Catalyzed Nitric Oxide Release Via Cu Nanoparticles Leads to an Increase in Antimicrobial Effects and Hemocompatibility for Short Term Extracorporeal Circulation

Abstract

Devices used for extracorporeal circulation are met with two major medical concerns: thrombosis and infection. A device that allows for anticoagulant-free circulation while reducing risk of infection has yet to be developed. We report the use of a copper nanoparticle (Cu NP) catalyst for the release of nitric oxide (NO) from the endogenous donor S-nitrosoglutathione (GSNO) in a coating applied to commercial Tygon S3^™ E-3603 poly(vinyl chloride) tubing in order to reduce adhered bacterial viability and the occurrence thrombosis for the first time in an animal model. Cu GSNO coated material demonstrated a nitric oxide (NO) release flux ranging from an initial flux of 6.3 ± 0.9 ×10⁻¹⁰ mol cm⁻² min⁻¹ to 7.1 ± 0.4 ×10⁻¹⁰ mol cm⁻² min⁻¹ after 4 h of release, while GSNO loops without Cu NPs only ranged from an initial flux of 1.1 ± 0.2 ×10⁻¹⁰ mol cm⁻² min⁻¹ to 2.3 ± 0.2 ×10⁻¹⁰ mol cm⁻² min⁻¹ after 4 h of release, indicating that the addition of Cu NPs can increase NO flux up to five times in the same 4 h period. Additionally, a 3-log reduction in S. aureus and 1-log reduction in P. aeruginosa was observed in viable bacterial adhesion over a 24 h period compared to control loops. A Cell Counting Kit-8 (CCK-8) assay was used to validate no overall cytotoxicity towards 3T3 mouse fibroblasts. Finally, extracorporeal circuits were coated and exposed to 4 h of blood flow under an in vivo rabbit model. The Cu GSNO combination was successful in maintaining 89.3% of baseline platelet counts, while the control loops were able to maintain 67.6% of the baseline. These results suggest that the combination of Cu NPs with GSNO increases hemocompatibility and antimicrobial properties of ECC loops without any cytotoxic effects towards mammalian cells.

Graphical Abstract

1. INTRODUCTION

Blood contacting devices used for extracorporeal circulation (ECC) are instrumental during medical procedures such as open-heart surgery, tissue oxygenation, and hemodialysis. However, despite the extensive use of these medical devices, thrombosis and infection still remain two major limitations.^1–2 When blood is introduced to a foreign surface, platelets can adhere, leading to thrombus formation. All current ECC strategies require systemic anticoagulation in order to maintain patency.³ However, administration of anticoagulants such as heparin requires a careful balance between under- and over-dosage in order to reduce thrombus formation while preventing excessive or uncontrolled bleeding.³ According to previous reports, bleeding and thrombosis occur at a rate of 38% and 31%, respectively, in extracorporeal life support patients.⁴ Moreover, despite systemic anticoagulation, platelet counts can still reduce to less than 40% of the normal count within the first few hours.⁵

Additionally, hospital acquired infections (HAIs) are the leading cause of complications for hospitalized patients.⁶ Of those obtained, approximately 15% of HAIs are primary bloodstream infections alone.⁷ Though antibiotics are currently being used to control infection, the Center for Disease Control and Prevention estimates that approximately 2 million people are infected with antibiotic-resistant bacteria yearly, and 23,000 die as a result.⁸ Frequent exposure to foreign devices and line movement result in life-threatening localized and bloodstream infection.⁹ Previous reports have indicated that biofilms can begin to form within a few hours, showing visible development within 5 hours after inoculation.^10–11 Moreover, extracorporeal membrane oxygenation (ECMO) devices have been reported to have a colonization rate of 32%, which was attributed to the artificial surfaces that comprise of the circuit allowing pathogen adhesion and colonization.¹² These adverse effects can complicate medical procedures relying on ECC, increasing hospital stay and risk of death. An anti-thrombogenic and antimicrobial ECC apparatus that allows for anticoagulant-free circulation with reduced rates of infection has yet to be developed.

