R-3 Repository :: Browsing by Author "Drezek, Rebekah A."

Browsing by Author "Drezek, Rebekah A."

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Adeno-associated virus capsid as a scaffold for metal binding and nanoparticle synthesis
(2014-04-24) Dempsey, Chris; Suh, Junghae; Drezek, Rebekah A.; Biswal, Sibani Lisa
Viruses, natural biological entities that have developed complex and compact mechanisms to deliver genetic material to target cells through natural evolution, can be repurposed for new nanoscale applications in a broad range of fields, including being used as biologically relevant therapeutics. Rationally designed genetic enhancements, chemical modifications, and hybrid linkages to other nanoscale materials can make viral vectors even more attractive as cargo-carrying compounds in cells. The motley array of amino acids on the surface of a virus capsid, which contains different side chains that have certain charge, hydrophobicity, and polarity properties, can be modified in order to bind inorganic metals and other metal ions for the purpose of synthesizing new compounds. Nanoscale metal and composite nanoparticles may have unique nanoscale properties that have relevance in a biological research setting, such as providing high signal over background contrast in crowded tissue compartments, Individual viruses can function as scaffolds, providing a surface for synthesis of these inorganic nanoparticles in order to combine the advantages of each individual element into a single hybrid compound. In this thesis, I first present my efforts to study a type of inorganic nanoparticle, which has been shown to generate high contrast nonlinear optical signal for biological imaging applications. Specifically, I created a hybrid labeling and delivery system by modifying the inorganic nanoparticles with a specific polymer compound, endowing them with the ability to condense DNA as well as to enter cells to deliver a genetic payload. Next, I detail a method of producing gold nanoparticles with variable morphology and dispersity using an adeno-associated virus as a scaffold for precursor nucleation. Finally, I describe how I generated mutant virus capsids that can bind metal ions after responding to an external stimulus that causes a conformational change in capsid subunits, externalizing metal binding domains. These detailed studies of hybrid molecules show that attractive properties of individual components of these nanomaterials can be combined or leveraged in a controlled manner in order to generate new materials for biologically relevant applications in the future.
Biodistribution of Cadmium Selenide/Zinc Sulfide Quantum Dots in Aquatic Organisms
(2011) Lewinski, Nastassja Aileen; Drezek, Rebekah A.
This thesis investigates the biodistribution and toxicological effects of amphiphilic polymer coated CdSe/ZnS quantum dots (QDs) in two aquatic species, Daphnia magna (daphnia) and Danio rerio (zebrafish). The use of QDs in the life sciences has become common practice over the past decade. In addition QDs are being incorporated in commercially available light emitting diodes and photovoltaic solar cells. As the widespread commercial use of QDs increases, environmental release is inevitable, and water will contain the highest environmental concentrations based on life cycle assessments. Despite increased attention to the aquatic toxicology of nanomaterials in recent years, little information exists on the biological fate of QDs in aquatic organisms. Quantitative data on the uptake and excretion of QDs from daphnia and zebrafish were collected using fluorescence imaging paired with metal analysis. First, daphnia were examined after aqueous and dietary exposure to amphiphilic polymer coated CdSe/ZnS QDs. Surface coating influenced QD acute toxicity and high particle aggregation correlated with daphnia mortality. QDs were readily ingested by daphnia and accumulated in the intestines. High body burdens of 150-200 μg/g were found in the daphnia, with intestinal QD concentrations significantly elevated above the exposure media concentration. The slow elimination observed in daphnia suggested that trophic transfer of QDs to higher organisms may occur. Using daphnia and zebrafish as a model food chain revealed that QDs can transfer to zebrafish through dietary exposure with body burdens of 8-9.5 μg/g found. However, no biomagnification between daphnia and zebrafish was observed and the biomagnification factor (BMF = 0.04) was significantly less than one. This work demonstrates that aqueous and dietary exposures to QDs can result in high total body concentrations in aquatic organisms with little to no gross toxicity. The low acute toxicity observed for some surface coated QDs encourages further design optimization to improve the biocompatibility and reduce the environmental impact of QDs.
Biological applications of novel fluorescent nanoprobes
(2007) Chang, Emmanuel Yih-Herng; Drezek, Rebekah A.
