Nitric Oxide-Generating Silicone as a Blood-Contacting Biomaterial

Coagulation upon blood-contacting biomaterials remains a problem for short and long-term clinical applications. This study examined the ability of copper(II)-doped silicone surfaces to generate nitric oxide (NO) and locally inhibit coagulation. Silicone was doped with 3-micron copper (Cu(0)) particles yielding 3 to 10 weight percent (wt%) Cu in 70-μm thick Cu/Silicone polymeric matrix composites (Cu/Si PMCs). At 3, 5, 8 and 10 wt% Cu doping, the surface expression of Cu was 12.1 ± 2.8%, 19.7 ± 5.4%, 29.0 ± 3.8%, and 33.8 ± 6.5% respectively. After oxidizing Cu(0) to Cu(II) by spontaneous corrosion, NO flux, JNO (mol*cm−2*min−1), as measured by chemiluminescence, increased with surface Cu expression according to the relationship JNO =(1.63 %SACu −0.81) ×10−11, R2 = 0.98 where %SACu is the percentage of surface occupied by Cu. NO flux at 10 wt% Cu was 5.35± 0.74 ×10−10 mol*cm−2*min−1. The clotting time of sheep blood exposed to these surfaces was 80 ± 13s with pure silicone and 339 ± 44s when 10 wt% Cu(II) was added. SEMs of coatings showed clots occurred away from exposed Cu-dendrites. In conclusion, Cu/Si PMCs inhibit coagulation in a dose-dependent fashion related to the extent of copper exposure on the coated surface

In Vitro Cytocompatibility of Antibacterial Levels of Polymer Nitric Oxide Release

Although the antibacterial property of nitric oxide (NO) has been well documented in gram positive and gram negative bacteria cultures, its cytotoxic effects are not completely clear. To limit potential in vivo cytotoxicity, our group recently investigated the effects of a range of NO fluxes on S. epidermidis and S. aureus to determine a minimum effective NO level. In this study, we report the antibacterial function of this minimum NO level also on gram negative Pseudomonas aeruginosa as well as its cytocompatibility effects on lung and kidney cells. Standardized bacterial cultures were treated with NO releasing PDMS substrates followed by plating, 24hr incubation, and colony analyses. Cytocompatibility or cell viability was conducted on WI-38 human lung fibroblasts and HEK-293 human embryonic kidney cells after their exposure to NO in vitro. NO flux of 21.18 ± 5.31 × 10−10 mol/ min/ cm2 significantly reduced P. aeruginosa growth compared to controls and PDMS-treated samples (p value \u3c 0.0001). No significant differences was seen between control and cells treated at this flux (total moles delivered in 24hrs = 0.76 ± 0.18), but a significant reduction was observed at 45.1 ± 2.55 ×10−10 mol/ min/ cm2 (total moles delivered = 1.6 ± 0.09). The results suggest that at the lower NO flux level pseudomonas aeruginosa growth is significantly inhibited while maintaining cell viability

Nitric Oxide Therapies for Local Inhibition of Platelets' Activation on Blood-Contacting Surfaces.

Blood-contacting devices interact with blood during their function much like the endothelium that modulates hemostasis. The surfaces of these devices however, lack endothelial-like properties, and consequently, upon blood contact, activate clotting factors to form clots. Systemic heparinization for inhibiting clot formation can cause bleeding and surface coatings show insignificant benefits. This research investigated nitric oxide (NO) production mimicry of the endothelium on artificial lungs (ALs) and pediatric catheters. Their surfaces were functionalized either by (1) entrapping NO donors inside their bulk, (2) incorporating catalysts to generate NO from NO-donors or (3) supplementing NO into sweep gas of artificial lungs. Pediatric catheters functionalized with NO-donor thin coats using method 1 is limited by short NO release duration. Method 2 has not been applied to large surface-area, low-flow devices like the AL. In this work NO-generating silicone membranes were synthesized and characterized to determine the relationship between surface properties, NO flux, and blood clotting time. These outcomes helped develop and optimize NO-generating gas-exchange silicone fibers that represent the majority of ALs surface area. The first NO-generating AL prototypes, using those fibers, were manufactured, incorporated into NO-generating circuits and tested for their non-thrombogenicity. To test for NO-release duration and non-thrombogenicity, catheters were fabricated to incorporate NO-donors inside their walls, characterized for NO flux and release duration by chemiluminescence, and tested for patency using a thrombogenicity model in rabbits. Methods 1-2 involve material modification using complicated and expensive chemical formulations and/or manufacturing. Method 3 however, functionalizes ALs by only adding NO into sweep gas. Decade-long anti-clotting testing using a wide range of NO concentrations has been conducted without knowledge of what concentration yields endothelial NO flux levels in the AL. This concentration was determined for the MC3 Biolung and the Terumo capiox rx25 ALs in vitro. All these ideas have shown positive results in short-term studies, and each may play a necessary role in inhibiting clot formation in future ALs. The sufficiency however, of each idea or of a combination for clot inhibition in long-term ALs remains to be determined.Ph.D.Biomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/89732/1/kagyaa_1.pd