Due to the essential role of the key signaling molecule nitric oxide (NO) in the nervous, immune, and cardiovascular systems, researchers have recently looked into incorporating synthetic NO donors into medical devices in order to alleviate side effects that commonly occur with implantation.^13–14 Mimicking NO release from endothelial cells can assist in achieving a truly hemocompatible surface. Nitric oxide is particularly useful for two specific areas of interest related directly to blood-contacting medical device applications: platelet activation regulation and immune response.¹⁵ Specifically, NO has shown to reduce platelet activity and is produced by macrophages to target both Gram-positive and Gram-negative bacterial pathogens.^16–19 To improve the hemocompatibility and antimicrobial behavior of these devices, both exogenous and endogenous NO donors such as S-nitrothiols (RSNOs), N-diazeniumdiolates, organic nitrates and nitrites, and metal nitrosyl complexes have been incorporated into natural and synthetic polymers to provide an source of localized nitric oxide from device surface to surrounding tissue.^20–21 While many NO donors have been developed, RSNOs have been of particular interest due to their straightforward synthesis and steady release, simulating physiological conditions.²² Amongst the RSNOs, S-nitrosoglutathione (GSNO) is one of the most prevalent naturally occurring S-nitrosothiols found physiologically, responsible for modulating vasodilation and inhibiting platelet aggregation.^{13, 23–24} Compared to their NO-releasing counterparts, endogenous RSNOs such as GSNO are superior due to their innate biocompatibility.²⁴ In the body, the endogenous NO donor GSNO behaves as a bioavailable reservoir for NO in the bloodstream and within cells.^{13, 24–25} In addition to its superior biocompatibility, GSNO has also been found to be very stable in comparison to other RSNOs.²⁶ This increased stability also gives a more consistent NO release profile over the tested time period, leading to less variation in flux as typically observed in SNAP coatings.²⁷ When devices are under flow conditions, the need for a stable donor only increases. For these reasons, there has been growing interest in utilizing GSNO as a therapeutic agent.²⁶ Therefore, incorporating GSNO into polymers used for blood-contacting applications to act as a stable, biocompatible NO reservoir is of significant interest.

The release of NO from GSNO is mediated through the cleavage of the S-NO bond present in all RSNO species, and can occur through several different mechanisms: light decomposition, thermal decomposition, and metal ion particle catalysis.^{13, 28} First, the S-NO bond can be cleaved photolytically by the irradiation of GSNO absorbance bands at 336 and 545 nm.^{13, 23} However, photocatalysis requires significant exposure to light, which limits medical applications. Secondly, although RSNOs can be stimulated at higher temperatures, extracorporeal devices are used at ambient temperatures. Therefore, the addition of a catalyst to the polymer to increase the nitric oxide flux from the polymer over the pertinent application period is of substantial interest. In addition to photolytic catalysis, the presence of transition metal ion particles have shown to have a strong catalytic effect on RSNOs.²⁹ Copper has been to shown to mediate GSNO decomposition through Cu⁺ interaction with the nitrosothiol, ultimately releasing NO.²³ Copper nanoparticles (Cu NPs), in addition to their potential catalytic effect on GSNO, can also interact with endogenous RSNOs found in the blood.³⁰ Cu NPs also possess an innate antimicrobial effect and are less cytotoxic than previously explored silver nanoparticles.^31–32

We report a unique multi-layer Cu GSNO coating applied to commercial Tygon S3^™ E-3603 poly(vinyl chloride) (PVC) tubing, a medical grade polymer commonly used in blood circulation applications, in order to reduce the occurrence of thrombosis and infection. The effect of the addition of Cu NP on GSNO in CarboSil, a medical grade polymer, is measured by the NO release over a 4 h period. In vitro viability of adhered bacteria was measured by counting the colony forming units (CFU) per cm² of polymer loops exposed to the Gram-positive strain Staphylococcus aureus (S. aureus) and the Gram-negative strain Pseudomonas aeruginosa (P. aeruginosa) for 24 h at 37° C. Cytotoxicity towards 3T3 mouse fibroblasts were assessed with a Cell Counting Kit-8 (CCK-8) after 24 h of leachate exposure. An in vivo 4 h ECC rabbit model was then used to measure hemocompatibility.

2. MATERIALS AND METHODS

2.1. Synthesis

2.1.1. Materials

Sodium nitrite, tetrahydrofuran (THF), ethylenediaminetetraacetic acid (EDTA), and the CCK-8 kit were purchased from Sigma Aldrich (St. Louis, MO 63103). Tygon S3^™ E-3603 poly(vinyl chloride) tubing was purchased from Fisher Healthcare (Houston, TX). Acetone was purchased from VWR (Radnor, PA). Carbosil-2080A (CarboSil) was purchased from DSM (Berkeley, CA). Cu NPs (99%, 40–60 nm) were obtained from SkySpring Nanomaterials, Inc. (Houston, TX). Phosphate-buffered saline (PBS), pH 7.4, was used for all in vitro experiments, which contained 138 mM NaCl, 2.7 mM KCl, and 10 mM sodium phosphate. Glutathione, Dulbecco’s modified Eagle’s medium (DMEM), and trypsin-EDTA were purchased from Corning (Manassas, VA 20109). The antibiotic Penicillin-Streptomycin (Pen-Strep) and fetal bovine serum (FBS) were purchased from Gibco-Life Technologies (Grand Island, NY 14072). The bacterial strains P. aeruginosa (ATCC 27853) and S. aureus (ATCC 5538), and mouse 3T3 fibroblast cells (ATCC 1658) were purchased from American Type Culture Collection (ATCC). LB broth was obtained from Fisher Bioreagents (Fair Lawn, NJ). LB Agar was obtained from Difco Laboratories Inc (Detroit, MI). Both the 16-gauge and 14-gauge IV polytetrafluroethylene (PTFE) angiocatheters were purchased from Exel International Co. (Redondo Beach, CA).

