sábado, 20 de marzo de 2010

Review of Quantum Dot Technologies for Cancer Detection and Treatment

Semiconductor quantum dots (QDs) are nanoparticles that have attracted
widespread interest in biology and medicine due to their unique
optical and electronic properties. These properties, especially their
reduced tendency to photobleach and the dependence of their
fluorescence wavelength on their size, make them suitable for
fluorescent probing applications to detect cancer biomarkers in vitro
and in vivo in cells/tissues/whole body. There is considerable
interest among researchers due to the recent developments in QD
technology. QDs have been encapsulated in amphiphilic polymers and
bound to tumor-targeting ligands and drug delivery vesicles for
targeting, imaging and treating tumor cells. Present efforts are
focussed on exploring the massive multiplexing capabilities of the QDs
for the simultaneous detection of multiple cancer biomarkers in blood
assays and cancer tissue biopsies. These advances in the QD technology
have unravelled a great deal of information about the molecular events
in tumor cells and early diagnosis of cancer.
Quantum Dots in Early Diagnosis of Cancer
Early screening of cancer is desirable as most tumors are detectable
only when they reach a certain size when they contain millions of
cells that may already have metastasized. Currently employed
diagnostic techniques such as medical imaging, tissue biopsy and
bioanalytical assay of body fluids by enzyme linked immunosorbent
assay (ELISA) are insufficiently sensitive and specific to detect most
types of early-stage cancers. Moreover, these assays are labour
intensive, time consuming, expensive and don't have multiplexing
capability. On the other hand, QD based detection is rapid, easy and
economical enabling quick point-of-care screening of cancer markers.
QDs have got unique properties which make them ideal for detecting
tumors. These include intense and stable fluorescence for a longer
time; resistance to photobleaching [1-5], large molar extinction
coefficients, and highly sensitive detection due to their ability to
absorb and emit light very efficiently. Due to their large surface
area-to-volume ratio, a single QD can be conjugated to various
molecules, thus making QDs appealing for employment in designing more
complex multifunctional nanostructures. Various types of biomarkers
such as proteins, specific DNA or mRNA sequences and circulating tumor
cells have been identified for cancer diagnosis from serum samples.
Therefore, QD based multiplexed approach [1] for the simultaneous
identification of many biomarkers would lead to more effective
diagnosis of cancer. QDs have been covalently linked to various
biomolecules such as antibodies, peptides, nucleic acids and other
ligands for fluorescence probing applications [6-19]. Some of the
applications of QDs in biology [20-32] along with their tremendous
potential for in vivo molecular imaging [33-37] have already been
Advantages of Inorganic Quantum Dots over Organic Fluorophores
Compared to traditional organic fluorophores used for fluorescence
labelling in biological experiments, inorganic QDs have wider
applications due to their high resistance to photobleaching, which
enables visualization of the biological material for a longer time.
Fluorophores are highly sensitive to their local environment and can
undergo photobleaching, an irreversible photooxidation process which
makes them non-fluorescent. This is the main limitation for all
studies in which the fluorophore labelled structure has to be observed
over extended periods of time. Fluorophores can be optically excited
only within a narrow range of wavelengths and fluorescent emission is
also restricted to a certain range of wavelengths. Whereas QDs can be
excited with a single light source having wavelength shorter than the
wavelength of fluorescence. The fluorescence spectra of QDs are
narrow, symmetric and have no red tail as observed in fluorophores.
Various colors can be observed and distinguished without any spectral
overlap. Therefore, multicolor labelling of different structures with
QDs of different colors became possible. This multiplexed approach [3,
38-40] is of great interest in wide ranging applications such as
disease diagnosis and drug delivery.
The field of QDs is of multidisciplinary as persons from different
scientific disciplines i.e. chemistry, physics, biology and medicine
are working together to harness their potential. Their employment for
the detection and treatment of cancer is one such application which is
of paramount importance.
Quantum Dot Technology
QDs are inorganic semiconductor nanocrystals having typical diameter
between 2-8 nm that possess unique luminescent properties. They are
generally composed of atoms from groups II and VI elements (e.g. CdSe
and CdTe) or groups III and V elements (e.g. InP and InAs) of the
periodic table. Their physical dimensions are smaller than the exciton
Bohr radius [1] that leads to quantum confinement effect, which is
responsible for their unique optical and electronic properties.
Synthesis of Quantum Dots
High quality QDs have been synthesized by various approaches [41-43].
But usually their synthesis is carried out in organic solvents such as
toluene or chloroform at higher temperatures in the presence of
surfactants. But the surfactant-coated particles are not soluble in
water as they have polar surfactant head group attached to the
inorganic core of QD and the hydrophobic chain protruding into the
organic solvent. Usually, all experiments with cells involve
water-soluble materials. Therefore, various strategies have been
developed to make them water-soluble, where either the surfactant
layer is replaced or coated with additional layer such as hydrophilic
or amphipathic polymers [44-45]. The hydrophobic coating of surfactant
is replaced by ligand molecules carrying functional groups at one end
that bind to the QD surface, and hydrophilic groups at other end that
make the QDs water soluble. The employment of amphiphilic polymers as
an additional coating on QD surface has also been reported [38,
46-48]. The hydrophobic tail of the polymer reacts with the
hydrophobic surfactant layer on QD surface whereas the hydrophilic
groups of the polymer on the outer end impart water solubility. QDs
have also been encapsulated in phospholipid micelles [8] to make them
water soluble.
Properties and Applications of Quantum Dots
The most commonly used QD system is the inner semiconductor core of
CdSe coated with the outer shell of ZnS. The ZnS shell is responsible
for the chemical and optical stability of the CdSe core. QDs can be
made to emit fluorescent light in the ultraviolet to infrared spectrum
just by varying their size. The wavelength of fluorescence of the QD
depends on its energy gap (i.e. the difference between the excited and
the ground state) which is determined by the size of the QD [49-52].
QDs have narrow spectral line widths, very high levels of brightness,
large absorption coefficients across a wide spectral range, high
photostability and capability of multiplexed detection. They are very
bright and stable even under complex in vivo conditions that make them
suitable for advanced molecular and cellular imaging, drug delivery
and for highly sensitive bioassays and diagnostics [53-54]. Highly
sensitive real-time imaging with greater resolution and tracking of
single receptor molecules on the surface of living cells have been
made possible by QD bioconjugates [13, 55]. Various applications of
quantum dots are stated in figure 1. In most of the cases, functional
QD conjugates for cancer detection are composed of a semiconductor
core (CdSe, CdTe); an additional shell such as ZnS in the case of CdSe
QDs having a higher band gap than CdSe to increase quantum yield; a
water soluble hydrophilic coating; and, functionalized antibodies or
other biomolecules complementary to the target cancer markers at the
tumor sites.
Figure 1. Applications of Quantum dots.

