KEYWORDS: Cell death, Tumors, Picosecond phenomena, Multispectral imaging, In vivo imaging, Breast cancer, Monoclonal antibodies, Tomography, Near infrared, Indocyanine green
Noninvasive and nonradioactive imaging modality to track and image apoptosis during chemotherapy of triple negative breast cancer is much needed for an effective treatment plan. Phosphatidylserine (PS) is a biomarker transiently exposed on the outer surface of the cells during apoptosis. Its externalization occurs within a few hours of an apoptotic stimulus by a chemotherapy drug and leads to presentation of millions of phospholipid molecules per apoptotic cell on the cell surface. This makes PS an abundant and accessible target for apoptosis imaging. In the current work, we show that PS monoclonal antibody tagged with indocyanine green (ICG) can help to track and image apoptosis using multispectral optoacoustic tomography in vivo. When compared to saline control, the doxorubicin treated group showed a significant increase in uptake of ICG-PS monoclonal antibody in triple negative breast tumor xenografted in NCr nude female mice. Day 5 posttreatment had the highest optoacoustic signal in the tumor region, indicating maximum apoptosis and the tumor subsequently shrank. Since multispectral optoacoustic imaging does not involve the use of radioactivity, the longer the circulatory time of the PS antibody can be exploited to monitor apoptosis over a period of time without multiple injections of commonly used imaging probes such as Tc-99m Annexin V or F-18 ML10. The proposed apoptosis imaging technique involving multispectral optoacoustic tomography, monoclonal antibody, and near-infrared absorbing fluorescent marker can be an effective tool for imaging apoptosis and treatment planning.
The transverse and longitudinal plasmon resonance in gold nanorods can be exploited to localize the photothermal therapy and influence the fluorescence to monitor the treatment outcome at the same time. While the longitudinal plasmon peak contributes to the photothermal effect, the transverse peak can enhance fluorescence. After cells take in PEGylated nanorods through endocytosis, autofluorescence from endogenous fluorophores such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) in the mitochondria is enhanced two times, which is a good indicator of the respiratory status of the cell. When cells are illuminated continuously with near infrared laser, the temperature reaches the hyperthermic region within the first four minutes, which demonstrates the efficiency of gold nanorods in photothermal therapy. The cell viability test and autofluorescence intensity show good correlation indicating the progress of cell death over time.
Fluorescence spectroscopy is a powerful and widely used technique, which finds extensive applications in biochemistry
and molecular biology. In many cases, detection of fluorescence from biological samples at low concentration demands
for a sensitive technique that can enhance the weak fluorescence signal. In order to realize this, metal enhanced
fluorescence technique is employed where an increase in fluorescence is observed when the fluorophore is in close
proximity to a metallic surface. In order to achieve this, nano-roughened silver surfaces such as fractal structures or
silver colloid-coated surfaces can be employed. However, preparation of such surfaces not only involves lengthy
chemical procedures but also result in poor reproducibility. To overcome these limitations, nanosphere lithography
(NSL) is proposed, which is an inexpensive, simple to implement and high throughput nanofabrication technique. In this
technique, polystyrene (PS) nano-beads are used to form a 2D monolayer of nanoparticle array followed by deposition of
silver to form a roughened metallic surface. The surface roughness of the silver coating is determined by the close
packing and arrangement of the nanobeads. In this work, substrates are fabricated using different sizes of PS beads to
result in different nanometric surface roughening for silver layer and its optimization to achieve better fluorescence
enhancement is carried out.
Optical profilometers for micrometer resolutions are getting increasingly popular for non-contact measurement of
surface profiles. Confocal profilometer is one such optical technique which can pick up sub-micron height variations of
the object surface. Though the system is a single point measurement scanning in the X-Y directions enables one to plot
the 3-D profile of the surface. However, the Z-scanning range is limited to ±1mm. In this work we report a system which
can adapt itself to widen the scanning range in the Z-direction. For increasing the scan range we have added another
translation stage in the Z axis which adapts itself when the system profiles a surface with step heights more than its
original range. The X-Y-Z translation stage is connected to the computer and is controlled using a LabVIEW program.
The 3-D plot is obtained by plotting the Z-values from the confocal system to the corresponding X-Y position of the
translation stage. When ever the system goes out of range the Z translation stage is increased or decreased thus adapting
itself to plot a 3D profile of the specimen.
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