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Portable Nitric Oxide (NO) Generator Based on Electrochemical Reduction of Nitrite for Potential Applications in Inhaled NO Therapy and Cardiopulmonary Bypass Surgery Yu Qin, Joanna Zajda, Elizabeth J. Brisbois, Hang Ren, John Toomasian, Terry Major, Alvaro Rojas-Pena, Benjamin Carr, Thomas Johnson, Jonathan Haft, Robert H. Bartlett, Andrew Hunt, Nicolai Lehnert, and Mark E. Meyerhoff Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00514 • Publication Date (Web): 11 Oct 2017 Downloaded from http://pubs.acs.org on October 13, 2017

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Molecular Pharmaceutics

Portable Nitric Oxide (NO) Generator Based on Electrochemical Reduction of Nitrite for Potential Applications

in

Inhaled

NO

Therapy

and

Cardiopulmonary Bypass Surgery a

a

a

a

b

b

Yu Qin , Joanna Zajda , Elizabeth J. Brisbois , Hang Ren , John Toomasian , Terry Major , b

b

b

b

b

Alvaro Rojas-Pena , Benjamin Carr , Thomas Johnson , Jonathan Haft , Robert H. Bartlett , a

a

Andrew Hunt , Nicolai Lehnert , and Mark E. Meyerhoff

a

Department of Chemistry and Department of Surgery

b

a*

, University of Michigan, Ann Arbor MI

48109 USA

KEYWORDS: nitric oxide, electrochemical reduction, copper(II)-ligand electron transfer mediator, inhaled nitric oxide therapy

ACS Paragon Plus Environment

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ABSTRACT. A new portable gas phase nitric oxide (NO) generator is described for potential applications in inhaled NO (INO) therapy and during cardiopulmonary bypass (CPB) surgery. In this system, NO is produced at the surface of a large area mesh working electrode by electrochemical reduction of nitrite ions in the presence of a soluble copper(II)-ligand electron transfer mediator complex. The NO generated is then transported into gas phase by either direct purging with nitrogen or via circulating the electrolyte/nitrite solution through a gas extraction silicone fiber-based membrane-dialyzer assembly. Gas phase NO concentrations can be tuned in the range of 5 - > 1,000 ppm (parts per million by volume for gaseous species), in proportion to a constant cathodic current applied between the working and counter electrodes. This new NO generation process has the advantages of rapid production times (5 min to steady-state), high Faraday NO production efficiency (ca. 93%), excellent stability, and very low cost when using air as the carrier gas for NO (in the membrane dialyzer configuration), enabling the development of potentially portable INO devices. In this initial work, the new system is examined for the effectiveness of gaseous NO to reduce the systemic inflammatory response (SIR) during CPB, where 500 ppm of NO added to the sweep gas of the oxygenator or to the cardiotomy suction air in a CPB system is shown to prevent activation of white blood cells (granulocytes and monocytes) during extracorporeal circulation with cardiotomy suction conducted with five pigs.


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INTRODUCTION Nitric oxide (NO) is endogenously produced and has important physiological functions that include: increasing vasodilation, preventing platelet adhesion/activation, promoting wound healing and angiogenesis, and serving as a potent antimicrobial agent released by macrophages and nasal epithelial cells to fight infection.1-8 Direct inhalation of nitric oxide (INO) therapy is a treatment approved by the US Food and Drug Administration (FDA) for persistent pulmonary hypertension of newborn babies (PPHN) 9,10 and has been demonstrated to improve oxygenation and reduce the need for higher-risk extracorporeal membrane oxygenation (ECMO) therapy.11 INO not only induces preferential pulmonary vasodilation and lowered pulmonary vascular resistance, but also has a beneficial effect on treatment of other illnesses including pneumonia,12 stroke,13 and acute respiratory distress syndrome (ARDS).14 Recent studies have reported the use of INO as an inhaled antiseptic agent in the treatment of cystic fibrosis15 and tuberculosis,16 and as an anti-inflammatory agent to modulate immune response and promote survival in patients with malaria,17 INO has also been demonstrated to provide neuroprotection and reduce brain damage.18 One other significant area for potential clinical use of gas phase NO is in the sweep gas of oxygenators and within the cardiotomy suction air used in cardiopulmonary bypass (CPB) surgery extracorporeal circuits. CPB causes in some patients a severe systemic inflammatory response (SIR) that is associated with multiple organ failure (MOF), where the severity is related to the length of CPB.19 It’s known that SIR is stimulated by blood-surface interactions and include leukocyte and platelet activation with release of cytokines.20 Further, in vitro studies have shown that the inflammatory response and associated hemolysis during cardiotomy suction is related to the air exposure in that portion of the circuit and that leukocytes are preconditioned


