Excessive production of superoxide (O2•-) in the central nervous system has been widely implicated in the pathogenesis of angiotensin II (AngII)-dependent neurogenic hypertension (HTN). Our group has tried to overcome the failed therapeutic potential of currently available antioxidants by utilizing nanoformulated copper/zinc superoxide dismutase (SOD1), so-called SOD1 nanozymes, that specifically scavenges intracellular O2•-. These nanozymes consist of SOD1 protein wrapped with cationic block copolymers followed by covalent cross-linking of the polycation template (cl-nano). We hypothesize that cl-nano delivers active SOD1 protein to neurons and can effectively decrease blood pressure in a mouse model of AngII-dependent neurogenic HTN. As determined by electron paramagnetic resonance (EPR) spectroscopy, cl-nano retains SOD1 activity and scavenges O2•- to levels comparable with native SOD1 protein in a cell-free environment (EPR arbitrary units: vehicle 1.12e6 ± 1.79e5; native SOD1 protein 4.45e4 ± 3.00e3; cl-nano 6.78e4 ± 1.74e3, p<0.05 vs. vehicle). Experiments to examine neuronal uptake of cl-nano, analyzed by western blot and SOD1 activity assays, reveal that cl-nano delivers active SOD1 to central neurons in culture (CATH.a neurons) more efficiently than native SOD1 protein following 1 hour treatment (SOD1 activity in units/mg protein: vehicle 336; native SOD1 protein 313; cl-nano 718). Furthermore, in vivo studies demonstrate that HTN established by chronic subcutaneous infusion of AngII (400 ng/kg/min) is significantly attenuated following a single intracerebroventricular (ICV) injection of cl-nano for up to 7 days (mean arterial pressure (MAP) in mmHg: pre-AngII 87 ± 3; 9 days post-AngII 138 ± 6; 7 days post-ICV injection of cl-nano 112 ± 4, p<0.05 vs. pre-ICV injection). These data provide evidence for the efficacy of nanoformulated SOD1 in counteracting excessive O2•- and decreasing MAP in AngII-dependent hypertensive mice when injected directly into the brain. Although further experiments must be performed with more clinically relevant routes of cl-nano administration, such as intravenous injection, this study supports the further development of cl-nano with SOD1 as an alternative therapeutic option for HTN.
Over-Expression of Copper/Zinc Superoxide Dismutase in the Median Preoptic Nucleus Attenuates Chronic Angiotensin II-Induced Hypertension in the Rat