Kaku Nakano

Formulation of nanoparticle-eluting stents by a cationic electrodeposition coating technology: efficient nano-drug delivery via bioabsorbable polymeric nanoparticle-eluting stents in porcine coronary arteries.

OBJECTIVES The objective of this study was to formulate a nanoparticle (NP)-eluting drug delivery stent system by a cationic electrodeposition coating technology. BACKGROUND Nanoparticle-mediated drug delivery systems (DDS) are poised to transform the development of innovative therapeutic devices. Therefore, we hypothesized that a bioabsorbable polymeric NP-eluting stent provides an efficient DDS that shows better and more prolonged delivery compared with dip-coating stent. METHODS We prepared cationic NP encapsulated with a fluorescence marker (FITC) by emulsion solvent diffusion method, succeeded to formulate an NP-eluting stent with a novel cation electrodeposition coating technology, and compared the in vitro and in vivo characteristics of the FITC-loaded NP-eluting stent with dip-coated FITC-eluting stent and bare metal stent. RESULTS The NP was taken up stably and efficiently by cultured vascular smooth muscle cells in vitro. In a porcine coronary artery model in vivo, substantial FITC fluorescence was observed in neointimal and medial layers of the stented segments that had received the FITC-NP-eluting stent until 4 weeks. In contrast, no substantial FITC fluorescence was observed in the segments from the polymer-based FITC-eluting stent or from bare metal stent. The magnitudes of stent-induced injury, inflammation, endothelial recovery, and neointima formation were comparable between bare metal stent and NP-eluting stent groups. CONCLUSIONS Therefore, this NP-eluting stent is an efficient NP-mediated DDS that holds as an innovative platform for the delivery of less invasive nano-devices targeting cardiovascular disease.

Nanoparticle-Mediated Delivery of Nuclear Factor κB Decoy Into Lungs Ameliorates Monocrotaline-Induced Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is an intractable disease of the small pulmonary artery that involves multiple inflammatory factors. We hypothesized that a redox-sensitive transcription factor, nuclear factor &kgr;B (NF-&kgr;B), which regulates important inflammatory cytokines, plays a pivotal role in PAH. We investigated the activity of NF-&kgr;B in explanted lungs from patients with PAH and in a rat model of PAH. We also examined a nanotechnology-based therapeutic intervention in the rat model. Immunohistochemistry results indicated that the activity of NF-&kgr;B increased in small pulmonary arterial lesions and alveolar macrophages in lungs from patients with PAH compared with lungs from control patients. In a rat model of monocrotaline-induced PAH, single intratracheal instillation of polymeric nanoparticles (NPs) resulted in delivery of NPs into lungs for ≤14 days postinstillation. The NP-mediated NF-&kgr;B decoy delivery into lungs prevented monocrotaline-induced NF-&kgr;B activation. Blockade of NF-&kgr;B by NP-mediated delivery of the NF-&kgr;B decoy attenuated inflammation and proliferation and, thus, attenuated the development of PAH and pulmonary arterial remodeling induced by monocrotaline. Treatment with the NF-&kgr;B decoy NP 3 weeks after monocrotaline injection improved the survival rate as compared with vehicle administration. In conclusion, these data suggest that NF-&kgr;B plays a primary role in the pathogenesis of PAH and, thus, represent a new target for therapeutic intervention in PAH. This nanotechnology platform may be developed as a novel molecular approach for treatment of PAH in the future.

Local Delivery of Anti-Monocyte Chemoattractant Protein-1 by Gene-Eluting Stents Attenuates In-Stent Stenosis in Rabbits and Monkeys

