Novel Devices for the Predictable Delivery of Nitric Oxide to Aqueous

Two novel devices were developed to deliver controlled and predictable amounts of NO to aqueous solutions for studying the effects of NO on biological...
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Chem. Res. Toxicol. 1998, 11, 1346-1351

Novel Devices for the Predictable Delivery of Nitric Oxide to Aqueous Solutions Mahendra Kavdia, Shravan Nagarajan, and Randy S. Lewis* School of Chemical Engineering, Oklahoma State University, Stillwater, Oklahoma 74078 Received May 20, 1998

Nitric oxide (NO), a recently discovered biological molecule synthesized by many cells, has many physiological roles including blood pressure regulation, neurotransmission, and inhibition of platelet adhesion. However, NO and reactive species formed in the presence of oxygen and superoxide can also be cytotoxic, mutagenic, or carcinogenic. Two novel devices were developed to deliver controlled and predictable amounts of NO to aqueous solutions for studying the effects of NO on biological systems. The devices contained either a slit or a tube composed of a gas-permeable membrane. Aqueous solution flowed on one side of the membrane while the other side of the membrane was exposed to NO gas. Mathematical models were used to predict the bulk or mixing-cup NO concentration in the aqueous solution at the exit of each delivery device following exposure to NO gas. Model predictions were in good agreement with experimental values at both 25 and 37 °C. One of the delivery devices was also connected to a well-stirred and oxygenated chamber. Model predictions of the chamber NO concentration, which included the reaction of NO with O2, were in excellent agreement with experiments.

Introduction Nitric oxide (NO)1 is synthesized by many cells including macrophages, neutrophils, endothelial cells, and hepatocytes. Important physiological roles of NO include blood pressure regulation, neurotransmission, and inhibition of platelet adhesion (1). However, NO and reactive species formed in the presence of oxygen and superoxide can also be cytotoxic, mutagenic, or carcinogenic (1, 2). In light of both the physiological and pathophysiological actions of NO, controlled and quantitative delivery of NO would be beneficial for studying the effects of NO and its reactive products on biological systems. Current methods for delivering or generating NO in biological systems include the release of NO from NOdonor compounds, the stimulation of NO synthase enzymes in cellular systems, the addition of NO-saturated solutions, and the permeation of NO through polymeric membranes (3-8). The majority of research investigating the role of NO on biological systems utilizes the first three methods. An advantage of using NO-donor compounds is that the NO-release rates can be modified based upon the structure of the donor compound (3, 8). However, light, pH, and the aqueous medium can affect the NO-release rate. In addition, the nonconstant release rate of NO and the reactivity and/or toxicity of the NO-donor compound following the release of NO can be problematic (8, 9). Stimulation of NOS to produce NO is a viable approach for in vivo studies and for producing a near-constant release of NO. However, other species generated or * Address correspondence to this author at the School of Chemical Engineering, 423 EN, Oklahoma State University, Stillwater, OK 74078. Telephone: (405) 744-5280. Fax: (405) 744-6338. E-mail: [email protected]. 1 Abbreviations: NO, nitric oxide; sccm, standard cubic centimeters per minute.

existing within a cell, such as superoxide, can react either intracellularly or extracellularly with NO to form other potentially damaging species. Addition of NO-saturated solutions to biological solutions has the drawback of the inability to maintain steady-state concentrations of NO, especially in a reactive environment. Permeation of gaseous NO through polymeric membranes enables a constant NO delivery rate that leads to steady-state NO concentrations, even in the presence of species which react with NO. A previous study incorporating NO permeation through a membrane resulted in constant formation of NO2- in the presence of O2, suggesting that the NO concentration achieved steady state (7). However, the delivery rate was only semiquantifiable, and the aqueous NO concentrations were not predictable or measured. In all methods of NO delivery, it is often advantageous to deliver NO at a constant and controlled rate to maintain a desired and predictable NO concentration in a biological environment. Knowledge of the concentration is beneficial for assessing the effects of NO on biological systems, especially when assessing the physiological relevance of the NO exposure level. In view of the advantages of a constant NO delivery method in which predictable and steady-state NO concentrations can be maintained, two devices for delivering NO through permeable membranes and into a flowing solution have been developed and modeled. The advantages of these devices are that (1) a controlled amount of NO is delivered to maintain a steady-state NO concentration, (2) the NO concentrations are predictable from models, (3) the pH and light effects on the delivery rate are avoided, and (4) the addition of undesired species is eliminated to avoid undesired reactions. An application of the delivery device is described for maintaining a constant and predictable NO concentration in a wellstirred chamber.

