Antioxidant Response Activating NanoParticles (ARAPas): Treating Arterial Disease in 3D

Cardiovascular disease (CVD) remains the leading cause of mortality worldwide, with arterial diseases, namely atherosclerosis, being a major underlying contributor. Atherosclerosis arises from localized pro-oxidant redox imbalance. Nrf2 regulates the expression of numerous antioxidant genes to restore redox homeostasis. Electrophiles such as cinnamic aldehyde (CA) and bardoxolone methyl (CDDOMe) activate Nrf2 by reacting with its negative regulator, Keap1. Enzymatic antioxidants like catalase and superoxide dismutase directly neutralize excess reactive oxygen species (ROS). Novel therapies attenuating CVD progression by targeting pathological redox dysfunction are urgently needed. This dissertation explores targeted delivery of Nrf2 activators and antioxidant enzymes as nanotherapeutics for CVD.Chapter 1 presents an introduction to the pathogenic roles ROS play in CVD, highlighting antioxidant therapeutics of interest, Nrf2 activators and antioxidant enzymes. Additionally includes a comprehensive review of targeted nanotherapeutics approaches for CVD, including those that have successfully employed antioxidants. Chapter 2 demonstrates the feasibility of encapsulating CA into nanoparticles termed Antioxidant Response Activating nanoParticles (ARAPas). ARAPas can be efficiently engulfed by macrophages and subsequently transferred to vascular cells in culture, activating Nrf2, and where it has the potential to exhibit antioxidant effects as shown with CA-ARAPas treatment. Building on this, Chapter 3 describes the development of ARAPas encapsulating the potent Nrf2-activator CDDOMe for targeted delivery to atherosclerotic plaques. CDDOMe-ARAPas exploited macrophage biology to accumulate in plaques and activated cellular antioxidant defense mechanisms, decreasing atherosclerosis in mice. Chapter 4 establishes new analysis pipelines leveraging machine learning and virtual reality to efficiently quantify vascular lesions in animal models. Light sheet fluorescence microscopy, optical clearing and immunofluorescence enabled detailed 3D visualization of diseased vessels. Labkit, and syGlass decreased the hands-on analysis time while generating accurate volumetric renderings of disease features. Lastly, Chapter 5 sets forth a summary of the discussions, conclusions, and future directions.Overall, this dissertation establishes proof-of-concept on the feasibility of macrophage-mediated antioxidant drug delivery to vascular cells in diseased arteries. It also details the therapeutic effect of targeted CDDOMe-ARAPas in atherosclerotic mice. It puts forth efficient image analysis frameworks to quantify vascular lesions. The approaches described hold potential toward the development of targeted antioxidant nanotherapeutics for CVD treatment.