Objective—We have previously shown that the intramuscular transfer of the anti–monocyte chemoattractant protein-1 (MCP-1) gene (called 7ND) is able to prevent experimental restenosis. The aim of this study was to determine the in vivo efficacy and safety of local delivery of 7ND gene via the gene-eluting stent in reducing in-stent neointima formation in rabbits and in cynomolgus monkeys. Methods and Results—We here found that in vitro, 7ND effectively inhibited the chemotaxis of mononuclear leukocytes and also inhibited the proliferation/migration of vascular smooth muscle cells. We then coated stents with a biocompatible polymer containing a plasmid bearing the 7ND gene, and deployed these stents in the iliac arteries of rabbits and monkeys. 7ND gene-eluting stents attenuated stent-associated monocyte infiltration and neointima formation after one month in rabbits, and showed long-term inhibitory effects on neointima formation when assessments were carried out at 1, 3, and 6 months in monkeys. Conclusions—Strategy of inhibiting the action of MCP-1 with a 7ND gene-eluting stent reduced in-stent neointima formation with no evidence of adverse effects in rabbits and monkeys. The 7ND gene-eluting stent could be a promising therapy for treatment of restenosis in humans.

Nanoparticle-Mediated Delivery of Pitavastatin Inhibits Atherosclerotic Plaque Destabilization/Rupture in Mice by Regulating the Recruitment of Inflammatory Monocytes

Background— Preventing atherosclerotic plaque destabilization and rupture is the most reasonable therapeutic strategy for acute myocardial infarction. Therefore, we tested the hypotheses that (1) inflammatory monocytes play a causative role in plaque destabilization and rupture and (2) the nanoparticle-mediated delivery of pitavastatin into circulating inflammatory monocytes inhibits plaque destabilization and rupture. Methods and Results— We used a model of plaque destabilization and rupture in the brachiocephalic arteries of apolipoprotein E–deficient (ApoE−/−) mice fed a high-fat diet and infused with angiotensin II. The adoptive transfer of CCR2+/+Ly-6Chigh inflammatory macrophages, but not CCR2−/− leukocytes, accelerated plaque destabilization associated with increased serum monocyte chemoattractant protein-1 (MCP-1), monocyte-colony stimulating factor, and matrix metalloproteinase-9. We prepared poly(lactic-co-glycolic) acid nanoparticles that were incorporated by Ly-6G−CD11b+ monocytes and delivered into atherosclerotic plaques after intravenous administration. Intravenous treatment with pitavastatin-incorporated nanoparticles, but not with control nanoparticles or pitavastatin alone, inhibited plaque destabilization and rupture associated with decreased monocyte infiltration and gelatinase activity in the plaque. Pitavastatin-incorporated nanoparticles inhibited MCP-1–induced monocyte chemotaxis and the secretion of MCP-1 and matrix metalloproteinase-9 from cultured macrophages. Furthermore, the nanoparticle-mediated anti–MCP-1 gene therapy reduced the incidence of plaque destabilization and rupture. Conclusions— The recruitment of inflammatory monocytes is critical in the pathogenesis of plaque destabilization and rupture, and nanoparticle-mediated pitavastatin delivery is a promising therapeutic strategy to inhibit plaque destabilization and rupture by regulating MCP-1/CCR2–dependent monocyte recruitment in this model.

Nanoparticle-Mediated Delivery of Pitavastatin Into Lungs Ameliorates the Development and Induces Regression of Monocrotaline-Induced Pulmonary Artery Hypertension

Pulmonary artery hypertension (PAH) is an intractable disease of the small PAs in which multiple pathogenic factors are involved. Statins are known to mitigate endothelial injury and inhibit vascular remodeling and inflammation, all of which play crucial roles in the pathogenesis of PAH. We tested the hypothesis that nanoparticle (NP)-mediated delivery of pitavastatin into the lungs can be a novel therapeutic approach for the treatment of PAH. Among the marketed statins, pitavastatin was found to have the most potent effects on proliferation of PA smooth muscle cells in vitro. We formulated pitavastatin-NP and found that pitavastatin-NP was more effective than pitavastatin alone in inhibiting cellular proliferation and inflammation in vitro. In a rat model of monocrotaline-induced PAH, a single intratracheal instillation of NP resulted in the delivery of NP into alveolar macrophages and small PAs for up to 14 days after instillation. Intratracheal treatment with pitavastatin-NP, but not with pitavastatin, attenuated the development of PAH and was associated with a reduction of inflammation and PA remodeling. NP-mediated pitavastatin delivery was more effective than systemic administration of pitavastatin in attenuating the development of PAH. Importantly, treatment with pitavastatin-NP 3 weeks after monocrotaline injection induced regression of PAH and improved survival rate. This mode of NP-mediated pitavastatin delivery into the lungs is effective in attenuating the development of PAH and inducing regression of established PAH, suggesting potential clinical significance for developing a new treatment for PAH.