A thorough investigation to evaluate the biological application of quantum dots was performed. Quantum dots are novel semiconductor nanocrystals that are highly tunable in their spectral properties and exhibit strong photoluminescence and high photostability. They have been shown to be a promising optical contrast agent for biological applications, however further biological studies are needed to evaluate and characterize their applicability. In this thesis, we examined the various applications of quantum dots and leverage its optical properties for cancer imaging. First, we evaluated the effects of different nanoparticle surface coatings on the cellular uptake of quantum dots to understand quantum dot delivery into cells. Building on our knowledge of surface coating influences, we then evaluated the cytotoxicity of quantum dots, reporting new insight on the intracellular evaluation of quantum dot cytotoxicity. Next, we demonstrated the specificity of bioconjugated quantum dots in molecular targeting and imaging of cancer cell markers. Finally, we engineered a novel nanoparticle construct, an activatable quantum probe that activates in the presence of proteolytic activity as a potential method for early cancer detection based on increased metalloproteinase activity in the stroma of cancer tissue. Furthermore, we expanded our focus on developing 'smart' functional nanoprobes by developing a novel siRNA-based molecular beacon useful for gene target validation and silencing as a promising tool for dual imaging and therapy in cancer.
Cardiac MRI: Improved Assessment of Left Ventricular Function, Wall Motion, and Viability
(2013-09-16) Krishnamurthy, Ramkumar; Drezek, Rebekah A.; Muthupillai, Raja; Jacot, Jeffrey G.; Clark, John W., Jr.
Heart failure is the clinical syndrome accompanying the inability of the heart to maintain a cardiac output required to meet the metabolic requirements and accommodate venous return, and is one of the leading causes of mortality in United States. Accurate imaging of the heart and its failure is important for successful patient management and treatment. Multiple cardiac imaging modalities provide complementary information about the heart – LV function and wall motion, anatomy, myocardial viability and ischemia. In many instances, it is necessary for a patient to undergo multiple imaging sessions to obtain diagnostic clinical information with confidence. It would be beneficial to the individual and the health care system if a single imaging modality could yield reliable clinical information about the heart, leading to a reduced cost, anxiety and an increased diagnostic confidence. This thesis proposes methods that would make cardiac MRI perform an improved assessment of LV function, wall motion, and viability, such that cardiac MRI is taken one step closer to being a single stop solution for imaging of heart. Conventional cardiac MR imaging is performed at a temporal resolution of around 40 ms per cardiac phase. While the global left ventricular (LV) function can be reliably established at this temporal resolution, functional metrics characterizing transient function like peak filling and ejection rates are not accurately assessed. A high temporal resolution is necessary to characterize such transient LV function and wall motion mechanics. This thesis proposes methods to acquire cine-images of the heart at a higher temporal resolution (~ 6 ms) and algorithms to acquire the LV volume across all cardiac phases that would yield functional metrics characterizing LV function and wall motion mechanics. The validation of these algorithms was performed on human subjects. Cardiac MR imaging is the current gold standard of myocardial viability imaging, in which scarred regions of the heart following myocardial infarction are visualized. However viability imaging faces image quality challenges in patients with severe arrhythmias and in cases where a higher spatial resolution, and hence a longer acquisition time, is desired. This thesis also proposes an arrhythmia insensitive inversion recovery (AIIR) algorithm that would significantly reduce artifacts that degrade image quality, thereby extending viability imaging to higher spatial resolution and in patients with severe arrhythmia. Simulations, experimental validation on phantoms and clinical verification on patients are performed. Results from high temporal resolution imaging reveal that obtaining cine cardiac MR images at a temporal resolution of 6 ms per cardiac phase is feasible. Appropriate validated algorithms yield LV time-volume curve from which LV functional metrics are reliably extracted. A dependence on temporal resolution is revealed, and a temporal resolution cut-off of 12 ms is proposed to reliably capture the temporal dynamics of the LV. Also, results from cardiac viability imaging show that the AIIR algorithm performs significantly better than conventional imaging methods in both phantoms and human subjects, as shown by the blinded expert scores, leading to a better image quality. In conclusion, this thesis proposes and implements methods that help cardiac MRI yield 1) a better function and wall motion assessment of the heart through high temporal resolution imaging and 2) a better assessment of myocardial viability through the AIIR algorithm.