10.1021/tx980112s CCC: $15.00 © 1998 American Chemical Society Published on Web 10/15/1998

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Figure 1. Slit-flow delivery device. The device is composed of a stainless-steel central portion through which solution flows. A gas-permeable membrane, Plexiglas, gasket, and gas inlet/outlet chamber are attached to each side of the central portion using hex bolts. Gas that contains NO can permeate through the membrane and into the solution in the section where the flowing solution is separated from the gas stream by only the membrane. All dimensions are described in the text.

Materials and Methods Note: Due to the potential toxicity of NO, all NO gas was vented to a hood. Reagents. Ultrahigh pure nitrogen, after passage through an O2 trap, was mixed with a mixture of 10% NO, balance N2 using controlled gas flow meters (Porter Instrument Co., Hatfield, PA) to obtain the desired NO gas concentration. Phosphatebuffered saline (PBS, 0.01 M) was obtained from Life Technologies (Grand Island, NY). Potassium iodide and glacial acetic acid were obtained from Sigma (St. Louis, MO). Delivery Devices. Two devices, one composed of a permeable tube and one composed of a thin slit bounded by two permeable sheets, were designed for physical delivery of NO to a flowing solution. Advantages of these devices are that the physical dimensions can easily be modified to vary the NO delivery rate and they are simple to fabricate. The slit-flow device is shown in Figure 1. The central portion of the device is made of stainless steel and contains a rectangular opening through which aqueous solution flows. The opening is 15.2 cm long with a cross section 0.5 cm wide and 0.3 cm high. A membrane, Plexiglas, gasket, and gas inlet/outlet chamber are attached to each side of the central portion using hex bolts, although only attachments on one side are shown in the 3-D profile. A semipermeable poly(dimethylsiloxane) membrane (MEM-100, 0.005 in. thick; Membrane Products, Albany, NY) is laminated to a 0.2 cm thick rectangular sheet of Plexiglas using silicone adhesive. The Plexiglas contains a rectangular opening of 2.5 cm length and 0.5 cm width to expose the flowing solution to gas permeating through the membrane. The Plexiglas opening is 10.2 cm downstream of the flow inlet. The Plexiglas opening can be lengthened to allow for a greater gas exposure area if required. The membrane, following compression between the Plexiglas and stainless steel, prevents the liquid from leaking upon initiation of flow through the device. A rubber gasket is placed between the Plexiglas and the gas inlet/outlet chamber to prevent gas leaks. The chamber contains

a section (15.2 cm long, 0.5 cm wide, 0.3 cm deep) through which gas flows. Tube fittings are threaded into the chamber for attachment of gas lines. As depicted in the side profile of the delivery device, solution flowing through the central portion is separated from the gas stream by only the membrane in one section of the device. In this section, a gas containing NO can permeate through the membrane and into the solution. The tube delivery device is shown in Figure 2. The device consists of Silastic tubing (VWR Products, 0.147 cm i.d., 0.196 cm o.d.) attached to Teflon tubing (0.132 cm i.d., 0.193 cm o.d.) placed inside a stainless-steel Swagelock cross. Heat shrink tubing (made of polyolefins), which is significantly less permeable to gas as compared to Silastic, is utilized to attach the Teflon tubing to the Silastic tubing. A section (3.0 cm) of the Silastic tubing is left exposed such that gas flowing across the tube permeates through the exposed Silastic tube and into a flowing solution. The exposed section or the NO gas concentration can easily be adjusted to permit more or less gas from permeating into the solution. Delivery Device Experiments. For both delivery devices, PBS was continuously pumped through the slit or tube at a flow rate of 3 mL/min using a peristaltic pump (Masterflex R, Model 77390-00, Cole-Palmer Instrument Co., Vernon Hills, IL). Thus, the residence times (volume divided by volumetric flow rate) in the region of gas exposure were 1.0 and 7.5 s for the tube device and slit-flow device, respectively. A gaseous mixture of NO and N2 of a specified concentration continuously flowed through the gas chamber or across the exterior of the Silastic tubing. The NO concentration was measured at the delivery device outlet and compared to model predictions. Experiments were performed at both 25 °C and 37 °C. For the 37 °C experiments, both delivery devices were autoclaved for 25 min at 250 °F prior to each experiment. The devices were autoclaved to assess the predictability of NO delivery for applications in which sterile delivery devices are desired. Nitric Oxide Analysis. The aqueous NO concentration, following exposure to NO gas, was measured using either a

