K22 - Dual-action Nanomatrix Coated Stent for Improving Endothelialization while Suppressing Restenosis and Inflammation

Introduction: : The most common cause of cardiovascular disease (CVD) is atherosclerosis. Atherosclerosis involves the initiation of endothelial dysfunction and recruitment of inflammatory cells. It is characterized by the development of plaques and proliferation of smooth muscle that lead to progressive arterial narrowing, known as restenosis. Bare metal stents (BMS) were developed to hinder stenosis and prevent ischemia. However, issues with BMS have been observed following deployment, primarily, restenosis of the blood vessels. Sustained smooth muscle cell proliferation and endothelial dysfunction have been cited as major drivers of this phenomenon. Drug-eluting stents (DES) have been developed to mitigate this, by releasing anti-proliferative drugs to suppress smooth muscle cell proliferation. Although DES can suppress restenosis in this manner, the anti-proliferative drugs also suppress endothelial growth and function. Another issue is the limitations of preclinical stent evaluation. Stent animal studies often involve the induction of endothelial dysfunction via denudation of the endothelium with a balloon. These ballon injury models are typically performed in healthy rabbit and porcine models and thus lack key characteristics of atherosclerosis seen in patients clinically. This can lead to overly optimistic results that do not accurately predict clinical outcomes. To address these issues, we have developed a dual action nanomatrix coated stent comprised of a nitric oxide (NO) releasing peptide amphiphile (PA) and everolimus-encapsulated liposomes to improve endothelial proliferation and suppress smooth muscle cell proliferation and inflammation. We plan to test our dual action nanomatrix coated stent in a high fat diet rabbit model to better predict its efficacy clinically.

Materials and

Methods: : The nanomatrix coating contains a PA consisting of a poly-lysine nitric oxide donor and a cell adhesive ligand (PA-KKKKK-YIGSR). The sequence was generated via solid phase peptide synthesis, and NO was charged to the PA. The everolimus-encapsulated liposomes were prepared with DPPC, DOTAP, DSPE-PEG, and cholesterol via thin film rehydration, followed by encapsulation with everolimus. TEM was performed on liposomes to gain information on their stability. To assess the long-term release kinetics, these liposomes were placed in a cartridge with a 10 kDa pore size dialysis membrane, allowing released everolimus to diffuse into a separate compartment containing only PBS. This PBS was collected, and everolimus content was measured via UV-vis spectroscopy to evaluate the release. To evaluate the dual effect of the nanomatrix coating, NO and everolimus were used to treat endothelial and smooth muscle cells. An MTT assay was used to evaluate the effects on metabolic activity. The high fat diet rabbit model will be generated in Male and Female New Zealand White rabbits. They will be fed an atherogenic diet consisting of cholesterol and peanut oil to induce hypercholesterolemic conditions. After this balloon injury will be induced prior to stent implantation.

Results, Conclusions, and Discussions:: Results and

Discussion: TEM images show that liposomes maintained stability over the course of 2 weeks. The everolimus release kinetics show 60% of encapsulated everolimus was released by day 30 following loading. The MTT assay showed that NO was able to recover endothelial cell metabolic activity despite the anti-proliferative effects of everolimus. It also showed that NO and everolimus combined were able to maintain and even lower smooth muscle cell metabolic activity compared to everolimus alone. For the high fat diet rabbit model, we are optimizing the conditions to induce conditions similar to fibroatheromas exhibited by atherosclerotic patients. This includes optimizing atherogenic diet (e.g., cholesterol and fat supplementation), balloon injury procedure, stent implantation, and the ideal timelines for the diet and procedures.

Conclusions: Everolimus-encapsulated liposomes have the potential to be an effective method of delivering everolimus due to their sustained release and long-term stability. NO and everolimus have a synergistic effect of improving endothelial function and suppressing smooth muscle cells. A high fat diet rabbit model has the potential yield outcomes that are more clinically relevant than balloon injury alone.

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