Stent-Based Local Delivery of Nuclear Factor-κB Decoy Attenuates In-Stent Restenosis in Hypercholesterolemic Rabbits

Background— Nuclear factor-&kgr;B (NF-&kgr;B) plays a critical role in the vascular response to injury. However, the role of NF-&kgr;B in the mechanism of in-stent restenosis remains unclear. We therefore tested the hypothesis that blockade of NF-&kgr;B by stent-based delivery of a cis-element “decoy” of NF-&kgr;B reduces in-stent neointimal formation. Methods and Results— Stents were coated with a polymer containing or not containing NF-&kgr;B decoy, which represented a fast-release formulation (<7 days). Bare, polymer-coated, and NF-&kgr;B decoy–eluting stents were implanted in iliac arteries of hypercholesterolemic rabbits. Increased NF-&kgr;B activity was noted at early stages after stenting, which was suppressed by stent-based delivery of NF-&kgr;B decoy. NF-&kgr;B decoy–eluting stents also reduced monocyte infiltration and monocyte chemoattractant protein-1 expression and suppressed CD14 activation on circulating leukocytes. Importantly, NF-&kgr;B decoy–eluting stents attenuated neointimal formation on day 28. There was no evidence of an incomplete healing process (persistent inflammation, hemorrhage, fibrin deposition, impaired endothelial regeneration) at the site of NF-&kgr;B decoy–eluting stents. Transfection of NF-&kgr;B decoy suppressed proliferation of human coronary artery smooth muscle cells in vitro. No systemic adverse effects of NF-&kgr;B decoy were detected. Conclusions— Stent-based local delivery of NF-&kgr;B decoy reduced in-stent neointimal formation with no evidence of incomplete healing. These data suggest that this strategy may be a practical and promising means for prevention of in-stent restenosis in humans.

Therapeutic Neovascularization by Nanotechnology-Mediated Cell-Selective Delivery of Pitavastatin Into the Vascular Endothelium

Objective—Recent clinical studies of therapeutic neovascularization using angiogenic growth factors demonstrated smaller therapeutic effects than those reported in animal experiments. We hypothesized that nanoparticle (NP)-mediated cell-selective delivery of statins to vascular endothelium would more effectively and integratively induce therapeutic neovascularization. Methods and Results—In a murine hindlimb ischemia model, intramuscular injection of biodegradable polymeric NP resulted in cell-selective delivery of NP into the capillary and arteriolar endothelium of ischemic muscles for up to 2 weeks postinjection. NP-mediated statin delivery significantly enhanced recovery of blood perfusion to the ischemic limb, increased angiogenesis and arteriogenesis, and promoted expression of the protein kinase Akt, endothelial nitric oxide synthase (eNOS), and angiogenic growth factors. These effects were blocked in mice administered a nitric oxide synthase inhibitor, or in eNOS-deficient mice. Conclusions—NP-mediated cell-selective statin delivery may be a more effective and integrative strategy for therapeutic neovascularization in patients with severe organ ischemia.

Angiotensin II Type 1 Receptor Blockade Attenuates In-Stent Restenosis by Inhibiting Inflammation and Progenitor Cells