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Figure 2. Tube delivery device. The device is composed of Silastic tubing (0.147 cm i.d., 0.196 cm o.d.) attached to Teflon tubing (0.132 cm i.d., 0.193 cm o.d.) and placed inside a Swagelok cross. Heat shrink tubing covers all but 3.0 cm of the Silastic tubing to allow for NO permeation into solution following exposure of the tubing exterior to NO gas. chemiluminescence analyzer (Model NOA 270B, Seivers Corp., Boulder, CO) or an amperometric probe (ISO-NOP, World Precision Instrument, Sarasota, FL). For the chemiluminescence method, aqueous samples were drawn using a gastight syringe (Hamilton Co., Reno, NV), and 0.1 or 0.25 mL was injected into 10 mL of solution composed of 0.2 M potassium iodide and glacial acetic acid mixed in a 1:3 volumetric ratio. The solution was contained in a glass vial and was continuously stirred and bubbled with N2 at 200 sccm1 to purge NO from the solution and transport the NO into the chemiluminescence detector. The concentration of NO in the sample was obtained by comparison with NO2- standards since NO2- is instantaneously converted to NO in the solution (10). The calibration curve was linear over the range of concentrations studied. The minimum detection limit is 25 pmol. For the amperometric probe measurements, the probe was inserted into a tee at the point of measurement. For experiments at 37 °C, the probe was located in an incubator since the probe response is sensitive to temperature. The probe was calibrated at both 25 °C and 37 °C. The calibration consisted of bubbling known concentrations of NO gas into deoxygenated PBS. The saturated NO aqueous concentrations were obtained from NO solubility data which are 2.53 and 2.14 µM/mmHg for NO at 25 and 37 °C, respectively (11). The saturated solution was pumped at 3.0 mL/min through the tee containing the probe to obtain the calibration curve. For 37 °C experiments, the solution was recirculated through the tee. At both temperatures, linear calibration curves were obtained over the range of concentrations studied. Model for Prediction of the NO Concentration. The bulk (or mixing-cup) NO concentration exiting the delivery device (Cb) was modeled and compared with experiments. The aqueous NO concentration (C) in the delivery device is obtained from the steady-state dimensionless continuity equation for NO which is

[

2

]

∂θ ∂θ ∂θ )A 3+B ∂ξ ∂η ∂η

(1 - η2)

) 0. The value of Co is the product of the NO solubility (H) and the gas partial pressure of NO. The dimensionless parameter ξ is z/L where the z coordinate represents the direction of flow (z ) 0 at the flow inlet) and L is the length of the membrane through which NO gas permeates. Equation 1 is based on fully developed laminar flow with a homogeneous fluid. The reaction of NO with aqueous O2 was not included in eq 1 since the reaction is slow for the NO concentrations of this study compared to the residence time of the solution in the slit chamber. For the tube device, η is r/R where the r coordinate represents the radial direction (r ) 0 at tube center) with a tube inner radius of R. The parameter A is DL/UmR2, where D is the aqueous NO diffusivity and Um is the maximum velocity. The value of Um is twice the average velocity. The parameter B is 1/η. For the slit-flow device, η is y/h where the y coordinate represents the slit height direction (y ) 0 at the slit center) with a slit height of 2h. The value of B is 0. The dimensionless parameter A is DL/Umh2. For a slit in which the width-to-height ratio is .1, the slit can be approximated as having an infinite width. For such an approximation, the velocity profile [which is Um(1 - η2) with Um equal to 3/2 of the average velocity] is independent of the width of the slit. Thus, the two-dimensional form of eq 1 where the width dimension is omitted is valid for predicting the NO concentration profile. The slit-flow device for this study has a width-to-height ratio of 1.7. Thus, the above assumption that the velocity profile is independent of the slit width is not entirely valid. The velocity profile has been solved for a rectangular slit in which the slit width is wide enough that only the nearest edge influences the velocity profile (12). The velocity profile resembles Um(1 - η2), but Um is a function of the slit width with values between 0 and 3/2 the average velocity. For this study, the model predictions for Cb shown in the figures for the slitflow device were obtained by assuming Um is 3/2 of the average velocity across the entire slit width (the infinite width approximation). Analysis using the velocity profile as a function of the slit width results in model predictions for Cb estimated to be approximately 25% higher. Thus, the best design for a slit-flow device would have a width-to-height ratio >10 for accurately predicting Cb or the NO concentration profile based upon eq 1 with Um equal to 3/2 the average velocity. The initial and boundary conditions to solve eq 1 are

