Nanotechnology in Cancer Imaging (ESSAY 1)

Background
Nanotechnology is basically the study of the design, characterization, production,
and application of structures, devices, and systems by controlled manipulation of
size and shape at the nanoscale (atomic, molecular, and macromolecular scale) that
produces structures, devices, and systems with a unique property. Nanotechnology
has rapidly developed over the past decade in the fields of biology, engineering and
materials science. In recent years, nanotechnology is increasingly being applied for
the treatment of cancer in two main areas: the development of nanovectors, such
as nanoparticles, to be loaded with drugs or imaging agents and then targeted to
cancer tumours, and nano-sensor devices for detecting the biological signatures of
cancer. Together, these technologies can allow earlier diagnosis and hence quicker
and more efficient treatment for patients with cancer. Currently, among the
breakthroughs in nanotechnology, cancer treatment ranks among one of the top
five.

The first article (1) analyses how Iron oxide nanoparticles can be used in the
Magnetic Resonance Imaging (MRI) of the tyrosine kinase Her-2/neu receptor in
breast cancer cells, which plays a significant role in
diagnosing and curing breast cancer. To test this, three
human breast cell lines, namely AU-565, MCF-7, and
MDA-MB-231, which had different expression levels of
the Her-2/neu protein were used. The test was done in
two steps, firstly attaching the Her-2/neu antibody
Herceptin to the receptors and then selectively binding
streptavidin-super paramagnetic iron oxide (SPIO)
magnetic resonance contrast agent to those receptors.
This contrast agent is 50nm in diameter, containing an SPIO core coated with
polysaccharide conjugated to streptavidin molecules to provide specific binding to
the receptors. The cells were then grown, and using laser to excite the FITC
fluorophore, a significant shift of fluorescence intensity for all breast cancer cell
lines was observed, showing that the SPIO nanoparticles with the antibody
Herceptin is able to efficiently bind to the cells, with it being most effective in AU565 cells and least effective in MDA-MB-231 cells. Hence this antibody would be
able to target the Her-2/neu receptor to potentially cure breast cancer.
The second article (2) analyzes the use of circumin (natural derivative of Curuma
longa with different chemical groups that have been formed to increase solubility of
curcumins and make them suitable for drug delivery, etc.) in breast cancer imaging

and also therapy. This plant possesses quite a number of beneficial biological
characteristics, which include those that fight against cancer. MDA-MB-231 cancer
cells are a type of breast cancer cell and have the ability to spread from one organ
to another. CUR can effectively upregulate p53, p21, and p27 and downregulate
cyclin E; thus, cell cycle arrest occurs at the G1 phase in these cancer cells, and
hence these cells cannot multiple and cause harm. CUR also causes damage to the
DNA of these cancer cells, and synergistic cell growth inhibition was found when
used with chemotherapy, radiation, and other drugs. Moreover, CUR is not
particularly directly toxic or harmful
to normal non-cancerous human cells.
However, CUR has problems of solubility degradation in human body, and rapid
metabolism by body cells. This problem can be solved by using nanocarriers, such
as polymer nanoparticles, nanoassemblies, and self-assemblies. This enhance drug
delivery to and thus induce cytotoxicity and apoptosis in cancer cells. Magnetic
Nanoparticles (MNPs) are considered one of the important materials for theranostic
applications due to their small particle size, high magnetization values, etc. To
improve the functioning of MNPs, an iron oxide nanoparticle core was made in the
presence of CD and then coated with pluronic polymer F68 (poly[ethylene-copropylene glycol]). Results were that nanoparticle aggregation in dynamic light
scattering particle size analysis was decreased , but the size of the individual
nanoparticle remained the same. This would improve the accuracy of cancer
imaging and improve diagnostics, hence bringing about early detection.
The third article(3) uses Bioresorbable calcium phosphate nanoparticles (CPNPs) in
which molecules of the near-infrared (NIR) emitting fluorophore, indocyanine green
(ICG), are embedded, and is one of the least toxic contrast agents administered to
humans. The objective of the research was to report a novel carrier system for
sensitive deep-tissue NIR imaging using sub-50 nm, biocompatible CP nanoparticles
(CPNPs), colloidally stable in physiological solutions, which exploit the matrixshielding effect and thereby impart improved fluorescence properties to the
encapsulated ICG suitable for sensitive, early state diagnostic imaging.
ICG has a low fluorescence quantum yield due to internal conversion and is prone to
photobleaching, solvatochromic effects, and nonspecific quenching, all of which
limit its utility in sensitive and prolonged in vivo imaging applications. Fluoroprobes,
for example, are being developed to achieve this selective imaging sensitivity,
particularly those that work in the near-infrared (NIR, 700900 nm) spectrum, a
wavelength region of low absorptivity by tissue chromophores. This permits
fluorescence signals relatively free of intrinsic background interference with
detectable signal intensities through several centimeters of tissue. This improves
various fluoroprobe molecules for in vivo applications, where a fluoroprobe is able to
retain its state of dispersion in physiological environments, sustain a strong,
prolonged signal intensity, accumulate in targeted regions of interest, and passively
be absorbed into the body upon completion of function.