2.1.2. Synthesis of GSNO

GSNO was synthesized by dissolving 900 mg of glutathione in a solution containing 12 M of HCl and DI water and chilled in an ice bath for 10 min. NaNO₂ was then added to the solution and was chilled for 40 min. Acetone was stirred into the solution for 10 min. The precipitate was collected via filtration and dried in a desiccator in the dark overnight.

2.1.3. Fabrication of Cu GSNO polymeric coatings ECC loops

Preparation of ECC loops used for animal testing was done following a previously describe protocol.^33–35 Briefly, the fully constructed loop configuration consisted of two 16 cm length pieces of PVC loops (1/4 inch ID) connected on either side of one 8 cm length piece of PVC loop (3/8 inch ID), which creates the thrombogenicity chamber that creates a disturbed flow and recirculation within the loop. 16-gauge and 14-gauge IV PTFE angiocatheters (Exel International, Co., Redondo Beach, CA) were then placed on either end of the assembled loop using 2 luer-lock PVC connectors. The 3/8 inch and 1/4 inch loop pieces were solvent welded together using a diluted solution of Carbosil in THF (25 mg/mL).

A multilayered coating system was employed for all ECC loops where designated solutions are filled through the tubing lumen and drained according to Figure 1.

Figure 1 –

Schematic of the fabrication of ECC loops. Cross-sections shows the layer-by-layer composition of each ECC tubing. Cu GSNO ECC loops (A) were compared to GSNO (B), Cu (C), and CarboSil control (D) loops.

Solutions of CarboSil in THF at a concentration of 50 mg/mL were first prepared. A 10 wt% suspension of GSNO 3 wt% suspension of Cu NPs with respect to the CarboSil solution were then added to the CarboSil solutions. A 3 wt% Cu NP solution was used based on a previous report that optimized the concentration of Cu NPs incorporated with the RSNO S-nitroso-N-acetylpenicillamine (SNAP).³⁶ For Cu GSNO loops, two coats of the GSNO solution were first employed, followed by two coats of Cu NP solution, and a final topcoat of CarboSil solution. Each coat was allowed to dry for 1 h at room temperature in the dark to avoid any undesired light catalysis with GSNO prior to any subsequent layer being added. After the final layer was added, loops were allowed to dry for 48 h at room temperature before being kept in a desiccator for 24 h to ensure there was no THF remaining. Loops consisting of either only GSNO (GSNO), copper nanoparticles (Cu), or Carbosil (CarboSil control) were also prepared. GSNO loops contained two coats of GSNO solution, followed by three coats of CarboSil solution. Cu loops contained two coats of Cu NP solution and three coats of CarboSil solution. CarboSil control loops were prepared with five coats of CarboSil solution. Each loop was coated with an equal number of total coats (5 total coats) to ensure that the thickness of the final multi-layered loop remained the same, and scanning electron microscopy (SEM) was later deployed to ensure that the thicknesses remained the same. Based on drying times used in previous studies, the assembled and coated loops were allowed to dry under ambient conditions for 48 h followed by vacuum drying for 24 h.^{1, 36–38} Previous studies have indicated that layers ~50 microns in thickness are adequate to prevent leaching from ECC loops.³⁹ Prior to each rabbit experiment, loops were presoaked with saline for 1 h and drained.

2.2. Scanning Electron Microscopy (SEM) and Energy-Dispersive X-ray Spectroscopy (EDS).

Scanning electron microscopy (SEM, FEI Teneo, FEI Co.) was employed at an accelerating voltage of 10.00 kV to examine the morphology of the fabricated materials along with the dispersion of Cu NPs and GSNO particles throughout the sample surfaces. The SEM was equipped with a large detector energy dispersive X-ray spectroscopy (EDS, Oxford Instruments) system used for elemental analysis and mapping of the elements. All materials were coated with gold-palladium to a thickness of 10 nm using a Leica sputter coater.