Changes in Optical Properties of Plasmonic Nanoparticles in Cellular Environments are Modulated by Nanoparticle PEGylation and Serum Conditions
(Springer, 2016) Chen, Allen L.; Jackson, Meredith A.; Lin, Adam Y.; Figueroa, Elizabeth R.; Hu, Ying S.; Evans, Emily R.; Asthana, Vishwaratn; Young, Joseph K.; Drezek, Rebekah A.; Bioengineering; Electrical and Computer Engineering
When plasmonic nanoparticles (NPs) are internalized by cells and agglomerate within intracellular vesicles, their optical spectra can shift and broaden as a result of plasmonic coupling of NPs in close proximity to one another. For such optical changes to be accounted for in the design of plasmonic NPs for light-based biomedical applications, quantitative design relationships between designable factors and spectral shifts need to be established. Here we begin building such a framework by investigating how functionalization of gold NPs (AuNPs) with biocompatible poly(ethylene) glycol (PEG), and the serum conditions in which the NPs are introduced to cells impact the optical changes exhibited by NPs in a cellular context. Utilizing darkfield hyperspectral imaging, we find that PEGylation decreases the spectral shifting and spectral broadening experienced by 100 nm AuNPs following uptake by Sk-Br-3 cells, but up to a 33 ± 12 nm shift in the spectral peak wavelength can still occur. The serum protein-containing biological medium also modulates the spectral changes experienced by cell-exposed NPs through the formation of a protein corona on the surface of NPs that mediates NP interactions with cells: PEGylated AuNPs exposed to cells in serum-free conditions experience greater spectral shifts than in serum-containing environments. Moreover, increased concentrations of serum (10, 25, or 50 %) result in the formation of smaller intracellular NP clusters and correspondingly reduced spectral shifts after 5 and 10 h NP-cell exposure. However, after 24 h, NP cluster size and spectral shifts are comparable and become independent of serum concentration. By elucidating the impact of PEGylation and serum concentration on the spectral changes experienced by plasmonic NPs in cells, this study provides a foundation for the optical engineering of plasmonic NPs for use in biomedical environments.
Complexed Multifunctional Metallic and Chalcogenide Nanostructures as Theranostic Agents
(2013-12-03) Young, Joseph; Drezek, Rebekah A.; Hicks, Illya V.; Kono, Junichiro
Nanostructures have attracted substantial attention due to their distinctive properties and various applications. Nanostructures consisting of multiple morphologies and/or materials have recently become the focus of intense study with particular attention being paid to their optical and magnetic properties and the enhanced role of the interface between materials. Of particular interest are metallic-based plasmonic nanostructures, structures that support surface plasmon resonances that are sensitive to the environment, and ferrimagnetic-based nanostructures, structures that exhibit strong magnetic properties when exposed to an external field. These nanostructures provide theranostic potential in the context of cancer photothermal therapies, diagnostics and imaging. Additionally, chalcogenide based nanostructure complexes are particularly interesting. Metallic chalcogenides offer the ability to combine different types of linear and nonlinear optical properties, enable design of nanostructure complexes with surface plasmon resonance effects in new wavelength ranges, and act as photo-emitting agents for novel theranostic applications. In this thesis an in depth analysis of plasmonic, magnetic and photo-emitting nanostructures as theranostic agents is presented. We have created several multifunctional nanostructures and the factors contributing to the functional properties of these nanostructures are explored systematically through experimentation, theory, and simulations. Both in vivo and in vitro testing demonstrates the applicability of these nanostructures as theranostic agents.
Development of a Novel Class of Self-Assembling dsRNA Cancer Therapeutics: A Proof-of-Concept Investigation
(Cell Press, 2020) Asthana, Vishwaratn; Stern, Brett S.; Tang, Yuqi; Bugga, Pallavi; Li, Ang; Ferguson, Adam; Asthana, Anantratn; Bao, Gang; Drezek, Rebekah A.; Bioengineering
Cancer has proven to be an extremely difficult challenge to treat. Several fundamental issues currently underlie cancer treatment, including differentiating self from nonself, functional coupling of the recognition and therapeutic components of various therapies, and the propensity of cancerous cells to develop resistance to common treatment modalities via evolutionary pressure. Given these limitations, there is an increasing need to develop an all-encompassing therapeutic that can uniquely target malignant cells, decouple recognition from treatment, and overcome evolutionarily driven cancer resistance. We describe herein a new class of programmable self-assembling double-stranded RNA (dsRNA)-based cancer therapeutics that uniquely targets aberrant genetic sequences and in a functionally decoupled manner, undergoes oncogenic RNA-activated displacement (ORAD), initiating a therapeutic cascade that induces apoptosis and immune activation. As a proof of concept, we show that RNA strands targeting the EWS/Fli1 fusion gene in Ewing sarcoma cells that are end blocked with phosphorothioate bonds and additionally sealed with a 2′-deoxyuridine (2′-U)-modified DNA protector can be used to induce specific and potent killing of cells containing the target oncogenic sequence but not wild type.