The fourth article (4) makes use of the synthesis and in vivo characterization of
bismuth sulfide (Bi2S3) nanoparticles labeled with the cyclic nine amino acid
peptide, CGNKRTRGC (LyP-1)-targeted to 4T1 breast cancer in mice. Larger atomic
size of Bi atom as compared to Iodine or Gold atoms allow it to be more easily
detectable by scans (e.g. CT). Bi2S3 also shown to have a equal or greater contrast
as a contrast agent. Due to size, the nanoparticles appear to undergo clearance
from the mice through a fecal route during this time period. Although this limits the
period of imaging, but can be said to provide a safe mechanism for nanoparticle
clearance, so the test subjects were not harmed.

In the 5th article, synthesis and in vivo characterization of bismuth sulfide ( Bi2S3)
nanoparticles labeled with the cyclic nine amino acid peptide, CGNKRTRGC (LyP-1)targeted to 4T1 breast cancer in mice is presented. Tissue-specific nanoparticles
have shown great potential as contrast agents for in vivo medical imaging of several
cancer types in enabling the acquisition of high fidelity CT images of the orthotopic
tumor, particularly at the tumor margins. The size of the nanoparticles limits renal
clearance, favoring splenic and hepatic clearance mechanisms instead, and the
presence of targeting
peptide exhibited no significant impact on the accumulation of the nanoparticles
within these organs. A similar proportion of Bi accumulated in the lungs, indicating
that larger sized agglomerates formed during circulation in the bloodstream.
Following accumulation, the monodispersed Bi2S3 nanoparticles yield sufficient
contrast to provide quantitative, high fidelity CT images of tumors for ca. 1 week
after injection. Notably, the nanoparticles appear to undergo clearance from the
mice through a fecal route during this time period.
Discussion
Article 1: MR Molecular Imaging of the Her-2/neu Receptor in Breast Cancer Cells
Using Targeted Iron Oxide Nanoparticles
MR Molecular
Properties
Imaging of the Her2/neu Receptor in
Breast Cancer
Cells Using
Targeted Iron
Oxide
Nanoparticles
In vitro

Advantages

Disadvantages

Non-invasive,
Possibly harmful to
allows for multiple humans in vivo
screening
due
to
high
molecular weight

Fe2O3
Nanoparticles
(high
molecular
weight)
with
polysaccharide
coating and binded
to
Herceptin
(antibody)

Magnetic
High amount of
Resonance
antibodies
used
Imaging allows for might
trigger
3D imaging
harmful
immune
response
in
humans

Enhances
signal
amplifiction
as
SPIO nanoparticles
act as and active
transporter system

Article 1 uses MRI to target the Her-2/neu receptor of the breast cancer cells, which
is non-invasive and hence is a method that can allow for early detection as well as a
complete picture and repeated measurements. Significant signal amplification can
be achieved if the contrast agent is allowed to accumulate in the target cells by
passive endocytosis, or by an active transporter system such as the SPIO
nanoparticles used in this experiment. However, these studies were only carried out
in-vitro, as the cells were harvested and incubated in a laboratory. Hence, it is not
guaranteed that this treatment would work when tested in-vivo, as the delivery of
high molecular weight compounds such as iron oxide may potentially harm the
body. Moreover, a harmful immune response may arise in the host due to the large
amount of antibody and macromolecular imaging agent used in the MRI.

Article 2: Curcumin-loaded magnetic nanoparticles for breast cancer therapeutics

and imaging applications
Curcumin-loaded
magnetic
nanoparticles for
breast cancer
therapeutics and
imaging
applications

Properties

Use of MNPs with

CUR to enhance
drug delivery and

Advantages

Disadvantages

imaging

an
iron
oxide
nanoparticle core
was made in the
presence of CD
and then coated
with
pluronic
polymer
F68
(poly[ethylene-copropylene glycol]).
Results were that
nanoparticle
aggregation
in
dynamic
light
scattering particle
size analysis was
decreased, which
would improve the
accuracy of cancer
imaging
and
improve
diagnostics, hence
bringing
about
early detection.

plant
possesses
quite a number of
beneficial
biological
characteristics,
which
include
those that fight
against cancer.

problems
of
solubility
degradation
in
human body, and
rapid metabolism
by body cells (can
be solved using
nanocarriers)

CUR causes cell

cycle arrest at the
G1 phase in cancer
cells, and hence
these cells cannot
multiple and cause
harm.