Achieving Totally Local Anticoagulation on Blood Contacting Devices

The recent years have witnessed an increased activity in biocompatibility research aimed at limiting biomaterial-induced blood coagulation. From 2008 to 2016, a total of $36,946,764.00 USD was awarded in grants to 213 research proposals and as large as 50.4$18,627,854.00) of that award monies were distributed to 101 proposals over the fiscal years of FY14 to FY16 alone. However, the complexity in blood responses to biomaterials, variability in blood function between individuals and animal species, and differences in medical device application and test setting all continue to pose difficulties in making a breakthrough in this field. This review focuses on the remaining challenges in the context of biomaterial surface interaction with blood, biomaterial properties and their influence on coagulation, old and new surface anticoagulation methods, main test systems (complement and platelet function) for evaluating those methods, limitations of modification techniques, and the current state of systemic anticoagulation usage as adjunctive therapy for controlling blood coagulation on biomaterials. Finally, we propose ingredients necessary for advancing the field towards achieving totally local surface anticoagulation on blood contacting devices including standardization of in vitro and in-vivo test methods. Some highlights of recent forward-looking work and articles on local anticoagulation are also presented

Multi-Modal, Surface-Focused Anticoagulation Using Poly-2-methoxyethylacrylate Polymer Grafts and Surface Nitric Oxide Release

This study examines platelet adhesion on surfaces that combine coatings to limit protein adsorption along with “anti-platelet” nitric oxide (NO) release. Uncoated and poly-2-methoxyethylacrylate (PMEA) coated, gas permeable polypropylene (PP) membranes were placed in a bioreactor to separate plasma and gas flows. Nitrogen with 100/500/1000 ppm of NO was supplied to the gas side as a proof of concept. On the plasma side, platelet rich plasma (PRP, 1 × 108 cell/mL) was recirculated at low (60)/high (300) flows (mL/min). After 8 hours, adsorbed platelets on PP was quantified via a lactate dehydrogenase assay. Compared to plain PP, the PMEA coating alone reduced adsorption by 17.4 ± 9.2% and 29.6 ± 16.6% at low and high flow (p \u3c 0.05), respectively. NO was more effective at low plasma flow. At 100 and 500 ppm of NO, adsorption fell by 37.9 ± 6.1% and 100 ± 4.7%, (p \u3c 0.001), on plain PP. At high flow with 100, 500, and 1000 ppm of NO, adsorption reduced by 17.9 ± 17.8%, 46.4 ± 23.2%, and 100 ± 4.8%, (p \u3c 0.001), respectively. On PMEA-coated PP with only 100 ppm, adsorption fell by 69.7 ± 6.8 and 65.6% ± 16.9%, (p \u3c 0.001), at low and high flows respectively. Therefore, the combination of an anti-adsorptive coating with NO has great potential to reduce platelet adhesion and coagulation at biomaterial surfaces

Evaluating the Effect of Shear Stress on Graft-To Zwitterionic Polycarboxybetaine Coating Stability Using a Flow Cell