Development of Osteoinductive Tissue Engineering Scaffolds with a Bioreactor
(2013-07-24) Thibault, Richard; Mikos, Antonios G.; Grande-Allen, K. Jane; Zygourakis, Kyriacos; Drezek, Rebekah A.; Kasper, Kurt; Jansen, John
The conventional treatments for craniofacial bone defects currently are unsatisfactory due to several drawbacks. Replacement of lost bone by autografts typically causes donor site morbidity while allografts, xenografts, and demineralized bone matrix all have a chance of disease transmission. Current synthetic implants placed within the defect site generally lack osseointegration and biodegradability. There are several methods of generating a hybrid extracellular matrix (ECM) and synthetic material construct. These include coating the synthetic material scaffold with collagen and calcium phosphate, incorporating acellular biological tissue within the scaffold material, and using cells to generate an ECM coating on the synthetic material scaffold. The research performed for this thesis developed and characterized mesenchymal stem cell (MSC)-generated extracellular matrix poly(ε-caprolactone) constructs (PCL/ECM) for the replacement of bone tissue. The osteogenic potential of the PCL/ECM constructs was explored by culturing i) MSCs and ii) whole marrow cells combined with MSCs onto the construct with or without the osteogenic differentiation supplement, dexamethasone. It was established that the osteogenic differentiation of MSCs seeded onto ECM-containing constructs was maintained even in the absence of dexamethasone and that the co-culture of MSCs and whole bone marrow cells without dexamethasone and ECM enhances the proliferation of a cell population (or populations) present in the whole bone marrow. The osteogenicity of the constructs encouraged the characterization of the protein and mineral composition of the ECM coating on the PCL/ECM constructs. Characterization revealed that at short culture durations the MSCs used to generate the ECM deposited cellular adhesion proteins that are a prerequisite protein network for further bone formation. At the later culture durations, it was determined that the ECM was composed of collagen 1, hydroxyapatite, matrix remodeling proteins, and regulatory proteins. The prior studies on the PCL/ECM constructs persuaded exploration of the effect of various devitalization and demineralization processes on the retention of the ECM components within and the osteogenicity of the PCL/ECM constructs. Analysis demonstrated that the freeze-thaw technique is a milder method of devitalization of cell-generated ECM constructs as compared to other methods, but it reduced the osteogenicity of the constructs. In addition, it was elucidated that void spaces in the surface of the constructs are important for allowing access of MSCs into the interior of the constructs.
Elimination of Metastatic Melanoma Using Gold Nanoshell-Enabled Photothermal Therapy and Adoptive T Cell Transfer
(Public Library of Science, 2013) Bear, Adham S.; Kennedy, Laura C.; Young, Joseph K.; Perna, Serena K.; Almeida, Joao Paulo Mattos; Lin, Adam Y.; Eckels, Phillip C.; Drezek, Rebekah A.; Foster, Aaron E.; Bioengineering; Electrical and Computer Engineering
Ablative treatments such as photothermal therapy (PTT) are attractive anticancer strategies because they debulk accessible tumor sites while simultaneously priming antitumor immune responses. However, the immune response following thermal ablation is often insufficient to treat metastatic disease. Here we demonstrate that PTT induces the expression of proinflammatory cytokines and chemokines and promotes the maturation of dendritic cells within tumor-draining lymph nodes, thereby priming antitumor T cell responses. Unexpectedly, however, these immunomodulatory effects were not beneficial to overall antitumor immunity. We found that PTT promoted the infiltration of secondary tumor sites by CD11b+Ly-6G/C+ myeloid-derived suppressor cells, consequently failing to slow the growth of poorly immunogenic B16-F10 tumors and enhancing the growth of distant lung metastases. To exploit the beneficial effects of PTT activity against local tumors and on antitumor immunity whilst avoiding the adverse consequences, we adoptively transferred gp100-specific pmel T cells following PTT. The combination of local control by PTT and systemic antitumor immune reactivity provided by adoptively transferred T cells prevented primary tumor recurrence post-ablation, inhibited tumor growth at distant sites, and abrogated the outgrowth of lung metastases. Hence, the combination of PTT and systemic immunotherapy prevented the adverse effects of PTT on metastatic tumor growth and optimized overall tumor control.
Evaluating the Effects of Cell Sample Preparation on FTIR Cancer Detection
(2013-09-16) Noelck, Sterling; Drezek, Rebekah A.; Kelly, Kevin F.; Kono, Junichiro
This thesis examines some of the challenges involved with using FTIR spectroscopy for cancer detection including sample preparation and correcting for distortion from cell scattering. Sample preparation affects the spectra differently depending on the cell type, and can lead to significant changes in cancer biomarkers for a given cell type. Biomarkers derived from specific cancer types under one sample preparation are not reliable for other cancer types, and may not be suitable for the same cancer type using a different sample preparation. Cell scattering can also significantly affect the cell spectra, and as a result, correcting for the cell scattering distortion leads to changes in the biomarkers. For reliable cancer detection controlling variability is critical, especially in the complex spectra of biological samples. Standard sample preparation methods and scattering correction post-processing could improve comparison of cancer detection methods.
