Graphene nanocomposites for real-time electrochemical sensing of nitric oxide in biological systems

Nitric oxide (NO) signaling plays many pivotal roles impacting almost every organ function in mammalian physiology, most notably in cardiovascular homeostasis, inflammation, and neurological regulation. Consequently, the ability to make real-time and continuous measurements of NO is a prerequisite research tool to understand fundamental biology in health and disease. Despite considerable success in the electrochemical sensing of NO, challenges remain to optimize rapid and highly sensitive detection, without interference from other species, in both cultured cells and in vivo. Achieving these goals depends on the choice of electrode material and the electrode surface modification, with graphene nanostructures recently reported to enhance the electrocatalytic detection of NO. Due to its single-atom thickness, high specific surface area, and highest electron mobility, graphene holds promise for electrochemical sensing of NO with unprecedented sensitivity and specificity even at sub-nanomolar concentrations. The non-covalent functionalization of graphene through supermolecular interactions, including π–π stacking and electrostatic interaction, facilitates the successful immobilization of other high electrolytic materials and heme biomolecules on graphene while maintaining the structural integrity and morphology of graphene sheets. Such nanocomposites have been optimized for the highly sensitive and specific detection of NO under physiologically relevant conditions. In this review, we examine the building blocks of these graphene-based electrochemical sensors, including the conjugation of different electrolytic materials and biomolecules on graphene, and sensing mechanisms, by reflecting on the recent developments in materials and engineering for real-time detection of NO in biological systems

Dual-Modality Photoacoustic and Ultrasound Imaging for Murine Atherosclerosis Characterization

Atherosclerosis accounts of 50% of the deaths in the western world leading to a plethora of diseases that include myocardial infarction, stroke, and peripheral artery disease. Currently available imaging modalities have inherent limitations, including ionizing radiation, lack of compositional information, and difficulty acquiring volumetric data that constrain their use in studying cardiovascular disease. Photoacoustic Tomography (PAT) has emerged as a promising modality that could address these limitations to improve the characterization and diagnosis of atherosclerosis-related conditions. Non-ionizing pulsed laser light is delivered to tissue leading to thermoelastic expansion followed by propagation of a pressure transient that can be detected with an ultrasound transducer. The magnitude of the ultrasonic PAT signal is proportional to the optical absorption at that location, revealing physiologically relevant compositional information of the tissue. The objective of this work is to therefore develop advanced volumetric imaging techniques to characterize disease progression in a murine model of atherosclerosis. The novelty of this work lies in the methodology and validation presented towards characterization of small animal vascular lipid accumulation with a high-resolution PAT system that utilizes the second near-infrared window (1100-1300nm). Additionally, we utilized in situ PAT to cross-sectionally assess lipid deposition and in vivo ultrasound to longitudinally assess hemodynamic, kinematic, and morphological changes during atherosclerosis progression. Together, this dissertation lays the foundation towards utilizing dual-modality PAT and ultrasound for various applications including understanding atherosclerosis pathophysiology, evaluation of novel therapeutics, and translation of clinically relevant techniques

Adjustable photoacoustic tomography probe improves light delivery and image quality

One cause for suboptimal photoacoustic tomography (PAT) penetration depth is attenuation of incident light by soft tissue. To better understand this problem, we investigated the effects of illumination fiber optic bundle geometry on PAT penetration depth and signal-to-noise ratio. An adjustable, motorized PAT probe was used to reduce probe-skin reflection artifacts and improve light distribution in the image acquisition plane by tuning fiber orientation. We validated our motorized PAT probe through Monte Carlo simulations and ex vivo imaging of a tissue mimicking phantom, and in vivo imaging of murine periaortic fat. Overall, our ex vivo results showed a several millimeter improvement in penetration depth and in vivo results showed a >62% increase in lipid signal-to-noise ratio. Our PAT probe also utilized a 7-μm aluminum filter to block in vivo probe-skin reflection artifacts. Together, these findings showed the importance of optimizing illumination geometry to enhance PAT image quality. Keywords: Photoacoustic, Ultrasound, Monte Carlo, Artifact, Signal-to-noise, Imagin

Development of a Glycosaminoglycan Derived, Selectin Targeting Anti-Adhesive Coating to Treat Endothelial Cell Dysfunction.