2.3. Detection of Cu NP and GSNO leaching from ECC loops

Inductively coupled plasma mass spectrometry (ICP-MS) is a highly sensitive technique that can measure the presence of trace elements of interest. Detection of copper leaching from polymer coatings was done using a VG ICP-MS Plasma Quad 3 instrument. Cu GSNO polymer films were weighed, measured, and then soaked in DMEM for 24 h. Afterwards, the films were removed and the solution containing potential copper leachate was tested for ⁶⁵Cu isotopes following a previously established protocol.⁴⁰

GSNO leaching was measured during a 4 h incubation period under standard conditions. GSNO loops and Cu GSNO loops were soaked in 1 mL PBS (pH 7.4) and measured with a Thermo Scientific Genysis 10S UV-Vis Spectrophotometer (UV-vis). Known GSNO concentrations were dissolved in 1 mL of PBS to determine a standard curve. Absorbance was recorded at 340 nm for each sample throughout several timepoints in the 4 hour timespan, which corresponds to the absorbance maxima of the S-NO bond found in RSNOs.⁴¹ Pure PBS was used as a blank control.

2.4. NO-releasing kinetics

NO release was recorded using a Sievers Chemiluminescence NOA 280i (Boulder, CO). Samples were submerged in 4 mL of PBS buffer solution (pH = 7.4) maintained at 37 °C in a dark reaction vessel to prevent any light catalysis. With GSNO samples, 100 μm of EDTA was added to the PBS buffer solution to prohibit any metal ion activity in the buffer from interacting with the samples. In the PBS solution containing samples with Cu NPs, however, no EDTA was added so that the catalytic activity of the Cu NPs would not be interfered with. To measure the NO levels released from each sample, nitric oxide was constantly swept from the headspace of the chamber and purged from the buffer solution by a bubbler and nitrogen sweep gas at 200 mL min⁻¹ into the chemiluminescence detection chamber. In the chamber, NO reacts with ozone (O₃) to produce a nitrogen dioxide (NO²*) at an excited state. The excited nitrogen dioxide decays and emits a photon used to detect the original concentration of NO released measured in ppb. After taking in consideration the NOA constant (mol ppb⁻¹ s⁻¹) and the surface area of the sample, the data was converted to surface flux (x10⁻¹⁰ mol cm⁻² min⁻¹). Each sample was measured for 4 hours.

To calculate the cumulative release, loops were measured prior to and after coating with the GSNO layer. The weight of the GSNO layer was then used to calculate the mg of GSNO incorporated. The initial amount of GSNO was compared to the amount of NO released during the 4 h period, which was calculated through the flux measured to compute the % cumulative release according to Equation 1:

$$\%\mathit{\text{Cumulative Release}}=\frac{\mathit{\text{mg GSNO used}}}{\mathit{\text{mg initial GSNO }}}$$

2.5. Colony-forming units quantification on coating surface

Viable bacterial adhesion was measured using a modified version of a previously established protocol.^42–43 First, isolated colonies of bacteria (S. aureus and P. aeruginosa) were cultured in LB broth at 37 °C until reaching a concentration of ~10⁶ CFU per mL verified by optical density measured by UV-vis. The culture was then centrifuged at 2500 revolutions per minute (RPM) for 7 min. The broth was removed, and the culture was resuspended in PBS. In a 24-well plate, 1 mL of the PBS-bacteria suspension was transferred to each well, each containing a different sample type and then incubated for 24 h. Each sample was then immersed in 1 mL of PBS contained in a 15 mL centrifuge tube and homogenized for 60 s at 25000 rpm using an Omni-TH homogenizer (Omni, Kennesaw, GA) and subsequently vortexed for 60 s to ensure any bacteria attached to the surface of the sample was transferred to the PBS solution. Serial dilutions made from the resulting PBS solution were then transferred to LB agar plates and cultured in an incubator at 37 °C. CFUs per cm² of tested loops were hand-counted from each sample type to determine bacterial inhibition effectiveness.

2.6. In vitro 24 h 3T3 mouse fibroblast cytotoxicity

To establish that the Cu GSNO synthesized polymer does not have any cytotoxic effects towards mammalian cells, cytotoxicity assessment was measured according to ISO 10993 standards.

2.6.1. Cell Culture preparation

Before performing the toxicity assays, 3T3 mouse fibroblast cells were grown in a 75 cm² T-flask containing DMEM with supplements. The cells were kept in culture at 37°C in a humidified atmosphere with 5% CO₂. The medium was replaced every 2–3 days until a confluency of 80–90% was reached after which cells were sub-cultured with trypsin-EDTA (0.18% trypsin and 5 mM EDTA) and incubated for 5 minutes. The cell suspension was then collected and counted using a hemocytometer using trypan blue dye exclusion method. After cell counting, 100 μL cells were transferred to a 96 well plate with each of the well containing 5000 cells per mL with n=7 of each sample type. This was followed by a 24 h incubation.