Gold Nanoconstructs for Multimodal Diagnostic Imaging and Photothermal Cancer Therapy
(2013-09-16) Coughlin, Andrew; West, Jennifer L.; Drezek, Rebekah A.; Hafner, Jason H.
Cancer accounts for nearly 1 out of every 4 deaths in the United States, and because conventional treatments are limited by morbidity and off-target toxicities, improvements in cancer management are needed. This thesis further develops nanoparticle-assisted photothermal therapy (NAPT) as a viable treatment option for cancer patients. NAPT enables localized ablation of disease because heat generation only occurs where tissue permissive near-infrared (NIR) light and absorbing nanoparticles are combined, leaving surrounding normal tissue unharmed. Two principle approaches were investigated to improve the specificity of this technique: multimodal imaging and molecular targeting. Multimodal imaging affords the ability to guide NIR laser application for site-specific NAPT and more holistic characterization of disease by combining the advantages of several diagnostic technologies. Towards the goal of image-guided NAPT, gadolinium-conjugated gold-silica nanoshells were engineered and demonstrated to enhance imaging contrast across a range of diagnostic modes, including T1-weighted magnetic resonance imaging, X-Ray, optical coherence tomography, reflective confocal microscopy, and two-photon luminescence in vitro as well as within an animal tumor model. Additionally, the nanoparticle conjugates were shown to effectively convert NIR light to heat for applications in photothermal therapy. Therefore, the broad utility of gadolinium-nanoshells for anatomic localization of tissue lesions, molecular characterization of malignancy, and mediators of ablation was established. Molecular targeting strategies may also improve NAPT by promoting nanoparticle uptake and retention within tumors and enhancing specificity when malignant and normal tissue interdigitate. Here, ephrinA1 protein ligands were conjugated to nanoshell surfaces for particle homing to overexpressed EphA2 receptors on prostate cancer cells. In vitro, successful targeting and subsequent photothermal ablation of prostate cancer cells was achieved with negligible nanoshell binding to normal cells. In vivo however, ephrinA1-nanoshells did not promote enhanced therapeutic outcomes in mice bearing subcutaneous prostate cancer tumors treated with NAPT compared to nontargeted particles. Nonetheless, both treatment groups demonstrated effective ablation of prostate tumors, as evidenced by tumor tissue regression. Further investigation is warranted to overcome probable protein immunogenicity that offsets ephrinA1 targeting in vivo. With future study, photothermal therapy with multimodal gadolinium-conjugated and molecularly targeted nanoshells may offer a viable treatment option for cancer patients in the clinic.
Gold nanoshells for optical coherence tomography
(2006) Lee, Min-Ho; Drezek, Rebekah A.
Near infrared tuned gold nanoshells have been developed to enhance the contrast of optical coherence tomography (OCT) images, and we have completed a systematic study which quantifies and optimizes the specifications of nanoshells that provide improved efficacy of OCT imaging and photothermal ablation of cancer. The optical properties of gold nanoshells, such as scattering, absorption, and asymmetry values were calculated with Mie scattering theory. For comparison and experimental quantifications, scattering coefficients were extracted from OCT images using Extended Huygens-Fresnel (EHF) principle based algorithms. With the addition of Her2 conjugated nanoshells, ex vivo OCT images of human breast cancer tissue, which express signatures of Her2/neu, provide significant contrast in comparison to the normal and malignant controls. As an extended study of dual NIR absorbing/scattering nanoshells for integrated cancer imaging and therapy in vitro, combined OCT imaging and photothermal tumor ablation was performed in vivo. Results showed that gold nanoshells selectively accumulated in the tumorous regions and enabled clear differentiation of tumor. Tumor regression by the photothermal ablation using NIR tuned nanoshells was also reported. Our studies have demonstrated that nanoshells can be designed specifically for diagnostic and therapeutic purposes.
Gold nanoshells: Contrast agents for molecular imaging
(2006) Loo, Christopher Han-Yan; Drezek, Rebekah A.