Endothelial cell (EC) dysfunction is associated with many disease states including deep vein thrombosis (DVT), chronic kidney disease, sepsis and diabetes. Loss of the glycocalyx, a thin glycosaminoglycan (GAG)-rich layer on the EC surface, is a key feature of endothelial dysfunction and increases exposure of EC adhesion molecules such as selectins, which are involved in platelet binding to ECs. Once bound, platelets cause thrombus formation and an increased inflammatory response. We have developed a GAG derived, selectin targeting anti-adhesive coating (termed EC-SEAL) consisting of a dermatan sulfate backbone and multiple selectin-binding peptides designed to bind to inflamed endothelium and prevent platelet binding to create a more quiescent endothelial state. Multiple EC-SEAL variants were evaluated and the lead variant was found to preferentially bind to selectin-expressing ECs and smooth muscle cells (SMCs) and inhibit platelet binding and activation in a dose-dependent manner. In an in vivo model of DVT, treatment with the lead variant resulted in reduced thrombus formation. These results indicate that EC-SEAL has promise as a potential therapeutic in the treatment of endothelial dysfunction

Development of a Glycosaminoglycan Derived, Selectin Targeting Anti-Adhesive Coating to Treat Endothelial Cell Dysfunction

Endothelial cell (EC) dysfunction is associated with many disease states including deep vein thrombosis (DVT), chronic kidney disease, sepsis and diabetes. Loss of the glycocalyx, a thin glycosaminoglycan (GAG)-rich layer on the EC surface, is a key feature of endothelial dysfunction and increases exposure of EC adhesion molecules such as selectins, which are involved in platelet binding to ECs. Once bound, platelets cause thrombus formation and an increased inflammatory response. We have developed a GAG derived, selectin targeting anti-adhesive coating (termed EC-SEAL) consisting of a dermatan sulfate backbone and multiple selectin-binding peptides designed to bind to inflamed endothelium and prevent platelet binding to create a more quiescent endothelial state. Multiple EC-SEAL variants were evaluated and the lead variant was found to preferentially bind to selectin-expressing ECs and smooth muscle cells (SMCs) and inhibit platelet binding and activation in a dose-dependent manner. In an in vivo model of DVT, treatment with the lead variant resulted in reduced thrombus formation. These results indicate that EC-SEAL has promise as a potential therapeutic in the treatment of endothelial dysfunction

Imaging of Small Animal Peripheral Artery Disease Models: Recent Advancements and Translational Potential

Peripheral artery disease (PAD) is a broad disorder encompassing multiple forms of arterial disease outside of the heart. As such, PAD development is a multifactorial process with a variety of manifestations. For example, aneurysms are pathological expansions of an artery that can lead to rupture, while ischemic atherosclerosis reduces blood flow, increasing the risk of claudication, poor wound healing, limb amputation, and stroke. Current PAD treatment is often ineffective or associated with serious risks, largely because these disorders are commonly undiagnosed or misdiagnosed. Active areas of research are focused on detecting and characterizing deleterious arterial changes at early stages using non-invasive imaging strategies, such as ultrasound, as well as emerging technologies like photoacoustic imaging. Earlier disease detection and characterization could improve interventional strategies, leading to better prognosis in PAD patients. While rodents are being used to investigate PAD pathophysiology, imaging of these animal models has been underutilized. This review focuses on structural and molecular information and disease progression revealed by recent imaging efforts of aortic, cerebral, and peripheral vascular disease models in mice, rats, and rabbits. Effective translation to humans involves better understanding of underlying PAD pathophysiology to develop novel therapeutics and apply non-invasive imaging techniques in the clinic