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by air exposure, priming them for adhesion and activation on the circuit’s surfaces.21 The antiinflammatory properties of NO may also prove beneficial for reducing such complications with these procedures. At present, INO and other therapeutic biomedical applications of NO requires the use of a NO gas cylinder and a complex delivery device to regulate and monitor NO concentrations. Hence, the use of NO is considered one of the most expensive drugs in neonatal medicine since INO has a cost of ca. $3,000 per day per patient,22,23 but is still considered cost effective in terms of decreasing the need for ECMO and being essential to prevent death of some neonates.24 However, NO pressurized in a conventional gas cylinder can undergo a disproportionation reaction to form N2O and highly toxic NO2, limiting the use-life of a gas cylinders for medical applications that contain NO at levels > 800 ppm.25 Therefore, NO gas cylinders at ≤800 ppm levels are required for current INO distribution systems and these cylinders are heavy, cumbersome, and very expensive. Thus, there is a great need to develop an inexpensive yet portable source of relatively pure NO for use in INO and other biomedical applications to make use of gas phase NO more available to a greater number of patients, facilitating clinical trials of different gas phase NO therapies on more diseases, and ultimately making INO available for home use (e.g., for cystic fibrosis patients) and in remote areas. Alternate gas phase NO generation techniques have already been proposed. These include catalytic conversion of liquid NO2/N2O4 into NO26 and generation of NO from air via pulsed electrical discharges.27 However, in these techniques a large amount of highly toxic NO2 needs to be used as the starting material or is produced as byproducts, which induces significant safety concerns.28


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In earlier work reported by our group, a controllable and inexpensive method was developed to generate NO by electrochemical reduction of nitrite using a copper(II)-tri(2-pyridylmethyl)amine (Cu(II)TPMA) complex as a mediator, and this approach was applied to develop intravascular (IV) catheters and sensors that emit low levels of NO to prevent clotting and infection.29,30 We also demonstrated the temporal profile of NO generation in the gas phase in the 0-400 ppb range using a nitrogen purge of 0.2 L/min in a beaker of nitrite ions with added Cu(II)TPMA mediator, and the gas phase level could be modulated readily by applying different cathodic potentials to the working electrode. In this present work, the generation of gas phase NO at concentrations that are much more relevant for INO therapy and the oxygenator/cardiotomy suction air application, up to 500 ppm and at higher flow rates from 0.2–5 L/min, is explored using two different designs of the new electrochemical NO generation system in combination with a new Cu(II)-ligand complex that helps produce NO more efficiently than Cu(II)TPMA.

EXPERIMENTAL SECTION Sodium nitrite (99.99%), N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES) acid (99.5%), HEPES sodium salt (99.5%), copper(II) sulfate pentahydrate (99.999%), and 1,4,7trimethyl-1,4,7-triazacyclononane (Me3TACN) (97%) were purchased from Sigma-Aldrich (St. Louis, MO) and used as received. All aqueous solutions were prepared with deionized water (18 MΩ cm-1) from a Milli-Q system (Millipore Corp., Billerica, MA). Gold (Au) mesh electrodes (99.99%, 52 mesh, 5 cm × 10 cm) and Platinum (Pt) mesh electrodes (99.99%, 52 mesh, 5 cm × 5 cm) were purchased from Alfa Aesar (Ward Hill, MA). Teflon® PFA-coated platinum wires (0.125 mm OD) from A-M Systems (Sequim, WA) were used as lead wires for the mesh


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electrodes. Nitric oxide standard gas cylinders (45 ppm) were products from Cryogenic Gas Inc. (Detroit, MI). A continuous micro liquid pump TCS M200 S and a micro air pump TCS D3K were purchased from Servoflo Corp. (Lexington, MA).