The precise mechanism by which angiotensin II type 1 receptor blocker reduces in-stent restenosis in clinical trials is unclear. We, therefore, investigated the mechanism of in-stent neointima formation. Male cynomolgus monkeys and rabbits were fed a high-cholesterol diet and were allocated to untreated control and type 1 receptor blocker groups. Five days after grouping, multilink stents were implanted in the iliac artery. The type 1 receptor blocker reduced the development of in-stent neointima formation by ≈30% in rabbits and monkeys. To investigate potential mechanisms, we examined the expression of renin-angiotensin system markers, all of which increased in monocytes and smooth muscle-like cells in the neointima and media within 7 days. The type 1 receptor blocker attenuated increased oxidative stress, the enhanced expression of markers of the rennin-angiotensin system and monocyte chemoattractant protein-1, and macrophage infiltration. The effects of type 1 receptor blocker on the differentiation of peripheral blood mononuclear cells into vascular progenitor cells were also examined. Treatment with type 1 receptor blocker suppressed the enhanced differentiation to smooth muscle progenitor cells induced by stenting. The type 1 receptor blocker attenuated in-stent neointima formation by inhibiting redox-sensitive inflammatory changes and by reducing recruitment of the progenitor cells. These potential actions of type 1 receptor blocker on inflammation and progenitor cells constitute a novel mechanism of suppression of in-stent restenosis by type 1 receptor blocker.

Local Delivery of Imatinib Mesylate (STI571)-Incorporated Nanoparticle Ex Vivo Suppresses Vein Graft Neointima Formation

Background— Clinical outcome of surgical revascularization using autologous vein graft is limited by vein graft failure attributable to neointima formation. Platelet-derived growth factor (PDGF) plays a central role in the pathogenesis of vein graft failure. Therefore, we hypothesized that nanoparticle (NP)-mediated drug delivery system of PDGF-receptor (PDGF-R) tyrosine kinase inhibitor (imatinib mesylate: STI571) could be an innovative therapeutic strategy. Methods and Results— Uptake of STI571-NP normalized PDGF-induced cell proliferation and migration. Excised rabbit jugular vein was treated ex vivo with PBS, STI571 only, FITC-NP, or STI571-NP, then interposed back into the carotid artery position. NP was detected in many cells in the neointima and media at 7 and 28 days after grafting. Significant neointima was formed 28 days after grafting in the PBS group; this neointima formation was suppressed in the STI571-NP group. STI571-NP treatment inhibited cell proliferation and phosphorylation of the PDGF-R-β but did not affect inflammation and endothelial regeneration. Conclusions— STI571-NP–induced suppression of vein graft neointima formation holds promise as a strategy for preventing vein graft failure.

Nanoparticle-mediated endothelial cell-selective delivery of pitavastatin induces functional collateral arteries (therapeutic arteriogenesis) in a rabbit model of chronic hind limb ischemia

OBJECTIVES We recently demonstrated in a murine model that nanoparticle-mediated delivery of pitavastatin into vascular endothelial cells effectively increased therapeutic neovascularization. For the development of a clinically applicable approach, further investigations are necessary to assess whether this novel system can induce the development of collateral arteries (arteriogenesis) in a chronic ischemia setting in larger animals. METHODS Chronic hind limb ischemia was induced in rabbits. They were administered single injections of nanoparticles loaded with pitavastatin (0.05, 0.15, and 0.5 mg/kg) into ischemic muscle. RESULTS Treatment with pitavastatin nanoparticles (0.5 mg/kg), but not other nanoparticles, induced angiographically visible arteriogenesis. The effects of intramuscular injections of phosphate-buffered saline, fluorescein isothiocyanate (FITC)-loaded nanoparticles, pitavastatin (0.5 mg/kg), or pitavastatin (0.5 mg/kg) nanoparticles were examined. FITC nanoparticles were detected mainly in endothelial cells of the ischemic muscles for up to 4 weeks. Treatment with pitavastatin nanoparticles, but not other treatments, induced therapeutic arteriogenesis and ameliorated exercise-induced ischemia, suggesting the development of functional collateral arteries. Pretreatment with nanoparticles loaded with vatalanib, a vascular endothelial growth factor receptor (VEGF) tyrosine kinase inhibitor, abrogated the therapeutic effects of pitavastatin nanoparticles. Separate experiments with mice deficient for VEGF receptor tyrosine kinase demonstrated a crucial role of VEGF receptor signals in the therapeutic angiogenic effects. CONCLUSIONS The nanotechnology platform assessed in this study (nanoparticle-mediated endothelial cell-selective delivery of pitavastatin) may be developed as a clinically feasible and promising strategy for therapeutic arteriogenesis in patients.