Overcoming the Toxic Nature of Quantum Dots
The native QDs made up of semiconductor nanoparticles are toxic in
nature. It has been observed that CdSe QDs are highly toxic to cells
exposed to UV for a longer time [56] as UV dissolves the CdSe, thereby
releasing toxic cadmium ions. However, polymer-coated QDs are
non-toxic in the absence of UV as demonstrated by in vivo studies
[48]. It has also been shown that the micelle-encapsulated QDs
injected into the frog embryo did not affect its development [8].
Therefore, QDs are normally encapsulated inside the outer coating of
amphiphilic polymers [57-58] to make them water-soluble and resistant
to chemical or enzymatic degradation. They are typically synthesized
in organic solvents such as tri-n-octyl-phosphine oxide (TOPO) [59-62]
and hexadecylamine, having long alkyl chains and high boiling points,
to prevent the formation of aggregates. In the recent years, there has
been a great development to modify the surface chemistry of QDs to
make them water soluble [63-64]. Most commonly, QDs are linked to
polyethylene glycol (PEG) or similar ligands to make them
biocompatible and to reduce nonspecific binding. They are made
specific to the target site by conjugating them to various bioaffinity
ligands such as peptides, antibodies, oligonucleotides etc. using
different strategies. A possible schematic of the QD bioconjugate for
the detection of tumor cell biomarkers is shown in figure 2. Figure 3
describes in brief the various steps of the QD technology for the in
vivo diagnosis of cancer.
Figure 2. Multifunctional QDs usually employed for targeting tumor
cells. QDs are conjugated to various affinity ligands (peptide,
antibody, inhibitor, drug etc.) specific for the tumor cell
Figure 3. Various steps in employing QDs for in vivo diagnosis of
cancer. (a) Formation of QD bioconjugates, (b) Intravenous injection
of QD bioconjugates into mouse, (c) Active targeting of tumor cells by
QD bioconjugates.

Blinking Behavior of Quantum Dots
Nirmal et al. [65] discovered for the first time that QDs shows a
blinking behavior i.e. intermittent on–off emission upon continuous
excitation, which was attributed to Auger ionization [65-66]. The
principle of this behavior is not well understood even today. But it
is a concern only when a signal from individual QD is required during
the analysis such as flow cytometry applications. In such cases, it
may be possible that the emission from the individual QD might be off
due to 'blinking' thus leading to the missing of signal at the
detector. But generally in most of the applications such as in
cell-based assays, there are more than one QD involved and even if
some QDs are blinking, others are giving signal for the final
detection and thus, no signal will be missed by the detector. One way
of counteracting the reduced quantum yield due to blinking is to grow
a shell of a few atomic layers of a material with a larger band gap on
top of the QD core.
Effect of Surface Functionalization on the Optical Properties of Quantum Dots
Fundamental studies have revealed that luminescence of QD is very much
sensitive to the surface functionalization procedures as the
interactions of the molecule with the QD's surface change the surface
charges on the QD [67]. But many of the QD based probing applications
are based on the change in fluorescence of QD after the interaction of
the target analyte molecules with the biomolecules functionalized on
the QD surface. It has been well reported that the surface
functionalization of QDs improves their solubility. But it could
reduce their quantum efficiency as well. This has demonstrated in the
case of meracptoacetic acid-treated QDs where the quantum efficiency
was reduced drastically [7, 63]. But protein functionalized quantum
dots tend to retain their quantum efficiency and offer longer shelf
life. They can also be further functionalized with multiple functional
groups [7] without decreasing their quantum efficiency.
Measurement System for Observing and Tracking Quantum dots
Single QDs can be observed and tracked for greater time duration up to
a few hours with confocal microscopy, total internal reflection
microscopy or epifluorescence microscopy. The scheme of the
fluorescent imaging employing QDs as labels and its measurement has
been described by Gao et al. [68] and So et al [69]. Gao et al.
employed a whole-body macro-illumination system with
wavelength-resolved spectral imaging, which allows high sensitivity
detection of molecular targets in vivo. So et al. also employed the
wavelength-resolved spectral imaging system having software that
separated autofluorescence from quantum dot signals.
Active and Passive Quantum Dot Targeting Mechanisms
C.I : 16.959.604

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