The gas permeable membrane modules

PermSelect® PDMSXA-2500 were products of Medarray (Ann Arbor, MI). All the electrochemical experiments were performed on a Gamry 600 potentiostat (Warminster, PA). The generation of NO was measured in real time via chemiluminescence using a Sievers Nitric Oxide Analyzer (NOA 280i) from GE Analytics (Boulder, CO), a CLD 822 CM NO/NOx analyzer from ECO Physics, Inc. (Ann Arbor, MI) or a lab made gas phase amperometric NO sensor.31 Preparation of the copper catalyst Copper(II) sulfate pentahydrate (7 mM) and Me3TACN (7 mM) were added to a solution containing 0.5 M HEPES buffer (pH 7.3) with 1 M NaNO2, the solution was used directly for NO generation and testing. For characterization purposes, Cu(II)Me3TACN was isolated as a solid by the following procedure. Copper(II) sulfate pentahydrate (113.71 mg, 0.455 mmol) and Me3TACN (78.0 mg, 0.455 mmol) were dissolved in 10 mL methanol and stirred for 1 h, resulting in an intense blue colored solution. All solvent was then removed via reduced pressure. The resulting blue solid was recrystallized by dissolving in a minimal amount of methanol, layering with diethyl ether and placing in a freezer overnight. The resulting blue solid was collected via vacuum filtration, washed with additional diethyl ether and dried under vacuum. FT-IR (ATR): 2904, 1647, 1503, 1454, 1302, 1217, 1133, 1079, 1059, 1007, 983, 941, 924, 895, 786, 741, 656 cm-1 (Figure S2).


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UV-Vis (H2O) λmax: 270, 680 nm (Figure S3). EPR (H2O, 30% glycerol; 110 K): g┴ = 2.055, g║ = 2.92, A║ = 463 MHz (Figure S5). Cu(II)Me3TACN NO2− complex: The isolated Cu(II)Me3TACN solid was redissolved in H2O resulting in a bright blue solution and mixed with solid NaNO2 or NaNO2 dissolved in H2O to give the stated concentration, resulting in a green solution that was directly characterized. By spectroscopy: UV-Vis (H2O) λmax: 276, 354 (NaNO2), 652 nm (Figure S4). EPR (H2O, 30% glycerol; 110 K): g┴ = 2.047, g║ = 2.25, A║ = 503 MHz (Figure S6). Electrochemical cell configuration A homemade glass cell with ports for a bubbler and gas outlet was filled with a solution (80 mL) of 7 mM Cu(II)Me3TACN, 1.0 M NaNO2 and 0.5 M HEPES buffer (pH 7.3). A twoelectrode system employing a constant current mode was used for electrochemical reduction of nitrite. A 5 cm x 10 cm Au mesh was used as the working electrode, and a 5 cm x 5 cm Pt mesh served as the counter/reference electrode. Both electrodes were fully submerged in the solution and separated to prevent shorting the circuit. Gas NO generation and measurement In one mode of operation, a fritted glass bubbler was placed in the glass cell containing the solution for supplying a carrier gas (N2 or air) to purge the NO generated on the electrode surface into the gas phase. The flow rate of nitrogen or air was controlled by a digital mass flow controller.

In second design, the nitrite/Cu(II)-complex solution in the glass cell was

continuously pumped through a gas separation silicone fiber dialyzer (PDMSXA-2500) via a micro liquid pump, and circulated back to the glass cell. A nitrogen or air recipient stream was introduced into the dialyzer module through the gas inlet port. The carrier gas sweeps through