Cancer remains a significant health concern today. It is the 2 nd leading cause of death in the United States. Critical to controlling cancer-associated morbidity and mortality is early detection. Early detection strategies include detecting molecular-level changes prior to phenotypic changes, enabling a sufficient amount of time for effective therapies to be implemented. Not only is early detection critical, but issues such as patient safety and cost should be considered when implementing these strategies. This thesis examines work using nanoshell-based optical contrast agents for early cancer detection using scattering-based optical imaging systems. Metal nanoshells are a novel class of optically-tunable nanoscale material that are composed of a dielectric core (usually silica) surrounded by a metallic shell (usually gold). By systematically varying the ratio between core diameter and shell thickness, the absorption and scattering maxima can be tuned to different wavelengths including those in the visible and near infrared (NIR). Specific Aim 1 addresses the fabrication of NIR scattering nanoshells for use as optical contrast agents to enable scatter-based cellular imaging. Specific Aim 2 focuses on using dual NIR absorbing/scattering nanoshells for a nanoshell-based integrated cancer imaging and therapy application. Finally, Specific Aim 3 addresses the diagnostic capabilities of gold nanoshells ex vivo using reflectance confocal microscopy (RCM).
An inexpensive, customizable microscopy system for the automated quantification and characterization of multiple adherent cell types
(PeerJ, 2018) Asthana, Vishwaratn; Tang, Yuqi; Ferguson, Adam; Bugga, Pallavi; Asthana, Anantratn; Evans, Emily R.; Chen, Allen L.; Stern, Brett S.; Drezek, Rebekah A.; Bioengineering
Cell quantification assays are essential components of most biological and clinical labs. However, many currently available quantification assays, including flow cytometry and commercial cell counting systems, suffer from unique drawbacks that limit their overall efficacy. In order to address the shortcomings of traditional quantification assays, we have designed a robust, low-cost, automated microscopy-based cytometer that quantifies individual cells in a multiwell plate using tools readily available in most labs. Plating and subsequent quantification of various dilution series using the automated microscopy-based cytometer demonstrates the single-cell sensitivity, near-perfect R2 accuracy, and greater than 5-log dynamic range of our system. Further, the microscopy-based cytometer is capable of obtaining absolute counts of multiple cell types in one well as part of a co-culture setup. To demonstrate this ability, we recreated an experiment that assesses the tumoricidal properties of primed macrophages on co-cultured tumor cells as a proof-of-principle test. The results of the experiment reveal that primed macrophages display enhanced cytotoxicity toward tumor cells while simultaneously losing the ability to proliferate, an example of a dynamic interplay between two cell populations that our microscopy-based cytometer is successfully able to elucidate.
Inverse model for the extraction of biological parameters from ovarian tissue fluorescence spectra and multivariate classifiers for tissue diagnosis
(2007) Appiah, Benjamin; Drezek, Rebekah A.
We present an inverse model to decompose bulk fluorescence spectra and extract tissue biological parameters. By deconvolving the effects of absorption and scattering from measured spectra, we are able to extract the intrinsic contributions from cellular and stromal fluorophores. Ovarian fluorescence, acquired ex-vivo immediately upon removal from patients, are analyzed using this model. We test the validity of the inverse model, and show that it has the ability to improve our understanding of the biological changes that cause the observed differences in the fluorescence spectra. The outputs of the model are used to develop classifiers for tissue diagnosis. Classifiers based on PLS selected features are also developed. An accuracy of more than 93% for discrimination between normal and cancerous ovarian tissues is achieved.
A mechanistic investigation exploring the differential transfection efficiencies between the easy-to-transfect SK-BR3 and difficult-to-transfect CT26 cell lines
(BioMed Central, 2017) Figueroa, Elizabeth; Bugga, Pallavi; Asthana, Vishwaratn; Chen, Allen L.; Stephen Yan, J.; Evans, Emily R.; Drezek, Rebekah A.; Bioengineering
Abstract Background Gold–polyamidoamine (AuPAMAM) has previously been shown to successfully transfect cells with high efficiency. However, we have observed that certain cell types are more amenable to Au–PAMAM transfection than others. Here we utilized two representative cell lines—a “difficult to transfect” CT26 cell line and an “easy to transfect” SK-BR3 cell line—and attempted to determine the underlying mechanism for differential transfection in both cell types. Using a commonly established poly-cationic polymer similar to PAMAM (polyethyleneimine, or PEI), we additionally sought to quantify the relative transfection efficiencies of each vector in CT26 and SK-BR3 cells, in the hopes of elucidating any mechanistic differences that may exist between the two transfection vectors. Results A comparative time course analysis of green fluorescent protein reporter-gene expression and DNA uptake was conducted to quantitatively compare PEI- and AuPAMAM-mediated transfection in CT26 and SK-BR3, while flow cytometry and confocal microscopy were used to determine the contribution of cellular uptake, endosomal escape, and cytoplasmic transport to the overall gene delivery process. Results from the time course analysis and flow cytometry studies revealed that initial complex uptake and cytoplasmic trafficking to the nucleus are likely the two main factors limiting CT26 transfectability. Conclusions The cell type-dependent uptake and intracellular transport mechanisms impacting gene therapy remain largely unexplored and present a major hurdle in the application-specific design and efficiency of gene delivery vectors. This systematic investigation offers insights into the intracellular mechanistic processes that may account for cell-to-cell differences, as well as vector-to-vector differences, in gene transfectability.