2.6.2. Cytotoxicity measurements using WST-8 dye

The leachates from the samples were collected from DMEM medium that contains 1 mg/mL of sample (10 mg dry weight in 10 mL medium) incubated for 24 h at 37°C. Thereafter, 10 μL of leachate solution was added to each of the wells and incubated for a period of 24 h in a 96 well plate. After 24 h, 10 μL dye solution from WST-8 based CCK-8 kit was used. The manufacturer’s (Sigma-Aldrich) protocol was followed while using the CCK-8 kit which utilizes highly water-soluble tetrazolium salt. WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt] is reduced by dehydrogenases in live cells to give formazan (an orange color product) detected at 450 nm. Thus, the number of living cells is directly proportional to the amount of the formazan dye generated by dehydrogenases in cells. Results were reported as percentage cell viability (percentage of control) after subtracting the average absorbance of the medium (without cells) as follows according to Equation 2:

$$\%\mathit{\text{Cell Viability}}=\frac{\mathit{\text{Absorbance of the test samples}}}{\mathit{\text{Absorbance of the control samples }}}\times 100$$

2.7. In vivo assessment of hemocompatibility of NO-releasing extracorporeal circuits

The rabbit model to evaluate hemocompatibility of the ECC loops was used as previously reported.^{1, 37, 39, 41} The animal handling and surgical procedures were approved by the University Committee on the Use and Care of Animals in accordance with university and federal regulations. A total of 12 New Zealand white rabbits (Charles River) were used in this study. Initially, all rabbits (2.5–3.5 kg) were anesthetized with intramuscular injections of 5 mg kg⁻¹ xylazine injectable (AnaSed® Lloyd Laboratories Shenandoah, Iowa) and 30 mg kg⁻¹ ketamine hydrochloride (Hospira, Inc. Lake Forest, IL).

Isoflurane gas (maintenance anesthesia) was administered via inhalation at a rate of 1.5–3% via mechanical ventilation which was done via a tracheotomy and using an A.D.S. 2000 Ventilator (Engler Engineering Corp.Hialeah, FL). Peek inspiratory pressure was set to 15 cm of H₂O and the ventilator flow rate set to 8 L min⁻¹. In order to aid in maintenance of blood pressure stability, IV fluids of Lactated Ringer’s were given at a rate of 10 mL kg⁻¹ h⁻¹. For monitoring blood pressure and collecting blood samples, the rabbits’ right femoral artery was cannulated using a 16-gauge IV angiocatheter (Jelco®, Johnson & Johnson, Cincinnati, OH). Blood pressure and derived heart rate were monitored with a Series 7000 Monitor (Marquette Electronics Milwaukee, WI). Body temperature was monitored with a rectal probe and maintained at 37 °C using a water-jacketed heating blanket.

Prior to placement of the arteriovenous (A-V) custom-built ECC loops, the rabbit left carotid artery and right external jugular vein were isolated and baseline cell counts were drawn from the femoral catheter. Baseline blood samples were collected for platelet and total white blood cell (WBC) counts which were measured on a Heska Element HT5 Hemotology Analyzer. After baseline blood measurements, the custom-built ECC device was placed into position by cannulating the left carotid artery for ECC in flow and the right external jugular vein for ECC out flow. The flow through the ECC device was initiated by unclamping the arterial and venous sides of ECC loops and blood flow in the ECC loop was monitored with an ultrasonic flow probe and flow meter (Transonic HT207 Ithaca, NY). Occlusions of the ECC tubing was considered to be 100% if the flow through the circuit was observed to be ≤ 5 mL min⁻¹. Animals were not systemically anticoagulated during the experiments.

Following the initiation of ECC blood flow, whole blood samples were collected in 3.2% sodium citrate vacutainers (Becton, Dickinson. Franklin Lakes, NJ) in 3 mL volumes for cell counts each hour during the 4 h experiment.

After 4 h of blood flow or once the ECC circuit was fully occluded, the ECC loops were clamped, removed from the rabbit, and rinsed with 30 mL of saline and drained. Due to the opacity of the ECC loops, the thrombogenicity chamber was longitudinally cut open to observe the extent of thrombus formation. The thrombogenicity chamber was photographed and the thrombus was gently transferred to a formalin container to be preserved until massed for quantification. The rabbits were euthanized using a dose of Fatal Plus (130 mg kg⁻¹ sodium pentobarbital). Prior to euthanasia, all rabbits were given a dose of 400 U/kg sodium heparin to prevent post-mortem thrombosis.

2.8. Statistical Analysis

All measured data is reported as a mean ± standard deviation. A standard two-tailed t-test was performed to determine significance (p < 0.05).

3. RESULTS AND DISCUSSION

3.1. Surface Morphology and Leaching Characterization

SEM imaging and Elemental Mapping.