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the inside of silicone fibers while the circulating solution from the electrochemical cell flows over the outside of the silicone fibers. The NO in the solution phase was separated and received into the gas phase because of its high permeability through the walls of the silicone fibers. Nitric oxide in the carrier gas (N2 or air) was measured by a chemiluminescence NO analyzer or via an amperometric NO sensor developed in our laboratory.31 Both the CLD NO/NOx and NOA 280i are calibrated with zero NO (N2) and standard 45 ppm NO or 10 ppm NO2 in N2 cylinders. For the Sievers Nitric Oxide Analyzer (NOA 280i), a splitter was used if gas flow rate was higher than 0.2 L/min. To measure NO level with the electrochemical NO sensor, a gas stream was also split from the main stream at a flow rate of 0.05 L/min and flowed over the surface of the sensor, with excess NO at the outlet of the sensor being scavenged by an activated carbon cartridge. Amperometric NO sensor The amperometric Pt-Nafion gas phase nitric oxide sensor was fabricated according to our previous report,31 with minor changes. Briefly, Nafion 117 films (DuPont, Wilmington, DE) was cut into ca. 1.6 cm dia. circles and cleaned of impurities by boiling in 3 M nitric acid for 1 h, and then by boiling in deionized water for 1 h. Platinum was chemically deposited into/onto the solid-polymer electrolyte using the impregnation-reduction method. The Nafion 117 membrane was mounted between two glass cells with 0.92 cm dia. openings (0.66 cm2 apparent geometric area), and one side was exposed to 2 mM Pt(NH3)4Cl2 and incubated for 20 h at 37°C. After that time, impregnation solution was removed and cell was washed with deionized water. Next, 50 mM NaBH4 in 0.1 M NaOH was placed at the same side of the glass cell, and the chemical reduction was allowed to proceed for 1 h at 37°C. Subsequently, the Pt-Nafion membrane was


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boiled in deionized water for 1 h to remove any remaining Pt complexes and reducing agents. The Pt-Nafion membrane was mounted in glass sensor assembly with the metallic side of the membrane electrode facing the gas phase. A 10 mm x 2 mm piece of 50 µm thick Au foil was used as the working electrode lead, and secured between the SPE membrane electrode and the gas inlet/outlet section of the sensor. A single junction Ag/AgCl (saturated KCl) reference electrode and bare Pt auxiliary electrode were placed in the liquid chamber filled with 0.5 M H2SO4 internal electrolyte. A CHI1206B potentiostat (CH Instruments, Austin, TX) was used to apply a potential to the working electrode (1V vs. Ag/AgCl) and record the sensor’s output currents. A MC-200SCCM mass flow controller (Alicat Scientific, Tucson, AZ) was used to deliver calibration gas and the gaseous sample to the gas phase sensor at a constant flow rate. Porcine model of CPB studying the effect of nitric oxide The experimental procedure was performed in an ovine model following protocol approval by the University of Michigan Institutional Animal Care and Use Committee (IACUC). All pigs used for the experiment were treated in compliance with the Guide for Care and Use of Laboratory Animals, 8th edition32.

Pigs were anesthetized, surgically instrumented, and

connected to a CPB circuit. All animals were triaged prior to surgery to have normal preoperative white blood cell count between 14-20 K/uL, if an animal had WBC out of this range; the animal was not used in the study. In addition, per our laboratory protocols, all animals received a prophylactic dose of IV antibiotics (1 g nafcillin and 80 mg of gentamycin) 1 h prior surgical incision.

The blood in the control group (n=5) was not exposed to air since no

cardiotomy suction was applied, while the air-blood interface (ABI) group (n=5) was placed on a three-pump circuit, which exposed the blood to air with cardiotomy suction. The treating group followed the same procedure as the ABI group but with 500 ppm NO present in the sweep gas of


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the oxygenator or added to cardiotomy suction air. The schematics of the in vivo venoarterial (VA)-CPB circuit porcine models are shown in Figure 7, below. Under general anesthesia, a venous cannula was placed to drain blood from the right atrium (RA) and inferior vena cava (IVC) into an open venous reservoir of the heart-lung machine. The venous blood was then passed through a pump and oxygenator (blood flow 1 L/min), and was reinfused through a cannula placed in the ascending aorta via the carotid artery to complete the venoarterial (VA) circuit. In the treatment group, a dose of 500 ppm NO from the proposed Echem NO generator was added to the gas inlet of the oxygenator within the CPB system or directly purged into the blood stream at the cardiotomy suction/air interface. After 2 h on the circuit, the pigs were recovered and monitored for 96 h in order to assess the long-term effects of the blood’s exposure to air and negative pressure as compared to the baseline measurements. After 96 h, the pigs were euthanized and necropsies were performed. Systemic hemodynamics and blood serum samples were collected during the CPB procedure and during the recovery period to assess levels of blood activation and results were analyzed using an unpaired two-tailed Student t-test with a p-value 1,000 ppm) can be generated in the gas phase when N2 is used as the carrier gas. The time for the NO concentration to change from one level to another when changing the applied current is < 5 min, demonstrating a good temporal control of the system (Figure 2A). The fluctuation of the purging gas flow rate may lead to the observed spikes in the time trace, which can be eliminated by using a precise mass flow controller. Under a given purge gas flow rate, the gas phase NO concentration in N2 is proportional to the current applied as shown in Figure 2B, and the associated faradaic efficiency is relatively stable (fluctuation < 10%) for different currents. The electrochemical generation of NO is stable for continuous use for at least 1 week (Figure 2C).