Mie and Finite-Element Simulations of the Optical and Plasmonic Properties of Micro- and Nanostructures
(2012) Hu, Ying Samuel; Drezek, Rebekah A.
A Mie-based code is developed for multilayer concentric spheres. The code is used in conjunction with a finite-element package to investigate the plasmonic and optical properties of micro- and nanostructures. For plasmonic nanostructures, gold-silica-gold multilayer nanoshells are computationally investigated. A plasmon hybridization theory is used to interpret the optical tunability. The interaction between the plasmon modes on the inner core and the outer shell results in dual resonances. The low-energy dipole mode is red-shifted by reducing the spacing ( i.e. , the intermediate silica layer) between the core and the shell. This extra tunability allows the plasmon resonance of a multilayer nanoshell to be tuned to the near-infrared region from a visible silica-gold nanoshell whose gold shell cannot be further reduced in thickness. For multilayer nanoshells with reduced geometrical symmetry ( i.e. , the inner core is offset from the center), modes of different orders interact. The mixed interaction introduces the dipolar (bright) characteristic into the higher-order (dark) modes and improves their coupling efficiency to the excitation light. The excitation of the dark modes attenuates and red-shifts the dipole mode and gives it higher-order characteristics. For non-plasmonic structures, simulations have demonstrated that multilayered structures can either reduce or enhance the scattering of light. By adding an anti-reflection layer to as microsphere made of a high-index material, the scattering force can be dramatically reduced. The reduced scattering allows optical trapping of high-index particles. Additionally, the improved trapping is not largely sensitive to the refractive index or the thickness of the coating. The technique has the practical potential to lower the requirement on the numerical aperture of the microscope objectives, making possible the integration of the imaging and optical trapping systems. While the anti-reflection coating reduces scattering, the photothermal bubble (PTB) generated by gold nanoparticles by and large enhances the scattering of light. Transient PTBs are generated by super-heating gold nanoparticles with short laser pulses. Mie-based simulations predict that the scattering of PTBs strongly depends on the transient environment immediately surrounding the nanoparticles. A scattering enhancement of two-to-four orders of magnitude from PBT is demonstrated from both calculations and experiments. Lastly, the near-field coupling between different plasmonie structures for surface-enhanced Raman scattering is investigated. A gold-coated silicon-germanium nanocone substrate has been fabricated and characterized. Finite-element simulations reveal that individual nanocones generate strong tip enhancement with axially polarized light ( i.e. , light polarized along the vertical axis of the nanocone) while the enhancement from transversely polarized light ( i.e. , light polarized in the plane of the substrate) is relatively weak. By simply filling the valleys between nanocones with plasmonic gold nanoparticles, the performance of the substrate is improved with in-plane excitation. Simulations reveal strong coupling between nanoparticles and adjacent nanocones with transverse exactions. An over one order-of-magnitude improvement has been experimentally observed.
Modulating Gold Nanoparticle in vivo Delivery for Photothermal Therapy Applications Using a T Cell Delivery System
(2012) Kennedy, Laura Carpin; Drezek, Rebekah A.