ξ)0

all η

all ξ

η ) 0 ∂θ/∂η ) 0

θ )1

(3)

all ξ

η ) 1 ∂θ/∂η ) -NShwθ

(4)

(2)

The Sherwood number (NShw) at the wall is kwh/D and kwR/D for the slit-flow device and tube device, respectively. The mass transfer coefficient characterizing the transport of NO through the permeable membrane is kw. The solution to eq 1 yields θ ) f(ξ,η). The solution can be obtained using a numerical package such as Matlab. Thus, the NO concentration profile within the delivery device is obtained. The predicted value of the bulk (mixing-cup or velocityweighted) NO concentration (Cb) exiting the NO delivery device (at ξ ) 1) is

∫ (1 - η )θ dη C - C ) ) ∫ (1 - η ) dη C - C 1

θb

2

ζ)1

0

1

2

b

o

i

o

(5)

0

(1)

The dimensionless concentration (θ) is (C - Co)/(Ci - Co) where Co is the aqueous NO concentration in equilibrium with the gaseous NO to which the delivery device is exposed and Ci is the aqueous NO concentration at the inlet. For this study, Ci

Analytical solutions for Cb for both slit-flow and tube devices have previously been solved (13, 14). For the model, the average velocity was obtained from the geometric dimensions and the volumetric flow rate. The value of D in PBS was assumed similar to that in water which is 2.7 × 10-5 cm2/s at 25 °C and 5.1 × 10-5 cm2/s at 37 °C (15). The value of kw was obtained from the NO permeability (P) of poly-

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Figure 3. Stirred chamber. The stirred chamber is composed of stainless steel with a Plexiglas cover attached with hex bolts. The chamber contains a stir bar located 0.3 cm from the bottom of the chamber. The volume of the chamber, with the stir bar in place, is 21.0 mL. (dimethylsiloxane) membranes (i.e., Silastic) according to kw ) P/Hδ for slit flow and kw ) P/[HR ln(Ro/R)] for flow in a tube. The membrane thickness is δ, and the tube outer radius is Ro. The value of P for NO is 2.3 × 10-13 mol cm-1 s-1 mmHg-1 at 25 °C (16). The permeability at 37 °C is approximately twice the reported value at 25 °C in order to account for the effect of heating the membrane during autoclaving (17). The value of H for NO is 2.53 and 2.14 µM/mmHg at 25 and 37 °C, respectively (11). Delivery Device Application. The slit-flow delivery device was connected to a stirred chamber to demonstrate an application of the delivery device. The accuracy of predicting the NO concentration in an O2 reactive environment was also assessed. The stirred chamber, shown in Figure 3, is stainless steel and was designed to include a Teflon stir bar. The chamber internal dimensions are 5.5 cm in diameter by 0.8 cm high. The rectangular stir bar (4.4 cm long, 0.6 cm wide, 0.3 cm high) was fabricated by placing small magnets in each end of the bar and sealing each end with silicone adhesive. The stir bar rotated around a screw that held the stir bar in place. The stir bar was 0.3 cm from the bottom of the stirred chamber. Thus, a cell culture plate containing adhering cells could be placed in the bottom of the chamber if desired. The chamber could then be utilized to expose cells to steady-state aqueous NO concentrations for any specified length of time. The aqueous NO concentration could also be adjusted at any time by changing the NO gas concentration to which the delivery device is exposed. PBS was continuously pumped through the delivery device and stirred chamber at 3.0 mL/min while the chamber was stirred at 30 rpm. The device and chamber were connected with 15.5 cm of Teflon tubing (0.16 cm i.d.) such that the residence time of the tubing was 0.1 min. The theoretical residence time of the 21.0 mL chamber was 7.0 min. A residence time distribution study confirmed that the stirred chamber was well mixed and that the measured and theoretical residence times were the same. The delivery device was exposed to various NO gas concentrations at both 25 and 37 °C. The outlet NO concentration of the stirred chamber, which is the same as the concentration in the chamber for a well-mixed fluid, was measured using an amperometric probe as described above.

