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A major problem which stymies the use of some photochemical techniques for biomedical use is the requisite ultraviolet (UV) radiation to initiate chemical reactions. UV covers the range of the electromagnetic spectrum from 10 to 400 nm. The majority of UV initiated photochemistry occurs within the range of 280 to 380 nm which constitutes the UVA and UVB. However, this range of UV is affiliated with carcinomas, DNA damage, immune modulation and mitochondrial damage.[1–3] Upconversion nanoparticles (UNPs) have the ability absorb multiple near-infrared (NIR) photons to produce emission with a higher energy than any of the individual absorbed photons ranging from higher energy NIR to UVB emissions. By utilizing NIR excitation, one can drive photochemical reactions in biological environments at the site of the UNPs rather than direct UV radiation to the culture or tissue. The biological window includes the NIR between 700 and 1350 nm which fortunately includes the range of excitation wavelengths in UNPs occur. This presents a serendipitous opportunity to develop systems for NIR triggered photochemical reactions without the need for a continuous dose of UV irradiation. The major goal of the research presented in this dissertation was to synthesize functional upconversion nanoparticles for improved UV emission, explore their potential as a nano-tool for photochemistry while maintaining imaging capabilities, and developing a novel UNP that will increase the appeal of UNPs as biomedical technology. The first aim was to perform an exhaustive study of the available preparative mechanisms for controlling the UV emission of UNPs (chapter 2). The second aim was to successfully and reproducibly synthesize UV emitting UNPs, and to functionalize these nanoparticles for use in optical techniques (chapter 3) to understand their potential as both a bioimaging agent and photochemical tool (chapter 4). The third aim was to develop a new paradigm for UNPs which utilizes a kinetics-based a

In this research, a variety of nanosized polymeric derivatives, particularly for applications in drug delivery systems and enzyme nanocarriers, were synthesized and characterized. Most of the nanocarriers were formulated with a matrix consisting of poly(DL-lactic acid-co-glycolic acid) (PLGA) or poly(ethylene glycol)-PLGA and stabilized by polyvinyl alcohol (PVA) or Pluronic F-68 for the encapsulation of an insulin derivative, a model therapeutic enzyme, and anticancer drugs. The protein hormone release from biodegradable nanoparticles prepared using double emulsion solvent evaporation was quantified and release phases were identified. Insulin-like growth factor (IGF1)-loaded nanoparticles showed that the released protein was able to stimulate an important pathway for effective cell proliferation and cell survival. Several excipients that helped increase encapsulation efficiency of IGF1 were used, particularly, bovine serum albumin (BSA), poly(propylene glycol), and a triblock polymer combining poly(propylene glycol) and poly(ethylene glycol) — materials that act as adsorption enhancers of the protein in the PLGA nanoparticle. The proteolytic enzyme, trypsin, was encapsulated in PLGA nanoparticles and its behavior during the encapsulation process and release represented the characteristics of most enzymatic systems. Experimental kinetic experiments towards a synthetic substrate studied the activity of trypsin during encapsulation and after its release. The nanoparticle encapsulated enzyme maintained significant enzymatic activity and after its release, a good amount of the activity was evident from the kinetic work. Its proteolytic activity was also unmistakable since the free enzyme released from the nanoparticle lost significant activity within four hours after its release. PEGylation of the protein was also considered in this study and was encapsulated as well. However, the determination of the conjugate enzymatic activity was challenging since the PEG-enzyme com

Vascular malformations and cancer remain the major brain pathologies that often require surgical treatment. Glioblastoma, classified as a grade IV astrocytoma, is a highly malignant cancer of the brain that has a median survival of 1-2 years, with more aggressive forms having median survival less than 14 months. These statistics include standard treatment protocols that include surgery followed with adjunctive chemotherapy and radiation treatment. Vascular malformations encompass multiple, more specific pathologies including arteriovenous malformation, cavernous malformation, mixed malformations, telangiectasis, vein of Galen malformation, and venous malformations. Treatment of either vascular anomalies or cancer can benefit from improved image guided surgery technologies. Surgical image guidance has continually been improving over the decades since the introduction of the microscope. New technologies including endoscopic cameras and non-invasive imaging (e.g., computed tomography and magnetic resonance imaging) have led to paradigm shifts in how disease is diagnosed and treated. However, even with the numerous advancements in imaging technologies, the microscope still remains the most used and required imaging technology in the neurosurgical operating suite. There remains to be a system that can match ergonomics and visualization that the operative microscope provides. The operative microscope retains all aspects of 3-D vision while magnifying and focusing the field of view for the surgeon. Cameras, light sources, and other surgical equipment have been added to the plethora of attachments now available for the operative microscope. This work is organized into three specific aims that collectively present the development of a new technology focused on improving image guided surgery coupled with a novel contrast agent. The first aim will present the development of an augmented microscope that is capable of augmenting the field of view in the oculars with NIR fluoresc

For many types of cancers chemotherapy is the treatment of choice despite evidence that this treatment modality is contributing only about 2 % to the 5-year survival in all types of cancers. One of the main disadvantages of the use of chemotherapy is that it is administered throughout the entire body, which accounts for the associated side effects of cancer treatment. Drug delivery systems (DDS) are a safe and reliable method of getting drugs to a disease site. They can effectively protect the healthy areas of the body from the adverse effects of the drug while also preventing the degradation of the drug, due to enzymatic action, within the body. The Food and Drug Administration (FDA)-approved DDSs, such as Doxil, a liposomal formulation of doxorubicin, have been introduced to clinical practice to limit systemic exposure to such drugs and thereby reduce related toxicities. However, one of the main challenges that this, and many other drug delivery systems face, is the ability to successfully release content on demand at the target site. The work contained within is focused on the optimization of a liposomal drug delivery system. This was done by harnessing both the plasmon resonant capabilities of gold nanoparticles as well as the ability to use ligands as a mechanism to target specific cancers. We introduced liposome-supported plasmon resonant gold nanoshells, a DDS that works on the premise of the conversion of light energy to heat that in turn initiates drug leakage from the liposome core. This dissertation is focused on building upon this observed phenomenon to address the need for a drug delivery system that is targeted specifically to diseased sites and is also capable of releasing content on demand.

Ovarian cancer is the deadliest gynecologic cancer for women. Diagnosis at the local stage leads to 91% 5-year survival rates, but only 15% of cases are detected early. Existing screening methods have proven ineffective in large clinical trials. Screening is complicated by the heterogeneity of the disease with multiple types of ovarian cancer originating both on the ovary and in the fallopian tube. Early stage cancer is too subtle for non-invasive imaging techniques such as ultrasound or magnetic resonance imaging. This study evaluates the feasibility and design of dual modality, multispectral fluorescence imaging (MFI) and optical coherence tomography (OCT) endoscopes for improved ovarian cancer screening. The study is broken up into three sections. In the first study MFI is validated in an ex vivo imaging study of human ovarian and fallopian tube tissue samples. Tissue autofluorescence excited by ultraviolet and blue wavelengths is shown to be a promising discriminator between normal and cancerous tissue. The second study combines OCT and MFI into a sub millimeter diameter endoscope designed to screen for ovarian cancer by screening inside the fallopian tube and at the ovary. The small size is required for screening the full length of the fallopian tube. MFI is implemented as a wide-field navigational imaging technique with high sensitivity complemented by high resolution structural depth imaging of OCT over a limited field of view. The final study presents a novel lens design for a scanning fiber endoscope with forward-viewing navigation and side-viewing OCT. A piezo tube is used to scan an optical fiber providing both the navigation channel’s illumination and OCT imaging. The design spatially separates the forward-viewing illumination from the OCT. As the piezo fiber circularly scans at its maximum deviation the OCT beam focus is rotationally scanned out the side of the endoscope tip by a rotationally symmetric double reflection in the cover plate.

Cancer is a multistage, heterogeneous disease that develops through a series of genetic mutations. Early stage cancer is most responsive to treatment but can be the hardest to detect due to its small size, lack of definitive symptoms and potential location deep in the body. Whole body imaging methods, MRI/CT/PET, lack the necessary resolution to detect cellular level abnormalities. Optical methods, which have sufficient resolution, can be miniaturized into endoscopes, which are necessary to overcome limited penetration of light into tissue. By combining optical coherence tomography (OCT) and fluorescence imaging methods it is possible to create endoscopes sensitive to molecular and structural changes. I applied a dual-modality 2mm diameter rigid endoscope to the study of the natural history of colon cancer in a mouse model, and later applied this knowledge to the design and characterization of a 0.8 mm dual-modality flexible probe for use in human fallopian tubes. By using this endoscope, which is introduced through the natural orifice and is compatible with existing hysteroscopes, high-risk women could be screened in a procedure at a similar level of invasiveness as a colonoscopy. Therefore, the endoscope fills this gap in clinical care for women at high-risk for ovarian cancer.

Congestive heart failure represents one of the leading causes of death in industrialized countries. A subset of heart failure situations are linked to genetic mutations, many of which reside in sarcomeric proteins. With the prevalence of mutations as approximately 1:500, the location of a mutation plays a direct role in the severity and lethality of the resulting disease state. The point mutation Arg403Glu (R403Q) located on the myosin heavy chain molecule is no exception with a resulting development of left ventricular hypertrophy, myocyte disarray, and increased cellular fibrosis. Given the severity of this mutation in humans, a mouse model was developed to recapitulate these phenotypic characteristics. An additional confounding factor often overlooked when studying the effects of cardiac disease is the role of biological sex. We, therefore, tested the hypothesis that R403Q mice will display altered crossbridge kinetics, specifically an increase in functioning at the expense of energy efficiency, and that biological sex will impact the cardiac response to the R403Q mutation in terms of both crossbridge functioning and post-translational modifications. To do this, we used both male and female R403Q transgenic mice and quantified myofilament mechanical properties including Ca²⁺ sensitivity, crossbridge cycling kinetics, and tension cost of contraction. In addition, phosphorylation patterning was quantified for one of the central, sarcomeric regulatory proteins, cardiac troponin I. We used 2 month animals which do not display overt pathology in the form of hypertrophy in order to minimize downstream, confounding effects. We were able to find that disease and biological sex played an important role in defining these parameters and suggests females are able to better compensate to the presence of altered sarcomeric interactions. Future investigations should focus on altered kinase activity, possibly driven by interactions with estrogen, in order to better define how fe

