Long-term nitric oxide release and elevated temperature stability with S-nitroso-N-acetylpenicillamine (SNAP)-doped Elast-eon E2As polymer

Abstract

Nitric oxide (NO) is known to be a potent inhibitor of platelet activation and adhesion. Healthy endothelial cells that line the inner walls of all blood vessels exhibit a NO flux of 0.5-4 × 10(-10) mol cm(-2) min(-1) that helps prevent thrombosis. Materials with a NO flux that is equivalent to this level are expected to exhibit similar anti-thrombotic properties. In this study, five biomedical grade polymers doped with S-nitroso-N-acetylpenicillamine (SNAP) were investigated for their potential to control the release of NO from the SNAP within the polymers, and further control the release of SNAP itself. SNAP in the Elast-eon E2As polymer creates an inexpensive, homogeneous coating that can locally deliver NO (via thermal and photochemical reactions) as well slowly release SNAP. Furthermore, SNAP is surprisingly stable in the E2As polymer, retaining 82% of the initial SNAP after 2 months storage at 37 °C. The E2As polymer containing SNAP was coated on the walls of extracorporeal circulation (ECC) circuits and exposed to 4 h blood flow in a rabbit model of extracorporeal circulation to examine the effects on platelet count, platelet function, clot area, and fibrinogen adsorption. After 4 h, platelet count was preserved at 100 ± 7% of baseline for the SNAP/E2As coated loops, compared to 60 ± 6% for E2As control circuits (n = 4). The SNAP/E2As coating also reduced the thrombus area when compared to the control (2.3 ± 0.6 and 3.4 ± 1.1 pixels/cm(2), respectively). The results suggest that the new SNAP/E2As coating has potential to improve the thromboresistance of intravascular catheters, grafts, and other blood-contacting medical devices, and exhibits excellent storage stability compared to previously reported NO release polymeric materials.

Fig. 1

Structure of (A) S-nitroso-N-acetylpenicillamine (SNAP) and (B) scheme of S-nitrosothiol (RSNO) decomposition, which can be catalyzed by metal ions (e.g. Cu⁺), light, and heat, yielding the disulfide (RSSR) product and nitric oxide (NO).

Fig. 2

Percent of SNAP remaining in films (initially prepared with 10 wt% SNAP) after various durations of soaking in 4 mL PBS in the dark at room temperature, 22°C (A), or 37°C (B). Data is based on the difference between the amount of SNAP that leached from various polymers into the PBS, as monitored at 340 nm, and the initial amount of SNAP doped in the film. Data is the mean ± SEM (n=3).

Fig. 3

(A) NO release behavior of 10 wt% SNAP/E2As film at 37°C in the dark, ambient light, and 100W floodlight. (B) NO release from 10 wt% SNAP in silicone rubber (SR), CarboSil, and Elast-eon E2As films at 37°C and continuously irradiated with the 100W floodlight. (C) NO release from 5 and 10 wt% SNAP in Elast-eon E2As films at 37°C continuously under ambient light (amb) or the 100W floodlight. Data is the mean ± SEM (n=3).

Fig. 4

(A) UV-vis spectra of a 10 wt% SNAP/E2As film, 1.0 mM SNAP, and E2As dissolved in N,N-dimethylacetamide (DMAc). (B) Cumulative NO release from 10 wt% SNAP/E2As films incubated in PBS under various conditions: room temperature (22°C) with ambient light, 37°C in the dark, 37°C under ambient light, and 37°C under the 100W floodlight. Data is the mean ± SEM (n=3).

Fig. 5

(A) Diffusion of SNAP from 10 wt% SNAP-doped E2As films soaking in 1 mL PBS in the dark, as monitored at 340 nm, at room temperature (RT, 22°C) or 37°C. (B) Comparison of the cumulative SNAP leaching and cumulative NO release (based on NOA-based or SNAP-based NO release data) from the 10 wt% SNAP-doped E2As films soaking in PBS at 37°C in the dark. Nitric oxide release from SNAP-doped E2As films can occur from thermal and/or photochemical decomposition of SNAP within the polymer phase, or from SNAP that leached into the aqueous phase. For the SNAP-doped E2As films, approximately 27% of the total NO release is attributed to the SNAP leaching.

Fig. 6

Stability of 10 wt% SNAP in E2As films stored dry with desiccant under various temperature and light conditions. Films were dissolved in DMAc to rapidly determine the amount of SNAP remaining at various times (compared to the initial level) as monitored at 340 nm by UV-vis. Data is the mean ± SEM (n=3).

Fig. 7

Diagram of the extracorporeal circuit (ECC) tubing coated with 5 wt% SNAP/E2As followed by a top coat of E2As.

Fig. 8

Time-dependent effects of the 5 wt% SNAP/E2As coating on platelet count (e.g. consumption) during the 4 h blood exposure in the rabbit thrombogenicity model. Data is the mean ± SEM (n=4).

Fig. 9

Two-dimensional representation of thrombus formation on the SNAP/E2As and control ECCs after 4 h blood exposure in the rabbit thrombogenicity model, as quantified using ImageJ software from NIH. Data is the mean ± SEM (n=4).