Kiran Bhattacharyya

An Effective Strategy for the Synthesis of Biocompatible Gold Nanoparticles Using Cinnamon Phytochemicals for Phantom CT Imaging and Photoacoustic Detection of Cancerous Cells

ABSTRACTPurposeThe purpose of the present study was to explore the utilization of cinnamon-coated gold nanoparticles (Cin-AuNPs) as CT/optical contrast-enhancement agents for detection of cancer cells.MethodsCin-AuNPs were synthesized by a “green” procedure, and the detailed characterization was performed by physico-chemical analysis. Cytotoxicity and cellular uptake studies were carried out in normal human fibroblast and cancerous (PC-3 and MCF-7) cells, respectively. The efficacy of detecting cancerous cells was monitored using a photoacoustic technique. In vivo biodistribution was studied after IV injection of Cin-AuNPs in mice, and also a CT phantom model was generated.ResultsBiocompatible Cin-AuNPs were synthesized with high purity. Significant uptake of these gold nanoparticles was observed in PC-3 and MCF-7 cells. Cin-AuNPs internalized in cancerous cells facilitated detectable photoacoustic signals. In vivo biodistribution in normal mice showed steady accumulation of gold nanoparticles in lungs and rapid clearance from blood. Quantitative analysis of CT values in phantom model revealed that the cinnamon-phytochemical-coated AuNPs have reasonable attenuation efficiency.ConclusionsThe results indicate that these non-toxic Cin-AuNPs can serve as excellent CT/ photoacoustic contrast-enhancement agents and may provide a novel approach toward tumor detection through nanopharmaceuticals.

Photoacoustic Detection of Melanoma Micrometastasis in Sentinel Lymph Nodes

Melanoma is the deadliest form of skin cancer and has the fastest growth rate of all cancer types. Proper staging of melanoma is required for clinical management. One method of staging melanoma is performed by taking a sentinel node biopsy, in which the first node in the lymphatic drainage path of the primary lesion is removed and tested for the presence of melanoma cells. Current standard of care typically involves taking fewer than ten histologic sections of the node out of the hundreds of possible sections available in the tissue. We have developed a photoacoustic method that probes the entire intact node. We acquired a lymph node from a healthy canine subject. We cultured a malignant human melanoma cell line HS 936. Approximately 1 x 10(6) cells were separated and injected into the lymph node. We also had a healthy lymph node in which no melanoma cells were implanted. We used a tunable laser system set at 532 nm to irradiate the lymph nodes. Three piezoelectric acoustic detectors were positioned near the lymph node to detect photoacoustic pulses generated within the lymph nodes. We also acquired lymph nodes from pigs and repeated the experiments with increased amplification and improved sensors. We detected photoacoustic responses from a lymph node with as few as 500 melanoma cells injected into the tissue, while normal lymph nodes showed no response. Photoacoustic generation can be used to detect melanoma micrometastasis in sentinel lymph nodes. This detection can be used to guide further histologic study of the node, increasing the accuracy of the sentinel lymph node biopsy.

Enhanced photoacoustic detection of melanoma cells using gold nanoparticles

We tagged melanoma cells with gold nanoparticles to show their viability for increasing sensitivity in a photoacoustic detection system. Ultimately, this study models the detection of circulating tumor cells, which are an important prognostic factor in the progress of melanoma.

Capture of circulating tumor cells using photoacoustic flowmetry and two phase flow

Melanoma is the deadliest form of skin cancer, yet current diagnostic methods are unable to detect early onset of metastatic disease. Patients must wait until macroscopic secondary tumors form before malignancy can be diagnosed and treatment prescribed. Detection of cells that have broken off the original tumor and travel through the blood or lymph system can provide data for diagnosing and monitoring metastatic disease. By irradiating enriched blood samples spiked with cultured melanoma cells with nanosecond duration laser light, we induced photoacoustic responses in the pigmented cells. Thus, we can detect and enumerate melanoma cells in blood samples to demonstrate a paradigm for a photoacoustic flow cytometer. Furthermore, we capture the melanoma cells using microfluidic two phase flow, a technique that separates a continuous flow into alternating microslugs of air and blood cell suspension. Each slug of blood cells is tested for the presence of melanoma. Slugs that are positive for melanoma, indicated by photoacoustic waves, are separated from the cytometer for further purification and isolation of the melanoma cell. In this paper, we evaluate the two phase photoacoustic flow cytometer for its ability to detect and capture metastatic melanoma cells in blood.

Gold Nanoparticle–Mediated Detection of Circulating Cancer Cells

Photoacoustic flowmetry has been used to detect pigmented particles in body fluids, most notably circulating melanoma cells in blood samples of metastatic melanoma patients. Exploiting the plasmon resonance of gold nanoparticles and the ability to specifically target cancer cell surface proteins, photoacoustic flowmetry may be used to detect non-pigmented CTCs. We targeted the EpCAM receptors to attach 50nm gold nanoparticles to a breast cancer cell line, T47D. After determining the absorption peak and thus the most sensitive laser wavelength, we performed serial dilution trials to show detection of small numbers of breast cancer cells in suspension. While some cell clumping may have altered some of our results for cell counting, it is feasible to use gold nanoparticles to detect and capture CTCs in a photoacoustic flowmeter. This ability may allow an earlier clinical diagnosis and management of metastatic disease for a range of solid tumor types. Capture of CTCs may also allow cell specific molecular analysis and a new paradigm for personalized cancer therapy.