CUR
is
not
particularly
directly toxic or
harmful to normal
non-cancerous
human cells.

There is also the

chance
of
ROS
production which
causes
inflammation, DNA
and cell damage,
etc.
ROS
production in cells
with CUR was not
that different from
cells without CUR,
but
ROS
was
increased in CURMNP cells.

CUR also causes

damage
to the
DNA
of
these
cancer cells, and
synergistic
cell
growth
inhibition
was found when
used
with
chemotherapy,
radiation,
and
other drugs.

MNPs are used in this experiment to improve drug delivery and also imaging to
detect cancerous cells early. This procedure is preferred as it harps on the medicinal
properties of CUR and improves the functioning of the drug and also in imaging. In
vitro MRI was performed for these MNP-CUR formulas and the fluorescence property
of CUR was used to more effective analyze cellular uptake of the drug. However,
these experiments were only carried out in-vitro and no in-vivo and hence it cannot
be guaranteed that these results can be reproduced for in-vivo conditions.
Moreover, CUR has problems of solubility, etc., and it cannot be confirmed if MNPs
can fully fight these problems unless this experiment is carried out in-vitro. There is
also the chance of ROS production which causes inflammation, DNA and cell
damage, etc. ROS production in cells with CUR was not that different from cells
without CUR, but ROS was increased in CUR-MNP cells. Hence, this is a concern for
application of CUR-MNP cells.

Article 3: Near-Infrared Emitting Fluorophore

Nanoparticles
for In Vivo Imaging of Human Breast Cancer
Properties
Fluorophobes
developed
to
selective
sensitivity

Doped

Advantages
permits
fluorescence
being signals relatively free of
achieve intrinsic
background
imaging interference
with
detectable
signal
intensities
through
several centimeters of
tissue

Calcium

Disadvantages

Phosphate

Internal conversion of ICG

ICG
has
fluorescence
yield

ICG
is
prone
to
photobleaching,
solvatochromic
effects
and
nonspecific
quenching

ICGs use in sensitive and

prolonged in vivo imaging
applications is limited

ICG is optically unstable

in physiologically relevant
solutions (eg. water, salt
solutions, plasma, blood)

Decreased
absorption,
reduced fluorescence and
variability of maximum
absorbence
wavelength
due to oxxidation and
dimerization of original
molecule

ICG
often
proteins

Results
in
rapid
agglomeration
and
subsequent
elimination
from the body

binds

to

Inhibition of unfavourable
conformational
reorganisation;
reduced
interaction with solvent
molecules

Improved photostability of
the
encapsulate;
avoidance of dynamic
processes
resulting
in
nonradiative
energy
losses

Encapsulation
of
the Monomeric state of the
fluorophore
encapsulated
within the rigid CP matrix ICG is retained without
deleterious structural or
chemical alterations that
adversely
affect
absorption and emission
of
the
dye,
leading
to
undesirable
peak
transformations
or
spectral shifts
CP matrix is impermeable Shields
the
dopant
to
the
solvent fluorophore from solvent
environment
interactions;
prevents
oxidative and solventinduced
alterations

a
low
quantum

Analysis of the notable

increase in the half-life of
the encapsulated dye
versus the free dye when
subjected to continuous
illumination

Encapsulated
ICG
molecules are protected
from
environmental
oxygen in the CPNP
fluoroprobe system, which
permits prolonged periods
of
excitation
without
significant degradation in
emission intensity

Use of a commercial laser

diode for excitation and
an
inexpensive
commercial CCD camera
modified for NIR imaging

Emphasizes the potential

for
ICG-CPNP
imaging
applications
where
portability and
ease of operation, such as
in the surgical operating
theater,
are
critical
requirements.