Results and Discussion Aqueous NO Concentration in Exiting Perfusate. The bulk or mixing-cup NO concentration (Cb) in the perfusate at the exit of each delivery device was measured as a function of the NO gas concentration to which the semipermeable membranes were exposed. Figure 4 shows the measured aqueous NO concentrations at steady state exiting both delivery devices. The steadystate aqueous NO concentrations were obtained within 2 min of changing the NO gas concentration, of which part of the time was due to the time required for the NO gas concentration to obtain steady state. The measured aqueous NO concentrations are shown relative to the NO concentrations (Co) which would be in equilibrium with

Figure 4. Delivery device NO concentrations at the outlet (Cb) are shown relative to NO concentrations (Co) that would be in equilibrium with the NO gas exposed to the delivery device. Means ( SD are shown as discrete symbols for slit and tube delivery devices at 25 and 37 °C. The dashed lines represent model predictions as described in the text.

the NO gas. The equilibrium NO gas concentrations were obtained using NO solubility data as previously given. Model predictions are also shown which are described later. The average values of Cb/Co at all NO gas exposure levels were 0.043 ( 0.005 (n ) 28), 0.064 ( 0.008 (n ) 16), and 0.107 ( 0.011 (n ) 15) for slit flow at 25 °C, slit flow at 37 °C, and tube flow at 37 °C, respectively. The measured values were obtained using chemiluminescence and the amperometric probe for the tube and slit-flow devices, respectively. In addition, the NO concentration exiting the tube device at 37 °C was also measured using the amperometric probe, with an average value for Cb/ Co of 0.120 ( 0.019 (n ) 8) over a similar range of Co. This shows that the NO concentrations as measured using the amperometric probe or chemiluminescence are similar. Thus, bioavailable NO is exiting the delivery devices. As shown, Cb/Co is not a function of Co as expected from model predictions explained later. It is also evident that the aqueous NO concentration is not saturated at any of the gas exposure levels, with only a maximum of 10% saturation achieved. A higher percentage of the saturated conditions was obtained with the tube device as compared to the slit-flow device due to the difference in geometry, exposure area, and flow velocities. By increasing the membrane exposure area and/or decreasing the flow rate, the NO concentration relative to equilibrium can be increased. Although at the highest gas exposure level the aqueous NO concentration approached 2 µM, higher concentrations are obtainable by increasing the NO gas exposure level, adjusting the flow rate, or modifying the delivery device dimensions.

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NO concentration exiting the stirred chamber will be less than that coming out of the delivery device because of the reaction with O2. For a well-stirred chamber, the NO concentration in the chamber (Cc) is

dCc Cb - Cc ) - 4k[Cc]2[O2] dt τc

Figure 5. NO concentrations (Cc) at the outlet of a well-stirred chamber exposed to oxygen are shown relative to NO concentrations (Co) that would be in equilibrium with the NO gas exposed to the slit-flow delivery device. Means ( SD are shown as discrete symbols at 25 and 37 °C. Model predictions according to eq 6 are shown for both oxygenated (solid lines) and deoxygenated (dashed lines) solutions.

Model Predictions of Exiting NO Concentration. Although the bulk aqueous NO concentrations were measured in the exiting perfusate of both delivery devices, it is useful to predict the NO concentrations. Predictions would be beneficial for selecting a desired NO concentration without experimental measurements based on adjustments in the flow rate or device dimensions (i.e., membrane exposure area). The value of Cb at the delivery device exit was predicted using the models for both the slit-flow and tube geometries. The values of Cb/ Co predicted by the models are shown in Figure 4. The models show good agreement with experiments for both delivery devices at all temperatures studied, irrespective of the size and geometry of the delivery device. It is notable that the model parameters were obtained independent of the experiments. The general agreement of the model predictions with experimental results suggests that the models can be utilized to effectively predict the outlet NO concentrations of the delivery devices. If desired, the models can also be used to obtain the spatial concentration profiles within the slit or tube. Delivery Device Application. One application of the delivery device is the connection of the device to a stirred chamber to maintain a specified NO concentration within the chamber for a period of time. The chamber could be used for biological studies in which a specified NO concentration is desired. For any application, it is important to assess the effects of reacting species with NO, such as O2, to predict the NO concentrations to which biological systems are exposed. For a small stirred chamber connected downstream of the slit-flow delivery device, the steady-state NO concentration in the chamber (Cc) was experimentally measured as shown in Figure 5. The measured aqueous NO concentrations are shown relative to the NO concentrations (Co) which would be in equilibrium with the NO gas. The steady-state concentrations in the stirred chamber were obtained in approximately 30 min following the exposure of NO gas to the delivery device. This time is consistent with eq 6 shown below for a step change in the chamber inlet NO concentration. A model was developed to predict the NO concentration exiting the stirred chamber following exposure of the delivery device to a specified NO gas concentration. The

(6)

The value of Cb, predicted from the delivery device model, was assumed constant in the tubing between the delivery device and the stirred chamber due to the short residence time of 6 s. The rate constant (k) for the reaction of NO with O2 is 2.1 × 106 and 2.4 × 106 M-2 s-1 at 25 and 37 °C, respectively (18). The stirred chamber residence time (τc) is 7.0 min. At steady state when dCc/dt ) 0, Cc is predictable from eq 6 using the predicted value of Cb from the delivery device model. For the model, it is appropriate to assume that the aqueous O2 concentration remains constant due to its large excess at saturated conditions as compared to the NO concentration exiting the NO delivery device. The saturated O2 concentrations are 265 and 223 µM at 25 and 37 °C, respectively (11). The model results for the stirred chamber are also shown in Figure 5 for both an oxygenated and a deoxygenated solution. The model predictions are shown in the absence of O2 to illustrate the effect that reactive species can have on predicting the NO concentration. It is notable that the oxygenated and deoxygenated model predictions approach each other at low NO concentrations since the reaction becomes less significant with decreasing NO concentration. The excellent agreement between the predicted and experimental NO concentrations shows that NO concentrations are predictable in experimental systems as long as the kinetics of significant reactions involving NO are known. The NO concentrations can also be maintained at steady state.

Conclusions In view of the importance of delivering predictable quantities of NO to biological systems for investigating the biological effects of NO, two simple delivery devices were designed. For applications of the delivery device to study the effects of NO exposure to biological systems, several methods can be utilized which incorporate the delivery devices. Cell adhesion (such as platelets) to various proteins coated on the permeable membrane can be studied in the presence or absence of NO delivery to assess the effects of NO on the adhesion process. The delivery device can be included in a circulating or noncirculating loop connected to a stirred chamber to expose cells in the chamber to steady-state NO conditions. In all designs, it is important to assess the effects of reacting species with NO in order to predict the NO concentrations to which biological systems are exposed. NO is a highly reactive molecule and can react with species such as superoxide, metal-containing proteins, or oxygen. Previous studies have shown that NO concentrations resulting from the delivery of NO to oxygenated culture medium containing serum were predictable while only accounting for the reaction with O2 (9). However, if other unknown but significant reactions with NO exist, the models described in this work can be used to provide an upper estimate of the NO concentration.

Nitric Oxide Delivery to Aqueous Solutions

Acknowledgment. This work was supported by a grant from the National Institutes of Health (R15DK51327).

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TX980112S