Ultrasound-responsive dual-drug-loaded nanodroplets enhance revascularization in murine ischemic hindlimbs by activating the eNOS/NO pathway

Nitric oxide (NO)-based therapy represents a promising noninvasive treatment strategy for peripheral artery disease (PAD) by enhancing microvascular and endothelial function. However, the efficacy of NO is severely limited by vascular sparsity and dysfunction in PAD patients, which hinder efficient targeted delivery of highly unstable NO and reduce the conversion efficiency of NO donors at the lesion site, even with the assistance of NO carriers. To address this, we developed novel ultrasound-responsive dual-drug-loaded nanodroplets (A&C-NDs) using a modified ultrasonic emulsification method for co-loading and targeted delivery of L -arginine ( L -Arg) and cilostazol (CLZ). The A&C-NDs exhibited uniform size, high drug-loading efficiency, and favorable ultrasound imaging capability and ultrasound-responsive drug-release properties, enabling effective drug accumulation and penetration in ischemic limbs. Importantly, ultrasound-triggered vaporization and CLZ release synergistically activated endothelial nitric oxide synthase (eNOS), significantly enhancing local NO production. Through targeted modulation of the eNOS/NO pathway, this system successfully achieved limb preservation in mouse models of hindlimb ischemia. Non-invasive super-resolution ultrasound (SRUS) imaging and immunofluorescence staining further demonstrated that the combination of ultrasound and A&C-NDs promoted vasodilation and microvascular angiogenesis, while attenuating muscle injury in the ischemic limbs, resulting in comprehensive therapeutic improvement. Collectively, this study presents a novel ultrasound-activated nanoplatform that combines synergistic L -Arg/CLZ delivery with localized ultrasound stimulation to precisely activate the eNOS/NO pathway, promoting angiogenesis and tissue repair, thereby offering a new strategy for the noninvasive treatment of limb ischemia.