NFκB p65 siRNA was mixed with in vivo-jetPEI at a N/P ratio of 6 at room temperature for 15 minutes. The mixture (400 μL) was injected via the jugular or tail vein into 8-week old male mice at doses of 3-12 nmol. For jugular vein injection, animals were anesthetized with an intraperitoneal injection of 4% chloral hydrate at a dose of 1 mL/kg for operation procedures. Following exposure of the external jugular veins the mixtures were injected using a 25G syringe needle.

The nuclear factor-kappaB (NF-kappaB) in cardiac vascular endothelial cells (type II VEC) is a key factor that activates delayed xenograft rejection (DXR), and therefore inhibition of NF-kappaB gene expression may alleviate post-transplant rejection. siRNA technology was used to inhibit NF-kappaB p65 gene expression in ICR mice. After jugular vein injection of siRNA/in vivo-jetPEI complex, fluorescence levels of FAM-labeled siRNA in hearts and lungs were much higher after jugular vein injection than tail vein injection, suggesting more efficient siRNA delivery to the heart through the jugular vein. The amount of FAM fluorescence of hearts increased to the highest level between 48 and 72 hours after injection, and decreased gradually 1 week after injection. A minimum dose of 6 nmol NF-kappaB p65 siRNA and a siRNA/in vivo-jetPEI ratio of 6 (N/P = 6) were required for in vivo siRNA-mediated gene silencing in the heart. Under these conditions, application of siRNA/in vivo-jetPEI complexes from the jugular vein successfully suppressed NF-kappaB p65 expression in the heart. The same strategy can be applied to heart transplant animal models to protect against NF-kappaB gene-related type II VEC activation and xenograft rejection.