Images of the surface were investigated for uniform deposition of GSNO and copper particles in the different layers of the polymer coating. First, both PVC loops and CarboSil-coated loops were imaged (Figure 2A and B) prior to adding the Cu NP and GSNO layers. Neither of these materials exhibited sulfur or copper elements as expected, therefore only SEM images are presented (elements detected were C, O, Cl from the polymers Au and Pd from sputter coating). As shown in Figure 2C and 2E, the addition of Cu NPs and GSNO did not significantly alter the topography of the coating. As seen in Figure 2D, Cu NPs were present and evenly dispersed throughout the coating. For the detection of GSNO, sulfur was mapped on the surface as GSNO was the only sulfur-containing compound in the polymer coating. As observed in Figure 2F, the surface had an evenly dispersed coating of GSNO. It can be noted that each layer was mapped in the absence of CarboSil topcoats as the presence of a topcoat reduces the ability to detect the elements of interest. However, because the loops were assembled layer-by-layer, these results accurately demonstrate the distribution of GSNO and Cu NPs within their respective layers that overall compose the entire ECC device coating.

Figure 2 –

SEM (A, B, C, and E) and EDS (D and F) analysis of the inner surface of the ECC loops. Both PVC (A) and CarboSil (B) tubing were imaged as control for test samples. The copper layer (C) and GSNO layer (E) did not significantly alter the morphology of the Tygon tubing. EDS mapping of copper (D) and sulfur (F) demonstrate the presence and even distribution of Cu NPs (orange) and GSNO (pink) within their respective layers.

After the entire coating process was complete, to ensure uniform coating thickness, cross-sections of each multi-layered tubing were imaged using SEM (Figure 3). The thickness of the resulting layers were measured and averaged to confirm uniform coating (n=10). Measurements showed that the thicknesses between each sample type were similar, indicating that the addition of GSNO and Cu had no significant effect on the coating process (CarboSil – 56.6 ± 1.9 μm, Cu – 56.8 ± 3.9 μm, GSNO – 57.6 ± 1.5 μm, Cu GSNO – 57.1 ± 4.2 μm).

Figure 3 –

SEM analysis of the cross section of the final multi-layered ECC loops. The white arrows indicate the coating on the inner surface of the tubing. Measurements indicate uniform coating thickness between each sample (A – CarboSil coating, B – Cu coating, C – GSNO coating, D – Cu GSNO coating).

Copper and GSNO Leaching.

Controlling the amount of copper leaching from polymers is crucial due to the inherent cytotoxic effects it has when at high enough concentrations. In the presence of an aqueous environment, metallic Cu can oxidize, resulting in Cu ions leaching into the surrounding environment. Each ECC loop was top coated with a layer of 50 mg/mL concentration of CarboSil to prevent excessive copper and GSNO leaching. After 24 h of soaking 1 mg sections of both Cu and Cu GSNO loops in DMEM at 37° C, ICP-MS was performed to detect exact amounts of copper ions leached into the media (Table 1). Both types of loops saw less than 11 × 10⁻⁶ mg of copper ions leached per mg of tubing, which equates to less than 0.1% of the total copper stored within the films. Moreover, like Ag NPs, Cu NPs possess innate antimicrobial effects, but are less cytotoxic than Ag NPs.^31–32 Previously reported copper leaching at similar levels showed beneficial antimicrobial activity while maintaining high mammalian cell viability.³⁶

Table 1 –

Total amount of Cu leached after 24 hours of soaking in DMEM at 37° C. No significant difference was observed between Cu and Cu GSNO samples (p > 0.05). The data are means ± SD.

Sample	Cu leaching (mg Cu/mg tubing)
Cu	9.9 ± 0.5 ×10−6
Cu + GSNO	10.7 ± 0.5 ×10−6

GSNO leaching studies were performed to determine the amount of NO leached into PBS during a 4 h incubation period. Initial leaching can severely limit the total time duration and consistency of the NO release from polymeric surfaces.⁴⁴ Although the use of hydrophobic polymers minimize water uptake, and therefore minimize leaching, even when RSNOs are incorporated into a hydrophobic polymer and/or are top-coated with a hydrophobic polymer, low levels of leaching can still occur due to the water uptake into the film. Previous studies have shown that the majority of leaching occurs within the first few hours.^{38, 41, 45–46} Moreover, this initial leaching is most likely due to trace amounts of diffusion that occurs when the final top coat is applied. Since it is a THF based solution, it will partially dissolve the underlying GSNO-CarboSil layer and allow some of the GSNO to be mixed in with this top coat. Therefore, in this study, we examined GSNO leaching over the first 4 h of soaking in a PBS solution. GSNO leaching was measured with UV-vis. During the first hour, less than 0.3 mg of total GSNO coated had leached from both the GSNO loops and Cu GSNO loops per mL of PBS, which equates to less than 2% of total GSNO originally incorporated into the loops, indicating that the samples did not experience a “burst effect” that corresponds to a high amount of GSNO being leached when initially immersed in PBS (Table 2). After 4 h of incubation, ~ 1.0 mg of total GSNO, which corresponds to less than 7% of total GSNO initially coated, per mL of PBS had leached from the surface.