The effect of surface coatings on the performance of antifouling activity under flow can be influenced by the flow/coating interactions. This study evaluates the effect of surface coatings on antifouling activity under different flows for the analyses of coating stability. This was done by exposing DOPA-PCB-300/dopamine coated polydimethylsiloxane (PDMS) to physiological shear stresses using a recirculation system which consisted of dual chamber acrylic flow cells, tygon tubing, flow probe and meter, and perfusion pumps. The effect of shear stress induced by phosphate buffered saline flow on coating stability was characterized with differences in fibrinogen adsorption between control (coated PDMS not loaded with shear stress) and coated samples loaded with various shear stresses. Fibrinogen adsorption data showed that relative adsorption on coated PDMS that were not exposed to shear (5.73% ± 1.97%) was significantly lower than uncoated PDMS (100%, p \u3c 0.001). Furthermore, this fouling level, although lower, was not significantly different from coated PDMS membranes that were exposed to 1 dyn/cm2 (9.55% ± 0.09%, p = 0.23), 6 dyn/cm2 (15.92% ± 10.88%, p = 0.14), and 10 dyn/cm2 (21.62% ± 13.68%, p = 0.08). Our results show that DOPA-PCB-300/dopamine coatings are stable, with minimal erosion, under shear stresses tested. The techniques from this fundamental study may be used to determine the limits of stability of coatings in long-term experiments

Hemocompatibility Comparison of Biomedical Grade Polymers Using Rabbit Thrombogenicity Model for Preparing Nonthrombogenic Nitric Oxide Releasing Surfaces

Nitric oxide (NO) is an endogenous vasodilator as well as natural inhibitor of platelet adhesion/ activation. Nitric oxide releasing (NOrel) materials can be prepared by doping an NO donor species, such as diazeniumdiolated dibutylhexanediamine (DBHD/N2O2), within a polymer coating. The inherent hemocompatibility properties of the base polymer can also influence the efficiency of such NO release coatings. In this study, four biomedical grade polymers were evaluated in a 4 h rabbit model of thrombogenicity for their effects on extracorporeal circuit thrombus formation and circulating platelet count. At the end of 4 h, Elast-Eon E2As was found to preserve 58% of baseline platelets versus 48, 40, and 47% for PVC/DOS, Tecophilic SP-60D-60, and Tecoflex SG80A, respectively. Elast-Eon also had significantly lower clot area of 5.2 cm2 compared to 6.7, 6.1, and 6.9 cm2 for PVC/DOS, SP-60D-60, and SG80A, respectively. Based on the results obtained for the base polymer comparison study, DBHD/N2O2-doped E2As was evaluated in short-term (4 h) rabbit studies to observe the NO effects on prevention of clotting and preservation of platelet function. Platelet preservation for this optimal NO release formulation was 97% of baseline after 4 h, and clot area was 0.9 cm2 compared to 5.2 cm2 for controls, demonstrating that combining E2As with NO release provides a truly advanced hemocompatible polymer coating for extracorporeal circuits and potentially other blood contacting applications

Thromboresistance Characterization of Extruded Nitric Oxide Releasing Silicone Catheters

Intravascular catheters used in clinical practice can activate platelets, leading to thrombus formation and stagnation of blood flow. Nitric oxide (NO)-releasing polymers have been shown previously to reduce clot formation on a number of blood contacting devices. In this work, trilaminar NO-releasing silicone catheters were fabricated and tested for their thrombogenicity. All catheters had specifications of L = 6 cm, inner diameter = 21 gauge (0.0723 cm), outer diameter = 12 gauge (0.2052 cm), and NO-releasing layer thickness = 200 ± 11 µm. Control and NO-releasing catheters were characterized in vitro for their NO flux and NO release duration by gas phase chemiluminescence measurements. The catheters were then implanted in the right and left internal jugular veins of (N = 6 and average weight = 3 kg) adult male rabbits for 4 hours thrombogenicity testing. Platelet counts and function, methemoglobin (metHb), hemoglobin (Hb), and white cell counts and functional time (defined as patency time of catheter) were monitored as measured outcomes. Nitric oxide-releasing catheters (N = 6) maintained an average flux above (2 ± 0.5) × 10−10 mol/min/cm2 for more than 24 hours, whereas controls showed no NO release. Methemoglobin, Hb, white cell, and platelet counts and platelet function at 4 hours were not significantly different from baseline (α = 0.05). However, clots on controls were visibly larger and prevented blood draws at a significantly (p \u3c 0.05) earlier time (2.3 ± 0.7 hours) into the experiment, whereas all NO-releasing catheters survived the entire 4 hours test period. Results indicate that catheter NO flux levels attenuated thrombus formation in a short-term animal model

