Nitric oxide (NO) releasing catheters to reduce thrombosis and bacterial infection

摘要

Statement of Purpose: Two major clinical problems with implanted catheters are clotting and infection. One approach to improving the hemocompatibility of blood-contacting devices is to develop materials that release nitric oxide (NO), a known potent inhibitor of platelet adhesion/activation and also an antimicrobial agent. Healthy endothelial cells exhibit a NO flux of 0.5-4×10~(-10) mol cm~(-2) min~(-1), and materials that mimic this NO release are expected to have similar anti-thrombotic properties. Diazeniumdiolates have been one of the most widely studied NO donors, which release NO through proton or thermal driven mechanisms. While diazeniumdiolated dibutylhexanediamine (DBHD/N_2O_2) is an excellent donor for incorporation into polymers to create NO release coatings, the loss of NO from this molecule creates free lipophilic amine species within the polymer that react with water, thereby increasing the pH within the organic polymer phase which effectively turns off the NO production before a significant fraction of the total NO payload has been released. This study involves the use of poly- (lactide-co-glycolide) (PLGA) species as additives to help stabilize the pH within the organic phase polymeric coatings. The addition of PLGA can be used to control the flux of NO emitted from polymers containing diazeniumdiolate species by helping to control the pH within the polymer phase. In this study, DBHD/N_2O_2-doped Elast-eon E2As catheters were prepared and implanted in rabbit veins for 9 d to observe effects on thrombus and bacterial adhesion. Methods: Catheters were prepared by dip coating polymer solutions on stainless steel mandrels. The DBHD/N_2O_2-doped E2As catheters had a trilayer configuration consisting of 5 base coats of Elast-eon E2As, 25 active coats of 25 wt% DBHD/N_2O_2 in E2As with 10 wt% PLGA, and 5 top coats of E2As. Control catheters were prepared in a similar manner with E2As only (no DBHD/N_2O_2 added). Catheters were soaked in 10 mM PBS at 37°C. NO release from the catheters under physiological conditions was determined via a chemiluminescence NO analyzer (NOA) (Sievers, 280, Boulder, CO). A long-term (9 d) rabbit model was used to evaluate the effects on dot formation and bacterial infection. Catheter patency was tested each day by drawing blood. Pictures were taken of the whole catheter and the 2D representation of the thrombus area was determined with the NIH ImageJ software. To quantitate the adhered bacteria, 1 cm sections of catheters were homogenized in sterile PBS buffer, serially diluted in PBS, and plated on agar plates. The agar plates were incubated at 37°C for 24 h to determine the number of colony forming units per catheter surface area (CFU/cm~2). Results: The DBHD/N_2O_2-doped Elast-eon E2As polymer creates catheters that can release physiologically relevant levels of NO for up to 14 d. The DBHD/N_2O_2-doped catheters remain patent during the 9 d implantation, while E2As control catheters dot after 24 h. After 9 d implantation in rabbit veins, catheters were explanted. The explanted SNAP-doped catheters had a flux of ～3×10-10 mol cm-2 min-1. DBHD/N_2O_2-doped catheters have significantly reduced thrombus area in comparison to E2As controls (see Fig. 1). The DBHD/N_2O_2-doped catheters have -90% less bacterial adhesion in comparison to E2As controls. Conclusions: The NO release from the DBHD/N_2O_2-doped catheters exhibits both antithrombotic and antimicrobial properties. After 9 d implantation in rabbit veins, the DBHD/N_2O_2-doped catheters have significantly reduced thrombus area and bacterial adhesion, in comparison to the E2As control catheters, demonstrating its potential for improving the hemocompatibility of blood-contacting devices.