Abdominal Aortic Aneurysm (AAA) is a complex disease that leads to a localized dilation of the infrarenal aorta that develops over years. Longitudinal information in humans has been difficult to obtain for this disease, therefore mouse models have become increasingly used to study the development of AAAs. The objective of this study was to determine any changes that occur in the biomechanical response and fiber microstructure in the apolipoprotein E difficient (ApoE-/-) angiotensin II (AngII) infused mouse model of aneurysm during disease progression, as well as determine some of the underlying changes in biochemistry, and demonstrate a novel method of reducing any pathogenic protease activity. Using a Microbiaxial Opto-Mechanical Device (MOD), ex vivo studies included adult aortas of ApoE-/- AngII infused mice excised and tested for mechanical response simultaneously imaged using two-photon microscopy to assess the microstructure at multiple time points. In vitro and ex vivo studies have shown changes in protease concentrations with the use of FRET based proteolytic beacons able to provide a non-destructive method to quantify protease activity measured against mechanical and microstructural changes. In vitro studies have demonstrated protease activity can be reduced using a molecule providing a positive feedback mechanism for protease inhibition and possibly provide a reduction in aneurysm progression.

Advances in optical imaging have provided methods for visualizing molecular expression in tumors in vivo, allowing the opportunity to study the complexity of the tumor microenvironment. The development of fluorescent contrast agents targeted to molecules expressed in cancer cells is critical for in vivo imaging of the tumors. Contrast agents emitting in the near infrared (NIR) allow for an increased depth of penetration in tissue due to decreased absorption and scattering. There is also significantly less autofluorescence from tissue in the NIR. Quantum dots are nanoscopic particles of semiconductors whose fluorescent emission wavelength is tunable by the size of the particle with desirable fluorescent qualities such as a wide range of excitation wavelengths, a narrow emission band, high quantum efficiency, high photostablility, and they can be produced to emit throughout the NIR imaging window. It has been shown that vascular endothelial growth factor receptor 2 (VEGFR2) is upregulated in many cancers, including colorectal, as it is important in tumor angiogenesis and is considered a predictor for clinical outcome and, in some instances, is used for targeted therapy with anti-angiogenic drugs. For these reasons, quantum dots bioconjugated to VEGFR2 antibodies have the potential to provide contrast between normal tissue and cancer, as well as a mechanism for evaluating the molecular changes associated with cancer in vivo. In this dissertation, we present on the design of two contrast agents using quantum dots targeted to VEGFR2 for use in the molecular imaging of colon cancer, both ex vivo and in vivo. First, as a preliminary ex vivo investigation into their efficacy, Qdot655® (655nm emission) were bioconjugated to anti-VEGFR2 antibodies through streptavidin/biotin linking. The resulting QD655-VEGFR2 contrast agent was used to label colon adenoma in vivo and imaged ex vivo with significant increase in contrast between diseased and undiseased tissue, allowing for flu

Leader cells are a fundamental biological process that have only been investigated since the early 2000s. These cells have often been observed emerging at the edge of an artificial wound in 2D epithelial cell collective invasion, created with either a mechanical scrape from a pipette tip or from the removal of a plastic, physical blocker. During migration, the moving cells maintain cell-cell contacts, an important quality of collective migration; the leader cells originate from either the first or the second row, they increase in size compared to other cells, and they establish ruffled lamellipodia. Recent studies in 3D have also shown that cells emerging from an invading collective group that also exhibit leader-like properties. Exactly how leader cells influence and interact with follower cells as well as other cells types during collective migration, however, is another matter, and is a subject of intense investigation between many different labs and researchers. The majority of leader cell research to date has involved epithelial cells, but as collective migration is implicated in many different pathogenic diseases, such as cancer and wound healing, a better understanding of leader cells in many cell types and environments will allow significant improvement to therapies and treatments for a wide variety of disease processes. In fact, more recent studies on collective migration and invasion have broadened the field to include other cell types, including mesenchymal cancer cells and fibroblasts. However, the proper technology for picking out dynamic, single cells within a moving and changing cell population over time has severely limited previous investigation into leader cell formation and influence over other cells. In line with these previous studies, we not only bring new technology capable of dynamically monitoring leader cell formation, but we propose that leader cell behavior is more than just an epithelial process, and that it is a critical physiological p