In Vitro and in Vivo Study of Sustained Nitric Oxide Release Coating Using Diazeniumdiolate-doped Poly(vinyl chloride) Matrix with Poly(lactide-co-glycolide) Additive

Nitric oxide (NO) is an endogenous vasodilator as well as natural inhibitor of platelet adhesion and activation that can be released from a NO donor species, such as diazeniumdiolated dibutylhexanediamine (DBHD/N2O2) within a polymer coating. In this study, various Food and Drug Administration approved poly(lactic-co-glycolic acid) (PLGA) species were evaluated as additives to promote a prolonged NO release from DBHD/N2O2 within a plasticized poly(vinyl chloride) (PVC) matrix. When using an ester-capped PLGA additive with a slow hydrolysis time, the resulting coatings continuously release between 7 and 18 × 10−10 mol cm−2 min−1 NO for 14 days at 37 °C in PBS buffer. The corresponding pH changes within the polymer films were visualized using pH sensitive indicators and are shown to correlate with the extended NO release pattern. The optimal combined diazeniumdiolate/PLGA-doped NO release (NOrel) PVC coating was evaluated in vitro and its effect on the hemodynamics was also studied within a 4 h in vivo extracorporeal circulation (ECC) rabbit model of thrombogenicity. Four out of 7 control circuits clotted within 3 h, whereas all the NOrel coated circuits were patent after 4 h. Platelet counts on the NOrel ECC were preserved (79 ± 11% compared to 54 ± 6% controls). The NOrel coatings showed a significant decrease in the thrombus area as compared to the controls. Results suggest that by using ester-capped PLGAs as additives to a conventional plasticized PVC material containing lipophilic diazeniumdiolates, the NO release can be prolonged for up to 2 weeks by controlling the pH within the organic phase of the coating

The Hemocompatibility of a Nitric Oxide Generating Polymer that Catalyzes S-nitrosothiol Decomposition in an Extracorporeal Circulation Model

Nitric oxide (NO) generating (NOGen) materials have been shown previously to create localized increases in NO concentration by the catalytic decomposition of blood S-nitrosothiols (RSNO) via copper (Cu)-containing polymer coatings and may improve extracorporeal circulation (ECC) hemocompatibility. In this work, a NOGen polymeric coating composed of a Cuo-nanoparticle (80 nm)-containing hydrophilic polyurethane (SP-60D-60) combined with the intravenous infusion of an RSNO, S- nitroso-N-acetylpenicillamine (SNAP), is evaluated in a 4 h rabbit thrombogenicity model and the anti-thrombotic mechanism is investigated. Polymer films containing 10 wt.% Cuo-nanoparticles coated on the inner walls of ECC circuits are employed concomitantly with systemic SNAP administration (0.1182 μmol/kg/min) to yield significantly reduced ECC thrombus formation compared to polymer control + systemic SNAP or 10 wt.% Cu NOGen + systemic saline after 4 h blood exposure (0.4 ± 0.2 NOGen/SNAP vs 4.9 ± 0.5 control/SNAP or 3.2 ± 0.2 pixels/cm2 NOGen/saline). Platelet count (3.9 ± 0.7 NOGen/SNAP vs 1.8 ± 0.1 control/SNAP or 3.0 ± 0.2 × 108/ml NOGen/saline) and plasma fibrinogen levels were preserved after 4 h blood exposure with the NOGen/SNAP combination vs either the control/SNAP or the NOGen/saline groups. Platelet function as measured by aggregometry (51 ± 9 NOGen/SNAP vs 49 ± 3% NOGen/saline) significantly decreased in both the NOGen/SNAP and NOGen/saline groups while platelet P-selectin mean fluorescence intensity (MFI) as measured by flow cytometry was not decreased after 4 h on ECC to ex vivo collagen stimulation (26 ± 2 NOGen/SNAP vs 29 ± 1 MFI baseline). Western blotting showed that fibrinogen activation as assessed by Aγ dimer expression was reduced after 4 h on ECC with NOGen/SNAP (68 ± 7 vs 83 ± 3% control/SNAP). These results suggest that the NOGen polymer coating combined with SNAP infusion preserves platelets in blood exposure to ECCs by attenuating activated fibrinogen and preventing platelet aggregation. These NO-mediated platelet changes were shown to improve thromboresistance of the NOGen polymer-coated ECCs when adequate levels of RSNOs are present