8^th World Medical Nanotechnology Congress & Expo June 08 - 09, 2016 Dallas, Texas, USA

Miranda N Hurst received her Bachelor of Science in Cell and Molecular Biology from Missouri State University in 2015. She is currently working as a Research Assistant under Dr. Robert Delong at the Nanotechnology Innovation Center of Kansas State (NICKS). Her current research focuses on the stabilization and delivery of RNA via nanoparticles into human and mouse cell lines. Her research interests include the relationship between structure and function of RNA and protein in the complex cellular environment, specifically for the development of cancer therapies. She has 2 publications in peer reviewed journals and 1 patent submitted.

Two dimensional fluorescence difference spectroscopy (2D FDS) is an innovative technique to detect nanoparticles and validate interaction upon surface functionalization and biomolecule loading. 2D FDS detects emission while scanning multiple excitation wavelengths, generating a contour plot with fluorescent intensity reflected as color per an excitation and emission intersect, also called a spectral signature. Initially metal oxide nanoparticles of various compositions were compared, where 2D FDS revealed a unique spectral signature per material composition. To gauge detection of surface functionalization, zinc oxide nanoparticles (ZnO NP) were loaded with one of three polymers: glycol chitosan, polyacrylic acid (PAA), and methoxy polyethylene glycol (mPEG). 2D FDS revealed a shift in the spectral signature in the presence of the polymer. ZnO NP was loaded with one of three RNAs: Torula Yeast RNA (TYRNA), polyinosinic: polycytidylic acid (pIC), and splice switching oligonucleotide (SSO), to determine the efficacy of 2D FDS in identifying biomolecular interactions. The greatest spectral shift was observed for pIC, followed by SSO and TYRNA. Extending this technique from nanoparticles to proteins, Ras binding domain (RBD) is a derivative of B-Raf protein capable of binding Ras and thus a potential cancer therapeutic. RBD was shown to give a spectral signature, where a shift in the optimal excitation and emission intersect elucidates protein conformation. In the presence of ZnO NP and iron oxide nanoparticle (Fe2O3 NP), a shift was observed for both particles. Altogether, these data support 2D FDS as a novel technique in identifying nanoparticles and their surface interactions.

Ashley Gasiorowski is currently pursuing her Bachelor of Science in Biology at Kansas State University. She is enrolled in the Veterinary Scholars Early Admission Program and Honors Program. She has previously worked as a Laboratory Assistant in the Department of Plant Pathology, as well as at Kansas State’s Olathe campus. She is continuing her passion for research as a student researcher under Dr. Rob DeLong in the Department of Anatomy and Physiology. Her research interests include veterinary science and medicine with hopes of becoming a veterinarian.

Luciferase (Luc) and β-Galactosidase (β-gal) serve as model enzymes where we study the effects of physiologically relevant nanoparticles on the structure and function of proteins. The objective is to examine the extent to which each nanoparticle will enhance or deter the performance of β-gal and Luc. Two dimensional fluorescence difference spectroscopy (2D FDS) determined the optimal intensity per excitation and emission wavelengths to view a change in the intrinsic fluorescence of both enzymes in the presence of nanoparticles, indicative of protein conformation. The intrinsic fluorescence spectra were obtained for both enzymes in the presence of copper (Cu) and boron carbide (B4C) nanoparticles revealing fluorescence quenching as result of interaction. Luc activity was measured by photoluminescence through the conversion D-Luciferin substrate to oxyluciferin, where Cu alters and B4C eliminates this catalytic reaction. Limited proteolytic digestion experiments were conducted on Luc where Cu increased degradation and B4C protected against it. Luc allows for rapid kinetic analysis where Cu showed delayed onset of the reaction and iron oxide was less inhibitory initially. β-gal activity was measured by absorbance as a result of cleaving the substrate ortho-nitrophenyl- β-galactoside (ONPG). The nanoparticles effect on both enzymes was compared to determine if the effect was specific or a general phenomenon.

Jacqueline Nolan is a graduate student at the University of Arkansas for Medical Sciences in Little Rock, Arkansas. She is currently pursuing her Doctorate degree in Cancer Biology in Dr. Vladimir Zharov’s laboratory.

The detection and enumeration of apoptotic and necrotic cells in response to nanoparticles (NPs) is important to control nanoparticle toxicity and efficiency of anti-tumor therapy. Previous studies recorded in vitro are not able to be effectively translated into in vivo conditions, as invasive extraction of cells from a living system may alter cell properties (e.g., morphology or marker expression), induce artifacts, or prevent the long-term study of cell-NP and cell–drug interactions in their biological environment. Another limitation is the low sensitivity in detecting rare circulating apoptotic tumor cells (CTCs) that are indicators of metastatic progression. Here we show that these limitations can be overcome by the use of in vivo flow cytometry (FC), which allows real-time monitoring of circulating normal blood cells and CTCs in response to NPs and anti-tumor drugs. We introduce high speed, multicolor in vivo FC that integrates photoacoustic (PA) fluorescence FC (PAFFC) to demonstrate a promising preliminary application of this unique technique for detection of rare circulating apoptotic cells in a mouse model. The verification of this approach was performed initially in vitro by the induction of apoptosis in cancer cells using a chemical apoptotic inductor (H2O2) and compared with apoptosis induced by graphene NPs followed by the injection of these cells in the circulatory system of a mouse. PA and fluorescence channels were used to detect cells with graphene and apoptotic cells in micro-vessels of the mouse’s ear. The coincidence of PA and fluorescence signals indicated cells with graphene–induced apoptosis.