Better optical properties Sequesters the ICG from

compared to the free environmental influence ,
fluorophore
hence
providing
increased
brightness and prolonged
signal intensity vital for
sensitive
diagnostic
applications.
NIR dye-doped calcium phosphate nanoparticles (CPNPs) was synthesised and
characterised, while the results of the study was categorised into
1. Optical properties of NIR dye-doped CPNPs. Emission enhancement
The brightness or intensity does not provide evidence that the encapsulation
provides intrinsically different fluorescence performance than the free molecules. In
order to assess whether the elevated brightness is intrinsic to the encapsulated ICG,
the quantum efficiency per molecule is presented and analysed.
2. Quantum Efficiency (QE)
Measuring the QE provides a metric for the potential of a fluoroprobe in imaging
schemes, for sensitive or long-term imaging. Thus, CPNPs doped with ICG exhibit
intrinsically greater brightness and enhanced QE relative to the free dye for in vivo
and in vitro applications.
3. Solvent protection
The chemical stability of the encapsulated fluorophore was experimentally verified
by analyzing fluorescence spectra as a function of solvent environment. In contrast,
the emission spectra for the encapsulated dye in CPNPs are stable regardless of
solvent environment. The CP matrix is impermeable in to the solvent environment,

which shields the dopant fluorophore from solvent interactions and prevents
oxidative and solvent-induced alterations, effectively sequestering the ICG from
environmental influence.
4. Photostability
The encapsulated ICG molecules are protected from environmental oxygen in the
CPNP fluoroprobe system, hence ensuring photostability.
5. In Vivo Pharmacokinetic Distribution and Tumor Localization in Nude Mice
The relatively short in vivo fluorescence of the free dye is attributed to fluorescence
quenching of free ICG in physiological environments with rapid aggregation and
clearance from the body. The lipophilic character of ICG means it is taken up
exclusively by hepatic parenchymal cells where it is then secreted into the bile. .
Negligible movement of fluorescence across the blood-brain barrier was observed,
which means that the barriers are relatively impermeable to the carboxylate- or
PEG-CPNPs.
6. Tissue imaging
The signal intensity from the ICG-CPNPs also highlights the optically transparent
nature of the CPNP carriers to the emission wavelength, an essential criterion for
sensitive tissue imaging applications.

Article 4: Bioconjugated Gold Nanoparticles as a Molecular Based Contrast Agent:

Implications for Imaging of Deep Tumors Using Optoacoustic Tomography
Properties

Advantages

Combination
of
compelling properties of
light
and
ultrasound
(acoustic waves)

Abillity of generate highcontrast

high-resoltuoin
imges
of
malignant
tumours located deep
within
tissue
(high
resolution deep tissue
imaging)

Shape and structure of serve as a very strong

NPs can be designed
optoacoustic
contrast
agent
Inert material used for Colloidal gold is especially
therapeutic applications attractive for use as NPs
in the past
Mab
as
a
selective
targeting agent, gold NPs
as an image contrast
agent, and optoacoustic
tomography
as
an

Gold NPs have potential in

being used for imaging
deep tumours in early
stages
of
cancer
or
metastatic
lesions;

Disadvantages

imaging
system
for
diagnosis of small tumors
undetectable by current
imaging modalities.

potential to become a
powerful
method
for
diagnosis of small tumors
undetectable by current
imaging modalities.

Optical illumination and ultrasonic detection to produce deep tissue images based
on their
light absorption. Firstly, antibodies were conjugated to NPs, resulting in an
estimated 8.28 x 1013 biotin binding sites in each ml of the streptavidin-conjugated
gold NPs. The cell lines of human breast tumor SK-BR-3 cells, L6 rat myoblasts and
primary human fibroblast cells were maintained in DMEM supplemented with 5%
FBS and 1% PSA at 37 degrees celcius in a humidified air atmosphere with 5% CO2.
Next, fibroblast cells were grown on glass coverslips, then fixed and washed with
various solutions like Herceptin, Cy3 conjugated goat anti-human antibody before
DAPI containing mounting media was used to stain the cell nucleus. SK-BR-3 cells
were either grown on glass coverslips or culture slides and rinsed with different
solutions. Slides were examined using the Carl Zeiss LSM410 confocal microscope,
or examined by an electron microscope at 60 kV after samples were cut very thinly
and stained with lead citrate. For scanning electon microscopy, specimen stubs
were mounted with cover-slip pieces and sputter-coated with gold-palladium,
corresponding to a 5nm thick coating. To study the amplitude of optoacoustic
signals as a function of concentration of gold NPs, each implanted gel object
containing NPs was illuminated while a transducer connected to a digital
oscilloscope detected optoacoustic signals which were saved to a file for
subsequent analysis. For the optoacoustic imaging of targeted cancer cells, 2 gel
phantoms were prepared and illuminated with laser pulses for 10ns. 1 gel phantom
had 3 tubes embedded in the centre while the other was used to present an
optoacoustic image of SK-BR-3 cell clusters targeted with gold NPs.
Article 5: X-Ray Computed Tomography Imaging of Breast Cancer by using Targeted
Peptide-Labeled Bismuth Sulfide Nanoparticles
Properties

