Sustained release nitric oxide from long-lived circulating nanoparticles

Abstract

The current limitations of nitric oxide (NO) delivery systems have stimulated an extraordinary interest in the development of compounds that generate NO in a controlled and sustained manner with a heavy emphasis on the treatment of cardiovascular disease states. This work describes the positive physiological response to the infusion of NO-releasing nanoparticles prepared using a new platform based on hydrogel/glass hybrid nanoparticles. When exposed to moisture, these nanoparticles slowly release therapeutic levels of NO, previously generated through thermal reduction of nitrite to NO trapped within the dry particles. The controlled and sustained release of NO observed from these nanoparticles (NO-np) is regulated by its hydration over extended periods of time. In a dose-dependent manner, circulating NO-np both decreased mean arterial blood pressure and increased exhaled concentrations of NO over a period of several hours. Circulating NO-np induced vasodilatation and increased microvascular perfusion during their several hour circulation lifetime. Control nanoparticles (control-np; without nitrite) did not induce changes in arterial pressure, although a decrease in the number of capillaries perfused and an increase in leukocyte rolling and immobilization in the microcirculation were observed. The NO released by the NO-np prevents the inflammatory response observed after infusion of control-np. These data suggest that NO release from NO-np is advantageous relative to other NO-releasing compounds, because it does not depend on chemical decomposition or enzymatic catalysis; it is only determined by the rate of hydration. Based on the observed physiological properties, NO-np has clear potential as a therapeutic agent and as a research tool to increase our understanding of NO signaling mechanisms within the vasculature.

Figure 1. NO releasing nanoparticle synthesis and morphology

a) Schematic of NO releasing nanoparticle synthesis. The various components are prepared and combined to form a block gel consisting of a silica hydrogel matrix encompassing the other ingredients. The block gel is dried and the nitrite precursor converted to NO via thermal reduction, resulting in the formation of the nanoparticles. b) TEM of NO releasing nanoparticles. The scale bars represent 100nm, the bottom one representing the lower left and right panes and the upper one for the upper left image. c) NO gas levels were measured using a chemiluminescent NO analyzer. Upper panel, 1 mg of NO-np or Control-np at 7.4 pH. Lower panel, 1 mg of NO-np at 6.0, 6.5 and 7.3 pH.

Figure 2. Effects in blood pressure after infusion of NO releasing nanoparticles (NO-np), control nanoparticles (Control-np) and NONOates donors

a) Infusion of 10 mg/kg (n=5) NO-np and 20 mg/kg (n=5) NO-np. Maximal decrease pressure was measured 90 min after infusion. MetHb levels 4 h after infusion of NO-np were 9 ± 2% for 10 mg/kg and 14 ± 3% for 20 mg/kg, respectively. b) Infusion of 10 mg/kg (n=5) Control-np and 20 mg/kg (n=5) Control-np. No changes in blood pressure were measure after in fusion of Control-np. c) Infusion of 10 mg/kg (n=5) DPTA NONOate and 20 mg/kg (n=5) DPTA NONOate. d) Infusion of 10 mg/kg (n=5) DETA NONOate and 20 mg/kg (n=5) DETA NONOate.

Figure 3. Exhaled NO after infusion of NO releasing nanoparticles (NO-np) and control nanoparticles (Control-np)

a) Exhaled NO after infusion of 10 mg/kg and 20 mg/kg (n=5) of NO-np and Control-np. b) Intravascular fluorescence of NO-np and Control-np. †, P<0.05 to Baseline.

Figure 4. Microvascular changes and blood chemistry after infusion of NO releasing nanoparticles (NO-np) and control nanoparticles (Control-np)

a) Changes in arteriolar diameter after infusion of 10 (white bars) and 20 (gray bars) mg/kg of NO-np (no pattern) and Control-np (pattern upward diagonal). Diameters (μm, mean ± SD) at baseline were 61.7 ± 7.9, N = 68. N = number of vessels studied. †, P<0.05 to Baseline; ‡, P<0.05 to Control-np at same concentration. b) Changes in arteriolar blood flow after infusion of NO-np and Control-np. Flow (nl/s, mean ± SD) at baseline were 12.6 ± 3.8, N =68. †, P<0.05 to Baseline; ‡, P<0.05 to Control-np at same concentration. c) Changes in functional capillary density (FCD) after infusion of NO-np and Control-np. FCD (perfused capillaries/cm^-1, mean ± SD) at baseline were 106 ± 6, n =20. †, P<0.05 to Baseline; ‡, P<0.05 to Control-np at same concentration.

Figure 5. Rolling and immobilized leukocytes after infusion of NO releasing nanoparticles (NO-np) and control nanoparticles (Control-np)

a) Immobilized leukocytes after infusion of 10 (white bars) and 20 (gray bars) mg/kg of NO-np (no pattern) and Control-np (pattern upward diagonal). 6 animals were used in each group, 6-8 venules were selected in each animal. †, P<0.05 to Baseline; ‡, P<0.05 to Control-np at same concentration. b) Rolling leukocytes after infusion of NO-np and Control-np. Rolling leukocytes were quantified in the same animals and locations as Immobilized. †, P<0.05 to Baseline; ‡, P<0.05 to Control-np at same concentration. c) Immobilized and rolling leukocytes after infusion of 20 mg/kg of NO-np and Control-np. Red arrows point to immobilized leukocytes on the images, other leukocytes in the images are rolling of flowing.

Figure 6. Blood pressure vascular tone effects of NO-np and Control-np mediated by NO synthase (NOS) and guanylate cyclase (GCM) metabolism

a) Infusion of 10 mg/kg of NO-np (n=5) and Control-np (n=5) during normal physiological condition. Blood pressure (mmHg, mean ± SD) at baseline for each group were NO-np: 112 ± 9 (n=5) and Control-np: 114 ± 8 (n=5). Diameters (μm, mean ± SD) at baseline for each group were NO-np: 62 ± 10 (N=24) and Control-np: 64 ± 12 (N=24). b) Infusion of 10 mg/kg of NO-np (n=5) and Control-np (n=5) during NO synthase inhibition with L-NAME. Blood pressure (mmHg, mean ± SD) at baseline for each group were NO-np: 111 ± 8 (n=5) and Control-np: 110 ± 7 (n=5). Diameters (μm, mean ± SD) at baseline for each group were NO-np: 60 ± 12 (N=22) and Control-np: 65 ± 8 (N=20). c) Infusion of 10 mg/kg of NO-np (n=5) and Control-np (n=5) after ODQ. Blood pressure (mmHg, mean ± SD) at baseline for each group were NO-np: 110 ± 6 (n=5) and Control-np: 112 ± 7 (n=5). Diameters (μm, mean ± SD) at baseline for each group were NO-np: 58 ± 11 (N=26) and Control-np: 62 ± 10 (N=20). d) Infusion of 10 mg/kg of NO-np (n=5) and Control-np (n=5) during phosphodiesterase inhibitor Zaprinast. Blood pressure (mmHg, mean ± SD) at baseline for each group were NO-np: 109 ± 7 (n=5) and Control-np: 112 ± 6 (n=5). Diameters (μm, mean ± SD) at baseline for each group were NO-np: 64 ± 12 (N=20) and Control-np: 66 ± 10 (N=20).