Table 2 –

Concentration of GSNO leaching over 4 hours of soaking in PBS at 37° C. No significant difference was observed between GSNO and Cu GSNO samples (p > 0.05). The data are means ± SD.

Time	GSNO (mg/ml)	Cu + GSNO (mg/ml)
1 h	0.1 ± 0.2	0.2 ± 0.2
2 h	0.7 ± 0.3	0.4 ± 0.5
4 h	0.8 ± 0.2	1.0 ± 0.3

3.2. NO release from Cu GSNO ECC loops

In the past, NO-releasing materials have been proven effective in reducing viability of adhered bacteria and platelet activation.^47–48 Although light catalysis is a known method to increase the release of NO from NO donors, this method has its limitations in biomedical device applications. Moreover, increasing the concentration of the NO donor within the polymer does not simply increase the release rate of NO, and also can have negative effects on the mechanical integrity of the materials.^{38, 49} Therefore, other methods need to be explored in order to control the NO release to fit desired applications. In this study, Cu NPs were incorporated into the polymer to increase the nitric oxide flux from the polymer. GSNO loops were compared to the Cu GSNO loops to measure the catalytic effect of Cu on the NO release (Figure 4A). Over a 4 h period, the GSNO loops were found to release an average flux between 1.1 ± 0.2 ×10⁻¹⁰ mol cm⁻² min⁻¹ (after 1 h of release) to 2.3 ± 0.2 ×10⁻¹⁰ mol cm⁻² min⁻¹ (after 4 h PBS incubation) (Figure 4A). However, when 3 wt.% Cu was incorporated into the polymer coatings, the average flux increased to 6.3 ± 0.9 ×10⁻¹⁰ mol cm⁻² min⁻¹ (after 1 h of release) to 7.1 ± 0.4 ×10⁻¹⁰ mol cm⁻² min⁻¹ (after 4 h of PBS incubation) over the same 4 h period (Figure 4A). The Cu GSNO loops had a maximum flux of 10.3 ± 0.8 ×10⁻¹⁰ mol cm⁻² min⁻¹, while the GSNO loops only showed a maximum flux of 2.3 ± 0.2 ×10⁻¹⁰ mol cm⁻² min⁻¹ towards the end of the incubation period. Based on this data, it can be concluded that the presence of Cu NPs in an adjacent layer has a catalytic effect on the NO release without creating a burst effect that results in all of the NO being released at once. The cumulative release profile indicates that the Cu GSNO loops used ~5% more of the nitric oxide stored in the polymer when compared to the GSNO loops over the same 4 h period (Figure 4B). As can be observed, the presence of Cu NPs elevates the total amount NO being released over the 4 h application period, but the % of NO released over time remains consistent. No burst effect that quickly extinguishes the NO supply is observed.

Figure 4 –

Hourly average (A) and cumulative (B) NO release analysis of control GSNO tubing compared to Cu GSNO tubing over a 4 h period. P-values < 0.05 were used to determine statistical significance indicated by *. For the real-time flux measurements, the data are presented as means ± SD. Cumulative release is reported as means of cumulative NO release for each sample. NO release increases when Cu NPs are present in the sample. N=3 per sample.

The NO flux behavior over the 4 h period of the two sample types can be explained by two phenomena. First, CarboSil is a hydrophobic polymer, exhibiting a contact angle greater than 100° and a water uptake of 0.7 ± 0.2 wt.% in previous literature.^{38, 50} Therefore, when the samples are initially immersed in PBS, the water uptake time is extremely gradual, corresponding to the gradual increase of NO release in both the control and the Cu GSNO samples in the first two hours. Although the control continues to increase gradually after the first two hours, the Cu GSNO samples experience a slight decrease in NO release. This can be explained by the ability of Cu²⁺ to be reduced to Cu⁺. Only Cu⁺ contributes to the catalytic effect of NO release.⁵¹ According to literature, unlike other NO donors, the release of NO from RSNOs is governed by heat, moisture, light, and/or metal ion catalysis. Initially, RSNOs present can decompose with the assistance of heat, moisture, and/or light, resulting in the release of NO and a byproduct of a reactive RS⁻ species. The RS⁻ species produced from the passive decomposition of GSNO readily reduces Cu²⁺, which is present of Cu NP corrosion, resulting in Cu⁺ and RSSR. This mechanism has been previously detailed Burg et al and further discussed by Pant et al.^{36, 52} Increasing the levels of Cu NPs in the samples has previously been established to increase the levels of NO from other NO donors, which suggests that if more Cu²⁺ is initially available to diffuse through the film, more Cu²⁺ can be reduced to Cu⁺.³⁶ However, for this study, we limited our scope to Cu NP levels that have previously been described to give maximum levels of NO release before reaching a range that is beyond the physiological range.³⁶

