Magnetically Actuated Urokinase Nanoclusters for Deep-Vein Thrombolysis: A Rat Study

Abstract

This study investigated whether magneto-mechanical micro-stirring generated by urokinase-loaded anisotropic iron-oxide nanoclusters could improve thrombolytic efficacy in acute deep-vein thrombosis while preserving systemic safety. A randomized, parallel-group, blinded in vivo experiment was conducted in 50 male Sprague-Dawley rats with confirmed inferior vena cava thrombosis. Animals were allocated equally to a control group treated with free systemic urokinase under sham magnetic exposure or to a study group treated with urokinase-loaded anisotropic magnetic nanoclusters followed by a localized rotating magnetic field. Formulation development demonstrated progressive physicochemical optimization across fabrication stages, with significant increases in hydrodynamic size, marked shifts in zeta potential toward a less negative surface, and reduced hemolysis after coating and loading, while polydispersity remained stable. Protocol optimization also showed significant between-protocol differences in ex vivo thrombus reduction and temperature rise, supporting selection of a non-thermal locked actuation regimen of 25 mT, 10 Hz, and 30 minutes. In vivo efficacy strongly favored the investigational platform. Standardized thrombus burden was markedly lower in the study group, whereas thrombus mass reduction, local urokinase activity, and thrombus iron localization were all substantially greater, each with large standardized between-group differences and highly significant between-group results. Baseline body weight, age, and thrombosis confirmation variables were well balanced between arms, supporting internal comparability. The systemic biomarker profile further indicated enhanced fibrinolysis, with higher D-dimer and plasmin-antiplasmin complex levels in treated animals, while PT, INR, aPTT, and fibrinogen remained broadly unchanged. Bleeding indices and hepatic and renal safety markers were comparable between groups, whereas inflammatory mediators were lower in the study arm. The main scientific contribution of this work is the integration of urokinase-loaded anisotropic iron-oxide nanoclusters with a locked non-thermal rotating magnetic-field regimen to enhance local thrombolytic activity while preserving systemic coagulation safety. Overall, the findings support the concept that magnetically actuated anisotropic nanoclusters can enhance target-site urokinase delivery, improve thrombus resolution, and maintain an acceptable systemic safety profile. This platform therefore represents a promising non-thermal strategy for more efficient and locally focused thrombolytic therapy in acute venous thrombosis.