Optically absorbing nanoparticle mediated cell membrane permeabilization

Membrane permeabilization is imperative for gene and drug delivery systems, along with other cell manipulation methods, since the average eukaryotic cell membrane is not permeable to polar and large nonpolar molecules. Antibody conjugated optically absorbing gold nanospheres are targeted to the cell membrane of T47D breast cancer cell line and irradiated with 5 ns pulse, 20 Hz, 532 nm light to increase membrane permeability. Up to 90% permeabilization with less than 6% death is reported at radiant exposures up to 10 times lower than those of other comparable studies.

Nanoparticle Mediated Thermal Ablation of Breast Cancer Cells Using a Nanosecond Pulsed Electric Field

In the past, ablation of cancer cells using radiofrequency heating techniques has been demonstrated, but the current methodology has many flaws, including inconsistent tumor ablation and significant ablation of normal cells. Other researchers have begun to develop a treatment that is more selective for cancer cells using metallic nanoparticles and constant electric field exposure. In these studies, cell necrosis is induced by heating antibody functionalized metallic nanoparticles attached to cancer cells. Our approach to studying this phenomenon is to use similarly functionalized metallic nanoparticles that are specific for the T47D breast cancer cell line, exposing these nanoparticle cell conjugates to a nanosecond pulsed electric field. Using fluorescent, polystyrene-coated, iron-oxide nanoparticles, the results of our pilot study indicated that we were able to ablate up to approximately 80% of the cells using 60 ns pulses in increasing numbers of pulses and up to approximately 90% of the cells using 300 ns pulses in increasing numbers of pulses. These quantities of ablated cells were achieved using a cumulative exposure time 6 orders of magnitude less than most in vitro constant electric field studies.

Total internal reflection photoacoustic spectroscopy for the detection of β-hematin.

Evanescent field sensing methods are currently used to detect many different types of disease markers and biologically important chemicals such as the HER2 breast cancer receptor. Hinoue et al. used Total Internal Reflection Photoacoustic Spectroscopy (TIRPAS) as a method of using the evanescent field to detect an optically opaque dye at a sample interface. Although their methods were successful at detecting dyes, the results at that time did not show a very practical spectroscopic technique, which was due to the less than typical sensitivity of TIRPAS as a spectroscopy modality given the low power (≈ 1 to 2 W) lasers being used. Contrarily, we have used an Nd:YAG laser with a five nanosecond pulse that gives peak power of 1 MW coupled with the TIRPAS system to increase the sensitivity of this technique for biological material sensing. All efforts were focused on the eventual detection of the optically absorbing material, hemozoin, which is created as a byproduct of a malarial infection in blood. We used an optically analogous material, β-hematin, to determine the potential for detection in the TIRPAS system. In addition, four properties which control the sensitivity were investigated to increase understanding about the sensors function as a biosensing method.

Visual Threat Assessment and Reticulospinal Encoding of Calibrated Responses in Larval Zebrafish

All visual animals must decide whether approaching objects are a threat. Our current understanding of this process has identified a proximity-based mechanism where an evasive maneuver is triggered when a looming stimulus passes a subtended visual angle threshold. However, some escape strategies are more costly than others, and so it would be beneficial to additionally encode the level of threat conveyed by the predators approach rate to select the most appropriate response. Here, using naturalistic rates of looming visual stimuli while simultaneously monitoring escape behavior and the recruitment of multiple reticulospinal neurons, we find that larval zebrafish do indeed perform a calibrated assessment of threat. While all fish generate evasive maneuvers at the same subtended visual angle, lower approach rates evoke slower, more kinematically variable escape responses with relatively long latencies as well as the unilateral recruitment of ventral spinal projecting nuclei (vSPNs) implicated in turning. In contrast, higher approach rates evoke faster, more kinematically stereotyped responses with relatively short latencies, as well as bilateral recruitment of vSPNs and unilateral recruitment of giant fiber neurons in fish and amphibians called Mauthner cells. In addition to the higher proportion of more costly, shorter-latency Mauthner-active responses to greater perceived threats, we observe a higher incidence of freezing behavior at higher approach rates. Our results provide a new framework to understand how behavioral flexibility is grounded in the appropriate balancing of trade-offs between fast and slow movements when deciding to respond to a visually perceived threat.

Role of branchiomotor neurons in controlling food intake of zebrafish larvae

Abstract The physical act of eating or feeding involves the coordinated action of several organs like eyes and jaws, and associated neural networks. Moreover, the activity of the neural networks controlling jaw movements (branchiomotor circuits) is regulated by the visual, olfactory, gustatory and hypothalamic systems, which are largely well characterized at the physiological level. By contrast, the behavioral output of the branchiomotor circuits and the functional consequences of disruption of these circuits by abnormal neural development are poorly understood. To begin to address these questions, we sought to evaluate the feeding ability of zebrafish larvae, a direct output of the branchiomotor circuits, and developed a qualitative assay for measuring food intake in zebrafish larvae at 7 days post-fertilization. We validated the assay by examining the effects of ablating the branchiomotor neurons. Metronidazole-mediated ablation of nitroreductase-expressing branchiomotor neurons resulted in a predictable reduction in food intake without significantly affecting swimming ability, indicating that the assay is robust. Laser-mediated ablation of trigeminal motor neurons resulted in a significant decrease in food intake, indicating that the assay is sensitive. Importantly, in larvae of a genetic mutant with severe loss of branchiomotor neurons, food intake was abolished. These studies establish a foundation for dissecting the neural circuits driving a motor behavior essential for survival.