Abstract P032: Nanoparticle Mediated Nox2-siRNA Therapy for Preventing Cardiac Dysfunction Following Myocardial Infarction

Abstract

Myocardial infarction (MI) is the most common cause of heart failure in the developed world. Following MI, reactive oxygen species (ROS) play a key role in the pathogenesis of cardiac remodeling leading to impaired ventricular function. Oxidative stress at high levels lead to many of the injury associated changes: proinflammatory cytokine release, myocyte apoptosis, cardiac fibrosis and hypertrophy. Nicotinamide adenine denucleotide phosphate (NADPH) oxidase with Nox2 as the catalytic subunit, is a major source for cardiac ROS production. After MI, Nox2 expression is significantly increased in the infarcted myocardium. Moreover, mice lacking the Nox2 gene are protected from ischemic injury. We used polyketals, a new class of acid-degradable polymers, as delivery vehicles for Nox2-siRNA to the post-MI environment. When engaged by macrophages, present in high quantities during MI, these particles have been shown to be taken up by macrophages and contents released within cells in active form. Nox2-siRNA was ion-paired to the cationic lipid DOTAP, and spherical particles averaging 500nm diameter were made by a single emulsion procedure with polyketal PK3 using PVA as surfactant. While a commercially available transfection reagent yielded 7% uptake as measured by flow cytometery, 81% of macrophages were positive for fluorescently-labeled siRNA in PK3-siNOX2 treated cells. These data were confirmed with confocal microscopy. Macrophages treated with PK3-siNOX2 demonstrated a significant 43% knockdown in Nox2 gene expression at 24 hours, while no reduction was seen with scrambled siRNA or empty particles. Functional activity was assessed by a fluorescent dihyroethidium dye based HPLC quantification after phorbol 12-myristate 13-acetate stimulation following 72 hours of particle treatment to measure superoxide production. PK3-siNox2 treated cells exhibited a 41% reduction in activity, with no significant changes seen with scrambled siRNA or empty particle treatment. Currently, the therapeutic potential of the Nox2-siRNA particles is being evaluated in a mouse model of ischemia-reperfusion. Successful completion of these studies could lead to a novel treatment for post-infarction injury.