBM patients. Although in vitro cell culture experiments indicated
that the fluorescent intensity of QDs depended on the quantity of EGFR on cell
membrane, the possibility to determine low-grade brain tumors and normal brain
tissues based on EGFR expression level remains unknown. Additional studies are
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required to increase the knowledge of QDs immunostaining of various types of brain
tumors and EGFR-negative tissue as well as GBM specimens.
Since EGFR expression level is related to malignancy of brain tumor, radiotherapy
resistance and prognostic results after surgery, the ability to detect EGFR expression
level in frozen tissue section may help physician define the grade of brain tumors
and choose the after-surgery treatment.
4.4 Conclusions
In summary, we demonstrate the efficiency and specificity of the antibody
conjugated QDs for imaging EGFR overexpressed cancer cell lines, such as human
GBM cell lines, U87 and SKMG-3, and the ability for imaging the QDs loaded
cancer lines for a long timeframe. The fluorescent intensity of QDs inside cells
depends on the quantity of EGFR on the cell membrane, which provides the
possibility to differentiate tumor and normal cells by quantify the intensity of EGFR-
targeted QDs. In addition, frozen tissue samples from three high-grade gliablastoma
patients were immunostained with anti-EGFR conjugated QDs, offering an
alternative method to conventional immunohistochemistry detection of EGFRs.
Although further investigations concerning various brain tumor tissues and EGFR-
negative tissues are necessary to provide detailed knowledge of tumor diagnostics
with QDs, our results suggest the possibility for enhanced optical demarcation of
brain tumors with QDs in vivo.
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Asset Metadata

Creator Wang, Jingjing (author)

Core Title A fluorescence microscopy study of quantum dots as fluorescent probes for brain tumor diagnosis

School Viterbi School of Engineering

Degree Master of Science

Degree Program Biomedical Engineering

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag engineering, biomedical,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Marcu, Laura (committee chair), D'Argenio, David (committee member), Gundersen, Martin A. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-49160

Unique identifier UC11338332

Identifier 1435094.pdf (filename),usctheses-c16-49160 (legacy record id)

Legacy Identifier 1435094.pdf

Dmrecord 49160

Document Type Thesis

Rights Wang, Jingjing

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA