Melissa M. Reynolds

The attenuation of platelet and monocyte activation in a rabbit model of extracorporeal circulation by a nitric oxide releasing polymer

Nitric oxide (NO) has been shown to reduce thrombogenicity by decreasing platelet and monocyte activation by the surface glycoprotein, P-selectin and the integrin, CD11b, respectively. In order to prevent platelet and monocyte activation with exposure to an extracorporeal circulation (ECC), a nitric oxide releasing (NORel) polymeric coating composed of plasticized polyvinyl chloride (PVC) blended with a lipophilic N-diazeniumdiolate was evaluated in a 4 h rabbit thrombogenicity model using flow cytometry. The NORel polymer significantly reduced ECC thrombus formation compared to polymer control after 4 h blood exposure (2.8 +/- 0.7 NORel vs 6.7 +/- 0.4 pixels/cm(2) control). Platelet count (3.4 +/- 0.3 NORel vs 2.3 +/- 0.3 x 10(8)/ml control) and function as measured by aggregometry (71 +/- 3 NORel vs 17 +/- 6% control) were preserved after 4 h exposure in NORel versus control ECC. Plasma fibrinogen levels significantly decreased in both NORel and control groups. Platelet P-selectin mean fluorescence intensity (MFI) as measured by flow cytometry was attenuated after 4 h on ECC to ex vivo collagen stimulation (27 +/- 1 NORel vs 40 +/- 2 MFI control). Monocyte CD11b expression was reduced after 4 h on ECC with NORel polymer (87 +/- 14 NORel vs 162 +/- 30 MFI control). These results suggest that the NORel polymer coatings attenuate the increase in both platelet P-selectin and monocytic CD11b integrin expression in blood exposure to ECCs. These NO-mediated platelet and monocytic changes were shown to improve thromboresistance of these NORel-polymer-coated ECCs for biomedical devices.

Combating medical device fouling.

When interfaced with the biological environment, biomedical devices are prone to surface biofouling due to adhesion of microbial or thrombotic agents as a result of the foreign body response. Surface biofouling of medical devices occurs as a result of nonspecific adhesion of noxious substrates to the surface. Approaches for biofouling-resistant surfaces can be categorized as either the manipulation of surface chemical functionalities or through the incorporation of regulatory biomolecules. This review summarizes current strategies for creating biofouling-resistant surfaces based on surface hydrophilicity and charge, biomolecule functionalization, and drug elution. Reducing the foreign body response and restoring the function of cells around the device minimizes the risk of device rejection and potentially integrates devices with surrounding tissues and fluids. In addition, we discuss the use of peptides and NO as biomolecules that not only inhibit surface fouling, but also promote the integration of medical devices with the biological environment.

Metal Organic Frameworks as Nitric Oxide Catalysts

The use of metal organic frameworks (MOFs) for the catalytic production of nitric oxide (NO) is reported. In this account we demonstrate the use of Cu(3)(BTC)(2) as a catalyst for the generation of NO from the biologically occurring substrate, S-nitrosocysteine (CysNO). The MOF catalyst was evaluated as an NO generator by monitoring the evolution of NO in real time via chemiluminescence. The addition of 2, 10, and 15-fold excess CysNO to MOF-Cu(II) sites and cysteine (CysH) resulted in catalytic turnover of the active sites and nearly 100% theoretical yield of the NO product. Control experiments without the MOF present did not yield appreciable NO generation. In separate studies the MOF was found to be reusable over successive iterations of CysNO additions without loss of activity. Subsequently, the MOF catalyst was confirmed to remain structurally intact by pXRD and ATR-IR following reaction with CysNO and CysH.

Effect of varying nitric oxide release to prevent platelet consumption and preserve platelet function in an in vivo model of extracorporeal circulation.

The gold standard for anticoagulation during extracorporeal circulation (ECC) remains systemic heparinization and the concomitant risk of bleeding in an already critically ill patient could lead to death. Normal endothelium is a unique surface that prevents thrombosis by the release of antiplatelet and antithrombin agents. Nitric oxide (NO) is one of the most potent, reversible antiplatelet agents released from the endothelium. Nitric oxide released from within a polymer matrix has been proven effective for preventing platelet activation and adhesion onto extracorporeal circuits. However, the critical NO release (NO flux) threshold for thrombus prevention during ECC has not yet been determined.1 Using a 4-hour arteriovenous (AV) rabbit model of ECC,2 we sought to find this threshold value for ECC circuits, using an improved NO-releasing coating (Norel-b ). Four groups of animals were tested at variable NO flux levels. Hourly blood samples were obtained for measurement of arterial blood gases, platelet counts, fibrinogen levels and platelet function (via aggregometry). A custom-built AV circuit was constructed with 36 cm of poly(vinyl)chloride (PVC) tubing, a 14 gauge (GA) angiocatheter for arterial access and a modified 10 French (Fr) thoracic catheter for venous access. The Norel-b coating reduced platelet activation and thrombus formation, and preserved platelet function — in all circuits that exhibited an NO flux of 13.65 × 10— 10 mol·cm—2·min—1. These results were significant when compared with the controls. With the Norel-b coating, the NO flux from the extracorporeal circuit surface can be precisely controlled by the composition of the polymer coating used, and such coatings are shown to prevent platelet consumption and thrombus formation while preserving platelet function in the animal. Perfusion (2007) 22, 193—200.

Fabrication of biodegradable polymeric nanofibers with covalently attached NO donors.

Many common wound healing aids are created from biodegradable polymeric materials. Often, these materials are unable to induce complete healing in wounds because of their failure to prevent infection and promote cell growth. This study reports the development of therapeutic materials aimed at overcoming these limitations through the release of a naturally occurring antimicrobial agent from a porous, polymeric fiber scaffold. The antimicrobial character was achieved through the release of nitric oxide (NO) while the porous structure was fabricated through electrospinning polymers into nanofibers. Three variations of the polymer poly(lactic-co-glycolic-co-hydroxymethyl propionic acid) (PLGH) modified to include thiol and NO groups were investigated. Fibers of the modified polymers exhibited smooth, bead free morphologies with diameters averaging between 200 and 410 nm. These fibers were deposited in a random manner to create a highly porous fibrous scaffold. The fibers were found to release NO under physiological pH and temperature and have the capacity to release 0.026 to 0.280 mmol NO g(-1). The materials maintained their fibrous morphological structure after this exposure to aqueous conditions. The sustained morphological stability of the fiber structure coupled to their extended NO release gives these materials great potential for use in wound healing materials.

Metal ion-mediated nitric oxide generation from polyurethanes via covalently linked copper(II)-cyclen moieties

Polyurethanes are widely used in the manufacturing of biomedical catheters and other blood-contacting devices; however, thrombus formation still occurs, which renders these catheters ineffective unless systemic anticlotting agents are used. Nitric oxide (NO) is a well-known inhibitor of platelet activity. In the current study, two commercially available medical polyurethanes (Pellethane and Tecophilic) were derivatized to possess NO-generating Cu(II)-cyclen moieties pendant to the polymer backbone. A new three-step synthetic approach is used, that is simpler than a recently reported method to prepare Cu(II)-cyclen-polyurethane materials. Both derivatized polyurethanes were found to produce NO at levels at or above those of endothelial cells. A comparison between the modified commercial polyurethanes (hydrophobic vs. hydrophilic) is presented, including the synthetic scheme, extensive characterization, and coating application. These derivatized polymers may serve as useful coatings to prevent clotting on the surface of catheters and other blood-contacting biomedical devices.

Immobilization of Metal–Organic Framework Copper(II) Benzene-1,3,5-tricarboxylate (CuBTC) onto Cotton Fabric as a Nitric Oxide Release Catalyst

Immobilization of metal-organic frameworks (MOFs) onto flexible polymeric substrates as secondary supports expands the versatility of MOFs for surface coatings for the development of functional materials. In this work, we demonstrate the deposition of copper(II) benzene-1,3,5-tricarboxylate (CuBTC) crystals directly onto the surface of carboxyl-functionalized cotton capable of generating the therapeutic bioagent nitric oxide (NO) from endogenous sources. Characterization of the CuBTC-cotton material by XRD, ATR-IR, and UV-vis indicate that CuBTC is successfully immobilized on the cotton fabric. In addition, SEM imaging reveals excellent surface coverage with well-defined CuBTC crystals. Subsequently, the CuBTC-cotton material was evaluated as a supported heterogeneous catalyst for the generation of NO using S-nitrosocysteamine as the substrate. The resulting reactivity is consistent with the activity observed for unsupported CuBTC particles. Overall, this work demonstrates deposition of MOFs onto a flexible polymeric material with excellent coverage as well as catalytic NO release from S-nitrosocysteamine at therapeutic levels.

Enzymatically degradable nitric oxide releasing S-nitrosated dextran thiomers for biomedical applications

Herein we report the development and evaluation of enzymatically degradable nitric oxide (NO) releasing S-nitrosated dextran thiomers as potent biomedical materials. These materials are characterized by their specificity to release NO under arterial blood conditions, followed by their susceptibility to undergo enzymatic degradation by dextranase. The very specific conjugation chemistries we employed for the thiol incorporation resulted in characteristic stabilization of the resulting S-nitrosothiol moieties, and consequently yielded stable pro-drugs for the storage and controlled delivery of NO. We evaluated the extent of NO loading and release kinetics using multiple and independent analytical techniques, and related these to the structure and environment associated with the thiol moiety incorporated onto the dextran backbone. Finally, the enzymatic degradation kinetics was followed by monitoring the molecular weight profile using gel permeation chromatography, and the results were interpreted using well-established model predictions.

S-Nitrosated biodegradable polymers for biomedical applications: synthesis, characterization and impact of thiol structure on the physicochemical properties

A new class of nitric oxide (NO)-releasing biodegradable polymers has been synthesized by derivatizing poly(lactic-co-glycolic-co-hydroxymethyl propionic acid) (PLGH) polymers with structurally unique thiol functionalities followed by nitrosation with t-butyl nitrite to yield pendant S-nitrosothiol moieties. The extent of thiolation was found to be dependent on the thiol moiety itself with the efficiency of incorporation as follows: cysteamine > cysteine > homocysteine. Glutathione and penicillamine were not incorporated to any significant extent. The structure and polymer environment associated with the pendant thiol has been related to the physicochemical properties of the resulting polymers. To quantify the extent of S-nitrosation, chemiluminescence and UV-visible spectroscopy techniques were employed in combination. The cysteamine and homocysteine derivatives were found to have the highest extent of nitrosation at 93 ± 3% and 96 ± 3%, respectively, followed by 43 ± 1% for cysteine. Thermal decomposition led to near-complete recovery of NO based upon the quantification of the RSNO formation for each nitrosated polymer. Our ability to exert control over the thiol structure, extent of incorporation and the subsequent nitrosation is crucial to the resulting range of NO release kinetics that were yielded. The functional utility of these materials is demonstrated in that these non-toxic polymers release NO under physiological conditions, have degradation profiles that are appropriate for tissue scaffolds and can be prepared as electrospun nanofibers, commonly used in tissue and bone regeneration applications.

The artificial endothelium

As the world of critical care medicine advances, extracorporeal therapies (ECC) have become commonplace in the management of the high risk intensive care patient. ECC encompasses a wide variety of technologies from hemodialysis, continuous renal replacement therapy (CRRT) and plasmapheresis, to cardiopulmonary bypass (CPB), extracorporeal life support (ECLS) and hepatic support. The development of internal man made organs is the next step with ventricular assist devices and artificial lungs. As we advance the technologies with smaller devices, and more intricate circuitry, we lack the keystone necessary to control the blood-biomaterial interface. For the last 50 years much has been learned about surface induced thrombosis and attempts have been made to prevent it with alternative systemic anticoagulation, circuitry surface modifications, or a combination of both. Despite these efforts, systemic or regional anticoagulation remain necessary for both laboratory and clinical application of ECC. As such, the development of an endothelial-like, biomimetic surface to reduce or perhaps even eliminate the blood activation/thrombus formation events that occur upon exposure to artificial surfaces is paramount.