Mohan C Pereira is a final year Doctoral candidate at University of Rhode Island. He earned his BS from University of Colombo with Physics major and Pure and Applied Mathematics minors. In 2014, he received S Letcher scholarship for his academic and research achievements. His research interests include nanotechnology for medicine, targeted drug delivery and tumor targeting, pHLIP technology, hyperthermia for treatment, fluorescent imaging, radiation physics and physics and mathematics education.

Extracellular acidity is not only a universal biomarker for carcinoma and several other pathological conditions, but also a significant factor for pathological cell functioning and proliferation. Here, we report how we can exploit this biomarker to design novel, pHsensitive nanomedicine for therapeutics & diagnostics using pHLIP® technology. We formulated pHLIP® coated small, unilamillar vesicles with high stability and prolong shelf life to deliver hydrophobic agents to disease sites in more effective and safer way. For regular cell functioning, the right balance of ions in intracellular and extracellular spaces is vital. Any alterations done to this vital balance of ions could induce the cell death in both healthy and diseased cells. Apart from the extracellular acidity, the reverse pH gradient (intracellular pHi is higher than extracellular pHe) is another signature for cancerous cells. Here we report a stout mechanism to deliver nano-pores to cancer cells to disrupt the monovalent cation balance and induce apoptosis using pHLIP® coated liposomes. In this work, a gramicidin A is used to form monovalent cation conductive nano-pores. Another example is delivery of lipophilic antineoplastic chemotherapeutic drug, paclitaxel (PTX), by pHLIP® coated liposomes. New formulation of PTX in pHLIP® coated liposomes shows high efficiency of PTX encapsulation and high stability. We show that PTX could be delivered to various forms of malignant cancers by pHLIP® coated liposomes. In both applications, pH dependent fusion of the liposomes is facilitated by the pH (Low) Insertion Peptide (pHLIP® peptide) attached to the liposomes.

Walter Harrington is a second year graduate student at the University of Arkansas for Medical Sciences (UAMS) pursuing a PhD in the Interdisiplinary Biomedical Sciences. His current work focuses on developing novel photoswitchable nanoprobes for labeling and tracking of cells in vivo and use this diagnostic platform for study of biodistribution of bacteria (S. aureus) in the body. He works under the supervision of Vladimir Zharov, PhD, Professor, who is Director of Arkansas Nanomedicine Center at UAMS and who pioneered photothermal nanotherapy and in vivo photoacoustic flow cytometry.

The development of photoswitchable fluorescent proteins with controllable light–dark states and spectral shifts in emission in response to light led to breakthroughs in study of cell biology. Nevertheless, conventional photoswitching is not applicable for weakly fluorescent proteins and is limited by low penetration of UV and visible light used into tissue, strong autofluorescent background, and phototoxicity concerns. As an alternative, photoacoustic (PA) imaging has demonstrated a tremendous potential in biomedical study of nonfluorescent cells and proteins. However, little progress has been made in the development of photoswitchable PA contast agents. Here, we introduce a novel concept of photoswitchable PA probes consisting of capsulated thermochromic dyes and absorbing nanoparticles, which can control light–dark states and spectral shifts in absorption in response to laser light. In these hybrid probes, temperature-sensitive dye absorption is photoswitched through laser heating of doped nanoparticles. The proof-ofconcept was demonstrated using near-infrared laser and two thermochromic dyes with magnetic nanoparticles that are reversibly photoswitched either from a colored light state to colourless dark state or from one colour to another. PA imaging provided visualization of these probes in solution and cells. We also propose photoswitching of plasmonic resonances in gold nanoparticle clusters through interparticle distance changes. A potential application of new nonfluorescent photoswitchable multicolour probes are discussed with focus on cellular diagnostics, tracking circulating tumor cells, viruses and bacteria, and magnetic and NIR photothermal therapy of cancer and infections.

Kai A Carey is currently pursuing PhD at the University of Arkansas for Medical Sciences under the supervision of Dr. Vladimir Zharov in the Arkansas Nanomedicine Center. His work currently focuses on the use of near-infrared lasers for noninvasive early detection and treatment of malarial infection.

Unprecedented nanotechnological advances promise to revolutionize deadly disease diagnosis and therapy by enhancing imaging contrast and improving drug/vaccine delivery. Nevertheless, challenges still remain in treating malaria which kills over half a million people every year. Early disease diagnosis and accurate staging are crucial for good treatment outcomes. We show here that early noninvasive (needleless) label-free diagnosis and, hence well-timed treatment of malaria are feasible by the use of hemozoin nanocrystals as intrinsic high contrast photoacoustic (PA) agents in combination with in vivo PA flow cytometry (PAFC). Hemozoin, with the average size of 50-400 nm are created in infected red blood cells (RBCs) as a result of detoxification of the byproducts from hematophagous parasites, in particular, P. yoelii. We discuss the properties of these not yet well characterized NPs and demonstrate that they can provide very high levels of PA contrast in infected RBCs above hemoglobin background comparable to that of engineered artificial metal nanoparticles (NPs) used for targeting circulating tumor cells and bacteria. Moreover, laser-induced vapor nanobubbles around overheated hemozoin nanocrystals as a PA signal amplifier makes it possible to detect rare infected RBCs even in deep vessels that improve diagnostic speed and sensitivity. PA detection of hemozoin in combination with fluorescent detection of GFP-expressing parasites provide a detailed real-time picture of infection dynamics. Laser disruption of hemozoin containing RBCs may be used for destruction of infected cells. We are confident that natural hemozoin nanoparticles may find multiple applications in health care similar to those of metal engineered nanomaterials.