<p>Although various proof-of-concept studies have demonstrated the eventual potential of a multifunctional SERS-active metallic nanostructures for biological applications such as single cell analysis/measurement and drug delivery, the actual development and testing of such a system in vitro has remained challenging. One key point at which many potentially useful biomethods encounter difficulty lies in the translation of early proof-of-concept experiments in a clean, aqueous solution to complex, crowded, biologically-active environments such as the interior of living cells. The research hypotheses for this work state that multifunctional nanoconstructs can be fabricated and used effectively in conjunction with surface-enhanced Raman scattering (SERS) spectroscopy and other photonics-based methods to make intracellular measurements in and deliver treatment to single cells. The results of experimental work address the specific research aims, to 1) establish temporal and spatial parameters of nanoprobe uptake and modulation, 2) demonstrate targeting of functionalized nanoparticles to the cytoplasm and nucleus of single cells, 3) deliver to and activate drug treatment in cells using a multifunctional nanosystem, and 4) make intracellular measurements in normal and disease cells using external nanoprobes,</p><p>Raman spectroscopy and two-dimensional Raman imaging were used to identify and locate labeled silver nanoparticles in single cells using SERS detection. To study the efficiency of cellular uptake, silver nanoparticles were functionalized with three differently charged SERS/Raman labels and co-incubated with J774 mouse macrophage cell cultures for internalization via normal cellular processes. The surface charge on the nanoparticles was observed to modulate uptake efficiency, demonstrating a dual function of the surface modifications as tracking labels and as modulators of cell uptake. </p><p>To demonstrate delivery of functionalized nanoparticles to specific locations within the cell, silver nanoparticles were co-functionalized with the HIV-1 TAT (49-57) peptide for cell-penetrating and nuclear-targeting ability and p-mercaptobenzoic acid (pMBA) molecules as a surface-enhanced Raman scattering (SERS) label for tracking and imaging. Two-dimensional SERS mapping was used to track the spatial and temporal progress of nanoparticle uptake in PC-3 human prostate cells and to characterize localization at various time points, demonstrating the potential for an intracellularly-targeted multiplexed nanosystem. Silver nanoparticles co-functionalized with the TAT peptide showed greatly enhanced cellular uptake and nuclear localization as compared with the control nanoparticles lacking the targeting moiety. </p><p>The efficacy of targeted nanoparticles as a drug delivery vehicle was demonstrated with development and testing of an anti-cancer treatment in which novel scintillating nanoparticles functionalized with HIV-1 TAT (49-57) for cell-penetrating and nuclear-targeting ability were loaded with tethered psoralen molecules as cargo. The experiments were designed to investigate a nanodrug system consisting of psoralen tethered to a nuclear targeting peptide anchored to UVA-emitting, X-ray luminescent yttrium oxide nanoparticles. Absorption of the emitted UVA photons by nanoparticle-tethered psoralen has the potential to cross-link adenine and thymine residues in DNA located in the nucleus. Such cross-linking by free psoralen following activation with UVA light has previously been shown to cause apoptosis in vitro and an immunogenic response in vivo. Experimental results using the PC-3 human prostate cancer cell line demonstrate that X-ray excitation of these psoralen-functionalized Y2O3 nanoscintillators yields concentration-dependent reductions in cell number density when compared to control cultures containing psoralen-free Y2O3 nanoscintillators. </p><p>The development and demonstration of a small molecule-sensitive SERS-active fiber-optic nanoprobe suitable for intracellular bioanalysis was demonstrated using pH measurements in single living human cells. The proof-of-concept for the SERS-based fiber-optic nanoprobes was illustrated by measurements of intracellular pH in MCF-7 human breast cancer, HMEC-15/hTERT immortalized normal human mammary epithelial, and PC-3 human prostate cancer cells. Clinical relevance was demonstrated by pH measurements in patient biopsy cell samples. The results indicated that that fiber-optic nanoprobe insertion and interrogation provide a sensitive and selective means to monitor biologically relevant small molecules at the single cell level.</p> / Dissertation
| Identifer | oai:union.ndltd.org:DUKE/oai:dukespace.lib.duke.edu:10161/3110 |
| Date | January 2010 |
| Creators | Gregas, Molly K. |
| Contributors | Vo-Dinh, Tuan |
| Source Sets | Duke University |
| Detected Language | English |
| Type | Dissertation |
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