Nitric oxide and nanotechnology: A novel approach to inhibit neointimal hyperplasia

ObjectiveNitric oxide (NO) has been shown to inhibit neointimal hyperplasia after arterial interventions in several animal models. To date, however, NO-based therapies have not been used in the clinical arena. Our objective was to combine nanofiber delivery vehicles with NO chemistry to create a novel, more potent NO-releasing therapy that can be used clinically. Thus, the aim of this study was to evaluate the perivascular application of spontaneously self-assembling NO-releasing nanofiber gels. Our hypothesis was that this application would prevent neointimal hyperplasia.MethodsGels consisted of a peptide amphiphile, heparin, and a diazeniumdiolate NO donor (1-[N-(3-Aminopropyl)-N-(3-ammoniopropyl)]diazen-1-ium-1,2-diolate [DPTA/NO] or disodium 1-[(2-Carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate [PROLI/NO]). Nitric oxide release from the gels was evaluated by the Griess reaction, and scanning electron microscopy confirmed nanofiber formation. Vascular smooth muscle cell (VSMC) proliferation and cell death were assessed in vitro by 3H-thymidine incorporation and Personal Cell Analysis (PCA) system (Guava Technologies, Hayward, Calif). For the in vivo work, gels were modified by reducing the free-water content. Neointimal hyperplasia after periadventitial gel application was evaluated using the rat carotid artery injury model at 14 days (n = 6 per group). Inflammation and proliferation were examined in vivo with immunofluorescent staining against CD45, ED1, and Ki67 at 3 days (n = 2 per group), and graded by blinded observers. Endothelialization was assessed by Evans blue injection at 7 days (n = 3 per group).ResultsBoth DPTA/NO and PROLI/NO, combined with the peptide amphiphile and heparin, formed nanofiber gels and released NO for 4 days. In vitro, DPTA/NO inhibited VSMC proliferation and induced cell death to a greater extent than PROLI/NO. However, the DPTA/NO nanofiber gel only reduced neointimal hyperplasia by 45% (intima/media [I/M] area ratio, 0.45 ± 0.07), whereas the PROLI/NO nanofiber gel reduced neointimal hyperplasia by 77% (I/M area ratio, 0.19 ± 0.03, P < .05) vs control (injury alone I/M area ratio, 0.83 ± 0.07; P < .05). Both DPTA/NO and PROLI/NO nanofiber gels significantly inhibited proliferation in vivo (1.06 ± 0.30 and 0.19 ± 0.11 vs injury alone, 2.02 ± 0.20, P < .05), yet had minimal effect on apoptosis. Only the PROLI/NO nanofiber gel inhibited inflammation (monocytes and leukocytes). Both NO-releasing nanofiber gels stimulated re-endothelialization.ConclusionsPerivascular application of NO-releasing self-assembling nanofiber gels is an effective and simple therapy to prevent neointimal hyperplasia after arterial injury. Our study demonstrates that the PROLI/NO nanofiber gel most effectively prevented neointimal hyperplasia and resulted in less inflammation than the DPTA/NO nanofiber gel. This therapy has great clinical potential to prevent neointimal hyperplasia after open vascular interventions in patients.Clinical relevanceAtherosclerosis affects >79 million Americans, many of whom require arterial intervention; unfortunately, treatment modalities often fail secondary to the development of neointimal hyperplasia, necessitating reintervention. It is well established that nitric oxide (NO) inhibits neointimal hyperplasia, but no NO-based therapies have been clinically applied owing to various concerns. In this study, we demonstrated inhibition of neointimal hyperplasia in the rat carotid artery balloon-injury model using the local application of a gel made from NO and self-assembling nanofibers. By inhibiting neointimal hyperplasia and subsequent restenosis after arterial interventions, we aim to improve long-term patency rates and reduce the number of repeat interventions