Advantages

Large atomic number of

Bi (z=83) compared to
currently
available clinical or preclinical
alternatives,
such as iodinated
(z=53) or gold-based
systems (z=79)

well-suited as CT contrast agents,

allowing it to be more easily
detectable by scans; NPs of Bi2S3
have also been shown to be equal or
superior to iodinated CT contrast
agents

Disadvantages

Limited signs of acute

cytotoxicity
at
concentrations up to
0.03m Bi, and the LyP-1
peptide-labeled
nanoparticles
preferentially
accumulated into 4T1
tumors both in vitro and
in vivo

Enables acquisition
of high fidelity CT images of the
orthotopic tumor, particularly at the
tumor margins

Able to provide quantitative, high

Monodispersed Bi2S3
nanoparticles
yield fidelity CT images of tumors for ca. 1
week after injection
sufficient contrast
The
size
of
nanoparticles; presence
of targeting
peptide;
similar
proportion
of
Bi
accumulated in the
lungs

Renal clearance is limited, splenic

and hepatic clearance
mechanisms are favoured instead;
no significant impact on the
accumulation
of the nanoparticles within these
organs (spleen, liver) was exhibited;
larger sized agglomerates formed
during
circulation

The intestines of the

mouse also
showed
enhanced
contrast; nanoparticles
appear
to
undergo
clearance from
the mice through a fecal
route

The nanoparticles are removed

through a hepatobiliary/fecal route;
a safe
mechanism
for
nanoparticle
clearance is provided despite the
limitation on the period of imaging.

In this article, synthesis and in vivo characterization of bismuth sulfide ( Bi2S3)

nanoparticles labeled with the cyclic nine amino acid peptide, CGNKRTRGC (LyP-1)targeted to 4T1 breast cancer in mice is presented. Tissue-specific nanoparticles
have shown great potential as contrast agents for in vivo medical imaging of several
cancer types in enabling the acquisition of high fidelity CT images of the orthotopic
tumor, particularly at the tumor margins. The size of the nanoparticles limits renal
clearance, favoring splenic and hepatic clearance mechanisms instead, and the
presence of targeting
peptide exhibited no significant impact on the accumulation of the nanoparticles
within these organs. A similar proportion of Bi accumulated in the lungs, indicating
that larger sized agglomerates formed during circulation in the bloodstream.
Following accumulation, the monodispersed Bi2S3 nanoparticles yield sufficient
contrast to provide quantitative, high fidelity CT images of tumors for ca. 1 week

after injection. Notably, the nanoparticles appear to undergo clearance from the
mice through a fecal route during this time period.
Although this limits the period of imaging, it provides a safe mechanism for
nanoparticle clearance.
While the accumulation observed in the kidneys and lungs was small, more work is
needed to determine the roles of agglomeration and degradation on the toxicity and
pharmacokinetic profiles of the material.

Future work
Some modifications could have been done to the experimental procedure for article
1 and 2. Both only carry out tests in-vitro, hence tests could have been carried out
in-vivo on animals such as in articles 3, 4 and 5 as well after they produced
promising results in-vitro. They could have also tested out different types of
nanoparticles other than iron oxides one to test and see which type of nanoparticle
would better work as an active transporter system for the signal amplification of the
MRI.
For article 2, ways to combat ROS production could be analyzed in detail, while
keeping the benefits possible. More types of nanoparticles could be tested with
CUR and hence their effectiveness could be analyzed.
For the third article, combined with the established biocompatibility and facile
resorbability of calcium phosphates, the physical and optical properties of ICGencapsulating CPNPs represent an attractive new fluoroprobe for sensitive
diagnostic imaging applications. Hence, this could also have been tested
experimentally to see how effective it would be in the imaging of breast cancer.
For the fifth article, an optoacoustic contrast agent allowing for utilisation of the full
diagnostic power of OAT could be developed while nanotechnology and molecular
targeting of tumours could be deployed to design and fabricate smart contrast
agents that would significantly enhance specificity and sensitivity of OAT for
detection of cancerous lesions in vivo.
Moreover, the first article suggests that nanoparticles with a higher molecular
weight would be potentially harmful to the body while article 4 suggests that such
particle are larger hence more easily detected in scans to identify tumours. This is a
contradiction, hence it could be tested which is the optimal size and mass of a
nanoparticle such that the benefits are maximised and the disadvantages are
minimised as much as possible.

References
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Her-2/neu Receptor

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Jaggi, M.; Chauhan, S.C. Curcumin-loaded magnetic nanoparticles for breast cancer
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