Figure 2. NO data from the gas phase NO generator with direct bubbling of the solution. (A) Time trace of NO level (ppm) vs. applied current; (B) calibration of gas phase NO (ppm) vs. applied current; and (C) example of gas phase NO levels generated over 7 d of continuous generation/ measurement of


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NO from the same solution of nitrite/Cu(II)Me3TACN. Working electrode: 5 cm ×10 cm Au mesh; counter electrode: 5 cm × 5 cm Pt mesh; N2 bubbling rate: 0.2 L/min.

The NO/N2 stream can be mixed with another stream of air or O2 to provide desired NO levels for biomedical applications. As demonstrated in Figure 3, by mixing NO/N2 from the generator with different ratio of N2, air (21% O2), or pure O2, various gas phase NO concentrations can be obtained. The final NO concentration is proportional to the dilution ratio. Using air or pure O2 as diluting gas does not change the NO concentration because of fast mixing and short contact time of NO and O2.

Figure 3. Production of different gas phase NO concentrations (ppm) using different mixing ratios with N2 (green open triangle), air (black open circle) and 100% O2 (red open square). (A) the flow rate of NO/N2 from the generator is 0.1 L/min for set A; (B) flow rate set at 0.2 L/min; and (C) 0.5 L/min. Applied current is 10 mA; working electrode: 5 cm × 10 cm Au mesh; counter electrode: 5 cm × 5 cm Pt mesh. The NO generator system shown in Fig. 1A was used to obtain these data.


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It is desirable to demonstrate that instead of N2, air can also serve as a carrier gas. However, as shown in Fig. 4A, when purging the nitrite/Cu(II)Me3TACN solution with air, the NO measured in the emitted gas phase is decreased more than 80% at an applied current of 5 mA. This is because the electrolyte solution is saturated with O2 and the high concentration of NO generated locally at the surface of the Au electrode can react with the oxygen at an appreciable rate, producing oxidized products. It might also be possible that superoxide can be generated in this case by direct reduction of O2.

Superoxide reacts extremely fast with NO, at a diffusion

controlled rate (k ~ 1010 mol-1s-1).39 Although ppm levels of NO can still be produced with this configuration while the air is used as a purge gas, the levels > 50 ppm cannot be reached. Interestingly, simultaneous measurement of NO and NOx species with the CLD 822 CM NO/NOx analyzer demonstrated that in both conditions NO is still the predominant species in the gas phase. Other nitrogen oxides such as N2O and NO2 are < 1 ppm for NO concentrations from 5 to 500 ppm.

Figure 4. NO concentration comparison between air and N2 as purge gas. A: NO generator using the configuration shown in Fig. 1A; B: NO generator using the configuration in Fig. 1B. Working


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electrode: 5 cm ×10 cm Au mesh; counter electrode: 5 cm × 5 cm Pt mesh; applied current: 5 mA.

Electrochemical NO generator with gas separation silicone fibers In order to generate higher concentrations of pure NO and using air as the carrier gas, the solution in the electrochemical cell was pumped rapidly and continuously into a gas extraction device that contains thousands of silicone hollow fibers (see Figure 1B). By passing a recipient gas (air) on the other side of the silicone fibers, NO is extracted into the gas phase due to its high permeability through the silicone materials,30, 35 while the solution and all nongaseous species are circulated back to the glass electrochemical cell. This design allows for a fast removal of NO from the electrode surface and the solution so that the reaction of NO with oxygen (coming from the air) is minimized. Figure 4 shows that with design A in Figure 1, when N2 is replaced with air as the purge gas, this causes a dramatic decrease of NO production from 350 ppm to 30 ppm (Figure 4A). However, when design B with the silicone fiber-based gas separation module is employed, the NO level decrease in going from nitrogen to air as the recipient gas through the fibers is only from 380 ppm to 190 ppm under identical experimental conditions (Figure 4B). The solution enters the fiber module through the inlet port to the tube side and flows through the outside of the hollow silicone rubber fibers. In the fiber inner side, a sweep/carrier gas flows therein to carry away the permeating NO. The gaseous NO in the solution phase with higher permeability will efficiently transfer at a significant rate across the walls of silicone hollow fibers into the carrier gas. The solution circulation NO generator allows easy and reasonably rapid temporal control of the resulting gas phase NO concentration by adjusting the applied current or the recipient gas flow rate. At a fixed gas flow rate, the NO level can be easily tuned by


