P9 - Development of Dual Pro-Angiogenic Agents Loaded Nanoparticles for Treatment of Peripheral Arterial Disease

Introduction: : Peripheral arterial disease (PAD) affects approximately 8.5 million individuals in the United States, predominantly impacting older adults. It is primarily caused by atherosclerotic narrowing of the arteries in the lower limbs, resulting in reduced blood flow and an inadequate supply of oxygen and nutrients to muscle tissues. In its advanced form, known as critical limb ischemia, PAD is associated with high rates of morbidity and mortality. Current invasive treatment options are often accompanied by significant risks and limitations, highlighting the urgent need for safer and more effective therapeutic strategies. Therefore, we propose a non-invasive alternative utilizing nanocarriers for the targeted and controlled delivery of dual pro-angiogenic factors to ischemic tissues. This approach aims to stimulate cellular proliferation, support cell migration, inhibit apoptosis, and ultimately promote angiogenesis, offering a promising therapeutic pathway for the treatment of PAD.

Materials and

Methods: : Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) encapsulating dual pro-angiogenic factors were synthesized using a standard double emulsion technique. These NPs were further incorporated with peptides targeting ischemic regions via carbodiimide chemistry. The NPs size and polydispersity were characterized using a dynamic light scattering (DLS) method. The in vitro therapeutic effect of these NPs was investigated on endothelial progenitor cells (EPCs). Cell survival was assessed under a stress-induced condition to mimic PAD pathology. A transmigration study using a 24-well Transwell system was also used to investigate cell transmigration after 12 hours of NPs’ treatment. The in vivo pilot dose dependent therapeutic effect of the NPs then was evaluated on PAD mice model. The ischemic hindlimb injury was induced by ligating the left femoral artery and its branches, while the right hindlimb was left untreated as a control leg. The NPs were administered intravenously (IV) via the tail vein. Blood perfusion recovery and functional improvement were monitored using Doppler imaging and treadmill performance up to 21 days post-treatment. Gastrocnemius muscle tissues were harvested for histological analyses, including hematoxylin and eosin (H&E) staining to assess tissue integrity and inflammation, Masson’s Trichrome staining for fibrosis, and immunostaining for collagen IV to evaluate capillary density and angiogenesis.

Results, Conclusions, and Discussions:: Results and

Discussion: The NPs were successfully fabricated with average diameters of around 250 nm and had an acceptable range of polydispersity. In vitro results demonstrated that the dual-factor nanoparticles significantly enhanced EPC survival and proliferation under stress conditions compared to untreated controls. Transwell assays confirmed increased cellular migration after nanoparticle exposure. In vivo, treated limbs showed notable preservation of tissue and prevention of amputation, alongside improved blood perfusion. Histological analysis revealed protection of muscle architecture, reduced fibrosis, and significantly increased capillary density in muscles of the NPs-treated group. These findings suggest that the nanoparticle system effectively promotes vascular regeneration in ischemic environments.
Conclusions and Future Work: The dual pro-angiogenic factor-loaded nanoparticles successfully enhanced cell survival, migration, and angiogenesis in vitro and promoted tissue protection and perfusion recovery in vivo. Future work will include extended in vitro assays, such as tube formation on Matrigel, and comprehensive in vivo studies to further validate the therapeutic efficacy of this nanoparticle-based approach for PAD treatment.

Acknowledgements and/or References (Optional):: This work is supported by the American Heart Association Institutional Research Enhancement Award 25AIREA1357216 (TN) (https://doi.org/10.58275/AHA.25AIREA1357216.pc.gr.226923). It was also partly supported by the National Heart, Lung, and Blood Institute under award number NIH R01 HL158204 (KTN). The content is solely the responsibility of the authors and does not necessarily represent the official views of either AHA or NIH.