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18BIOPHOTONIC APPROACHES TOWARDS CIRCULATING TUMOR CELL DETECTION AND PHOTODYNAMIC CANCER THERAPY
HEMA SANTHAKUMAR, KUNNUMPURATHU JIBIN, RAMAPURATH. S. JAYASREE
Sree Chitra Tirunal Institute for Medical Sciences and Technology, BMT Wing, Thiruvananthapuram, Kerala, India jayasree@sctimst.ac.in
ABSTRACT
Although, cancer is considered as a localized disease in its pre-mature stages, in certain types of cells, it often is innate with patient becoming symptomatic and results in metastasis. Cancer cells are showered from the primary tumor cells into the circulating blood stream that finally forms metastasis. Thus, it becomes critical to identify and quantify the circulating tumor cells (CTC) during the early stages of tumorigenesis.
Here, we discuss about the design and fabrication of a user-friendly custom-designed nanotag enabled portable system for the selective separation and isolation of CTCs from whole blood. The detection should be followed by treatment. Gold nanoclusters of ultrasmall size comprising a few atoms are known for its unique feature of excellent fluorescent property. Herein, simple one step green synthesis method for the preparation of highly fluorescent silver doped gold nanoclusters using custom synthesized tripeptide Asp-Cys-Gly and its potential in generating ROS towards cancer treatment, and simultaneous imaging is discussed. Because invasive tissue biopsy data obtained from limited amount of collected tissues are often expensive and biased, "liquid biopsy" has gained significant attention as a promising diagnostic procedure for the identification and quantification of cancer driven materials present in the blood stream. The key targets employed in liquid biopsy include various circulating biomarkers such as circulating tumor cells, circulating vesicles, circulating nucleic acids and circulating proteins. Conventionally, these biomarkers are detected either by traditional protein or nucleic acid-based assays. Among various circulating biomarkers, CTC show great promise in the early detection of cancer and more specifically cancers of metastatic nature, since they are prognostic markers which are present in the blood plasma. However, it is extremely challenging to develop a system to count and detect the CTC in cancer patients as its presence is as low as 1-10 CTC/mL of blood plasma. Herein with the aid of nanotechnology, we present a custom-designed SERS nanotag enabled portable filter-based sensor platform which exploits both tumor cell target specificity and size based centrifugal force for the separation and isolation of circulating breast cancer cells (SKBR3 cells) from peripheral blood sample. Towards this goal, we designed a simple and easy-to-handle custom made centrifugal prototype comprising of three independent chambers (figure 1b), all of which are made transparent for clear internal visibility, detachability of components for ease of use, reusability and autoclavability. The single prototype can serve the purposes starting from collecting blood along with SERS nanotag, centrifugation, size and antibody-based cell separation and finally the detachable filter taken out for SERS and microscopic analysis.
Considering the unique roles that could perform within the single prototype, it is hereafter named as a 'lab -on-a-filter' system. The 'lab-on-a-filter' system is equipped with an anti-EpCAM immobilized flexible and transparent track-etched polycarbonate (PC) membrane filter with pore size 8^M. To rely on the SERS technology to detect breast cancer cells in peripheral blood, a highly sensitive and target specific sandwich system of SERS nanotag (Au-rGO@anti-ErbB2) is prepared and incubated with the collected blood before using it in the developed system. As we have used SKBR3 cells as model CTCs, which has both EpCAM and ErbB2 over expression, we have used two different antibodies, anti-EpCAM conjugated over the polycarbonate filter sheets and anti-ErbB2 antibody for the SERS tag preparation. This dual functionalization increases the specificity of the 'lab-on-a filter' system towards the capturing of the cancer cells for an efficient isolation and detection. The PC membrane filter is placed in the centrifugal prototype in such a way that, it can be detached and mounted for SERS imaging analyses after the selective isolation of CTCs on top of it. Taken together, the lab-on-a-filter system will serve as a potential candidate for the identification, isolation and accurate quantification of CTC, which may have huge impact in translational clinical research.
In another study we have utilized gold nanoclusters which has unique features arising from quantum size effect for the imaging and cancer therapy. Gold cluster is a material of interest with ultrasmall size with a few atoms (< 3 nm), has excellent fluorescent property, tremendous catalytic activities, two-photon absorptions and long lifetime. Its physico-chemical and biological properties are extremely favourable for drug delivery and therapeutic applications due to its high inertness, low toxicity with good renal clearance and long blood circulation time. The fluorescence of the gold nanoclusters can be tuned with different core size and ligand functionalization. Here, we have synthesized a novel cluster using simple one step method to get highly fluorescent silver doped gold nanoclusters (DCG-GNC) using custom synthesized tripeptide Asp-Cys-Gly (DCG) as reducing cum stabilizing agent. The peptides were designed to contain cysteine residue to facilitate interaction with gold ions, thereby to achieve biomineralization of gold clusters. Unlike the case of gold nanoparticles which is known for its surface plasmon resonance peak in the visible range, gold nano cluster shows no sharp absorption. Moreover, it exhibits strong fluorescence which can be tuned in the desired region (figure 2a). DCG-GNC displayed strong fluorescent property, large Stokes shift and good photostability, thereby making them promising bioimaging agents. The cytocompatibity and cellular uptake of DCG-GNC were investigated in MCF-7 breast cancer cells. In addition, the anti-cancer activity of DCG-GNC was also explored with the photoinduced generation of increased intracellular ROS followed by stimulation of mitochondrial apoptotic pathway in cancer cells. Photodynamic therapy (PDT) works with the accumulation of photosensitizer (PS) in tumours, which upon light irradiation generate singlet oxygen (1O2) and other reactive oxygen species (ROS) to induce cell death. ROS, a specific type of oxygen-
containing reactive molecules plays important roles in different cellular processes, including cell proliferation at basal level, but at high concentration, ROS can be cytotoxic and induce apoptosis/necrosis. The photodynamic potential of DCG-GNC by acting as a photosensitizer or induce ROS generation under laser irradiation was evaluated by H2-DCFDA staining method. H2-DCFDA is a nonfluorescent cell-permeable dye, which is activated by intracellular esterases through cleavage of acetate groups and becomes fluorescent (Xem = 525 nm) upon oxidation by ROS. The ROS production in the presence and absence of laser irradiation performed in breast cancer cells is shown in figure 2b. The study points to the fact that peptide stabilized silver doped gold cluster in general and specifically, DCG-GNC can act as a promising cancer theranostic agents and the potential of their use could be ventured into for future clinical applications.
Figure 1. a) Formation of Au-rGO@anti-ErbB2 nanotag. b) Design of centrifugal prototype and c) Detection specificity of SKBR3 cells in whole blood and d) Raman intensity against number of SKBR3 cells for limit of detection.
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Figure 2. UV-Vis Absorption, Emission & Excitation spectra of TPGNCsDCG-GNC and DCS-GNC. Insets show the photographic image of gold nanoclusters under visible (left) and UV light, 365nm (right) illumination. Microscopic images of ROS generation in DCG-GNC and DCS-GNC treated cancer cells with and without laser irradiation
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