This thesis reports new gold nanoparticle-based methods to treat chemotherapy-resistant and metastatic tumors that frequently evade conventional cancer therapies. Gold nanoparticles represent an innovative generation of diagnostic and treatment agents due to the ease with which they can be tuned to scatter or absorb a chosen wavelength of light. One area of intensive investigation in recent years is gold nanoparticle photothermal therapy (PTT), in which gold nanoparticles are used to heat and destroy cancer. This work demonstrates the utility of gold nanoparticle PTT against two categories of cancer that are currently a clinical challenge: trastuzumab-resistant breast cancer and metastatic cancer. In addition, this thesis presents a new method of gold nanoparticle delivery using T cells that increases gold nanoparticle tumor accumulation efficiency, a current challenge in the field of PTT. I ablated trastuzumab-resistant breast cancer in vitro for the first time using anti-HER2 labeled silica-gold nanoshells, demonstrating the potential utility of PTT against chemotherapy-resistant cancers. I next established for the first time the use of T cells as gold nanoparticle vehicles in vivo. When incubated with gold nanoparticles in culture, T cells can internalize up to 15000 nanoparticles per cell with no detrimental effects to T cell viability or function (e.g. migration and cytokine secretion). These AuNP-T cells can be systemically administered to tumor-bearing mice and deliver gold nanoparticles four times more efficiently than by injecting free nanoparticles. In addition, the biodistribution of AuNP-T cells correlates with the normal biodistribution of T cell carrier, suggesting the gold nanoparticle biodistribution can be modulated through the choice of nanoparticle vehicle. Finally, I apply gold nanoparticle PTT as an adjuvant treatment for T cell adoptive transfer immunotherapy (Hyperthermia-Enhanced Immunotherapy or HIT) of distant tumors in a melanoma mouse model. The results presented in this thesis expand the potential of gold nanoparticle PTT from only chemotherapy-sensitive or localized cancers to chemotherapy-resistant non-localized cancers that currently defy conventional therapies.
Multimodal Optical Imaging for Detection of Cervical Neoplasia
(2013-09-16) Bubi, Tefo; Richards-Kortum, Rebecca Rae; Drezek, Rebekah A.; Baraniuk, Richard G.
Despite being the most preventable cancer, cervical cancer remains the third leading cause of cancer death worldwide. Over 85% of cervical cancer incidence and mortality occurs in low-resource countries where screening programs for early detection are either inadequate or unavailable. In the developed world, where screening programs are well organized, incidence and mortality rates are greatly reduced. Recent advances in optical imaging have the potential to enable cervical cancer screening at the point-of-care, even in the hands of less experienced providers. High performance optical imaging systems can be constructed at relatively low cost, and image analysis can be automated; thus, these technologies may provide a way to bridge the gap to cervical cancer screening for developing countries. This work focuses on the design, construction, and clinical testing of a novel multimodal optical imaging (combination of wide-field imaging and high-resolution) for early detection of cervical neoplasia. The Multimodal Digital Imager (MDI) acquires in vivo images of cervical tissue in fluorescence, narrow band reflectance, and orthogonal polarized reflectance modes using multiple illumination wavelengths. The High Resolution Microendoscope (HRME) was used to interrogate clinically suspicious areas with subcellular spatial resolution, revealing changes in nuclear to cytoplasmic area ratio. In vivo image data from the wide-field system was combined with image data from a high- resolution microendoscope (HRME) in order to test the effectiveness of the multimodal optical imaging in discriminating between cervical neoplasia and non-neoplastic. Multimodal optical imaging coupled with computer aided diagnostic achieved a sensitivity of 82% and specificity of 85% for discriminating cervical neoplastic from non-neoplastic This work has demonstrated that multimodal optical imaging; combination of wide-field and high-resolution optical imaging of the cervix can assist in the detection of cervical neoplasia and can be implemented effectively in a low-resource setting.
Nanoengineered contrast agents for biophotonics: Modeling and experimental measurements of gold nanoshell reflectance
(2006) Lin, Alex Wei Haw; Drezek, Rebekah A.
The growing interest in using exogenous agents to enhance the subtle differences between optical signatures of normal and cancerous tissue, has spurred the development of novel nanoparticulate agents that exhibit desirable optical responses and at the same time, used to target biomolecular markers of diseases. Gold nanoshells are a class of core-shell nanoparticle, exhibiting an extremely agile peak optical resonance, ranging from the near-ultraviolet to the mid-infrared wavelengths. Although optical properties of gold nanoshells in transmission have already been well documented, the reflectance characteristics have not been elucidated. Yet, in order to use gold nanoshells as a contrast agent for scattering-based optical diagnostic tools, it is critical to study the reflectance behavior. Using a combination of experimental observations and Monte Carlo models, we investigated gold nanoshell reflectance characteristics and its effect on tissue phantoms. Gold nanoshells were shown to significantly alter reflectance signatures of tissue phantoms, both experimentally and in computer models. Monte Carlo simulations of gold nanoshell reflectance demonstrated the efficacy of using such methods to model diffuse reflectance and also reaffirm the experimental observations. Our studies suggest that gold nanoshells are an excellent candidate as an optical contrast agent and Monte Carlo methods can be a useful tool for optimizing nanoshells best suited for scattering-based optical methods to enhance the detection and imaging of cancers.

Browsing by Author "Drezek, Rebekah A."

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