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changing the applied current and the resulting NO concentration is proportional to the current as shown in Figure 5B. The time for the NO level to change while applying a different current is less than 5 min (Figure 5A). A long term stability experiment showed that over 24 h a stable NO with high concentrations up to 400 ppm can be produced continuously using the design B.

Figure 5. NO Data from the NO generator with solution circulation (Fig. 1B). , Different currents were applied for the stepwise change; 0.1 L/min and 0.2 L/min air flow rate through fibers were used. (A) Time trace of NO (ppm) vs applied current with different air flow rates; (B) calibration of NO (ppm) vs applied current at 0.1 L/min air flow rate. Working electrode: 5 cm x 10 cm Au mesh; counter electrode: 5 cm x 5 cm Pt mesh.

The purity of NO in the gas phase generated using the design B (Fig. 1B) was investigated with the ECO-Physics CLD 822 CM NO/NOx analyzer by measuring the NO and NO2 levels simultaneously. This analyzer has a highly efficient NOx converter that converts all NOx into NO, and the total NO is measured and compared to the NO level detected in the unmodified stream. The difference is identified with the NO2 level. The ECO-Physics NO2 detection system that we employed to quantitate the gas phase NO2 levels is a widely employed commercial system designed to detect NO2 levels with reasonably good accuracy. Hence, we are confident that the levels we have measured are accurate. The results shown in Figure 6 demonstrate a


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relatively low level of NO2 (< 1.5 ppm) during continuous generation of NO at ca. 60 ppm level with the air as the carrier gas. The NO2 level did not increase when the current was changed to increase the gas phase NO concentration. To validate the experimental data, a bag of 43 ppm NO from an NO cylinder mixing with air for 15 min, was measured with the analyzer at 16 min. Under these conditions 8 ppm of NO2 was produced which corresponds well to the reaction rate of NO with O2 in the air.33

Figure 6. NOx, NO and NO2 measured by the CLD 822 CM NO/NOx analyzer for the design B NO generator. Blue line: total NO (NOx) concentration in ppm after conversion; red line: NO concentration; green line: NO2 concentration by subtracting the NO concentration from the NOx concentration. At 16 min, the sample was switched to a bag of 43 ppm NO from an NO cylinder mixed with air for 15 min. Air flow rate from the gas pump: 1 L/min; the NO generating solution was circulated through a micro liquid pump and a PDMSXA-2500 silicone fiber module was used as gas separation device; applied current: 50 mA and 40 mA; working electrode: 5 cm × 10 cm Au mesh; counter electrode: 5 cm × 5 cm Pt mesh.


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Monitoring NO from the generator with amperometric NO sensor and application for in vivo animal studies The nitric oxide from the electrochemical NO generator was used in extracorporeal circuit procedures involving a porcine model. A constant current of 80 mA was applied in the generator of Design configuration A (Fig 1A) and nitrogen with flow rate of 0.55 L/min was used as the purge gas in the NO generator. This provides 1,000 ppm gaseous NO in N2. NO from the generator (1,000 ppm) was then split into two streams, one with 0.05 L/min NO in N2 was delivered to the amperometric NO sensor for continuous monitoring of NO levels; and the rest of the NO/N2 (0.5 L/min) stream was mixed with 100% O2 in 1:1 ratio immediately before being delivered to the animal to provide the 500 ppm level in a 50% oxygen background (so high oxygen is going into the oxygenator or air portion of the cardiotomy suction unit employed in the animal experiments), so there is very little time for the NO to react with the oxygen. The NO sensor exhibits rapid response to gas phase NO, with linear response from 5 ppb – 1,000 ppm NO and a response time of ~

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