3.3. In vitro analysis of bacteria eradication on ECC surface

Bacterial adhesion to a polymer surface is problematic for biomaterials, limiting the efficacy of a material and increasing the risk of infection and mortality for patients.⁵³ NO donors can avert risk of infection associated with medical devices, showing a reduction in viable bacteria adhered both in vitro and in vivo.^{16, 45, 54–55} In addition, heavy metals have been reported to have an innate oligodynamic effect, resulting in the reduction of viable bacteria.^56–57 To measure the antibacterial impact of a Cu GSNO combination, 24 h in vitro bacterial adhesion was measured by exposing a Gram-positive and Gram-negative bacterial strains commonly associated with hospital-acquired infections, S. aureus and P. aeruginosa, to prepared loops of each material type. Figure 5 shows a comparison of bacterial colony-forming units per cm² present on the bacteria in relation to the CarboSil control. Cu GSNO was found to have the most significant reduction, resulting in a 3-log reduction with S. aureus and a 1-log reduction with P. aeruginosa when compared to the CarboSil control.

Figure 5 –

CFU/cm² quantification of viable gram-positive (S. aureus) and gram-negative (P. aeruginosa) bacteria adhered to control and test surfaces after 24 h. The Cu GSNO combination showed the greatest reduction in viable bacteria of both strains, which can be explained by the catalytic increase in NO flux and oligodynamic role of the Cu nanoparticles. P <0.05 were considered significantly different. Note: *, $, % indicate significant difference in CFU/cm² of that sample to the CarboSil, 3% Cu, and GSNO controls, respectively. The data are means ± SD.

When isolating each material type, Cu- as well as GSNO-coated loops both demonstrated bacterial adhesion inhibition on both strains (Table 3). The Cu loop showed a 99.6% ± 0.1% and 74.1% ± 5.5% reduction in viable adhered S. aureus and P. aeruginosa, respectively. The antimicrobial effects of Cu NPs is well-documented and explains why the Cu loops showed increased antibacterial activity.^31–32 The GSNO loops showed a 99.74% ± 0.04% and 74.58% ± 12.3% reduction in viable adhered S. aureus and P. aeruginosa, respectively. However, when combining the two, the catalytic effect of copper on the nitric oxide release increased the nitric oxide flux from the surface, which corresponded with a decrease in viable bacterial adhesion to 99.94% ± 0.03% and 96.7% ± 0.5% for S. aureus and P. aeruginosa, respectively. These findings show that the NO flux from the surface of the loops is inversely proportional to the number of viable bacteria found on the surface, indicating that increasing the surface flux during the application period increases the likelihood that infection can be averted. Moreover, NO donors can be combined with other therapeutic materials such as quaternary ammonium ions, diatomaceous earth particle, and silicone oil in order to improve antimicrobial performance.^{44, 48, 58–59} Therefore, the combined bactericidal effects of the low concentration of copper with the nitric oxide donor GSNO provides an innovative way to amplify the reduction of bacterial adhesion to a polymeric surface for biomedical applications, a method that can in the future be combined with other antimicrobial materials.

Table 3 –

% of bacterial adhesion reduction in GSNO, Cu, and Cu GSNO samples compared to a CarboSil control. The data are means ± SD.

Sample	S. aureus	P. aeruginosa
GSNO	99.74% ± 0.04%	74.6% ± 12.3%
Cu	99.6% ± 0.1%	74.1% ± 5.5%
Cu GSNO	99.94% ± 0.03%	96.7% ± 0.5%

3.4. Cytotoxicity of Cu GSNO ECC loops

Antibacterial and antithrombic potential are important attributes to validate the success of a material for biomedical application, but not at the cost of any undesirable side effects like cytotoxicity towards healthy cells. Therefore, in order to establish that the antibacterial and antithrombic Cu GSNO coatings are not toxic towards mammalian cells, mouse fibroblast cells were exposed to 24 hours leachate collected from the sample. In the current study, the result showed that the material is completely safe as the viability of the mammalian cells exposed to Cu GSNO leachate was similar to that of Carbosil control as shown in Figure 6.

Figure 6 –

Cytotoxicity measurements of each sample type against 3T3 mouse fibroblast cells (n=7). The samples were soaked for 24 h in DMEM, and the resulting leachates were then added to the cells to measure for cytotoxicity. All samples showed >95% cell viability, indicating that none of the samples had cytotoxic effect. The data are means ± SD. P-values < 0.05 were used to compare.

This is in agreement with the previous report where copper nanoparticles assisted NO release from another NO donor, SNAP, was non-cytotoxic to mammalian cells while maintaining its antibacterial and antithrombotic effect.³⁶ Other studies have also tested NO donors on the mammalian system and found them to be innocuous at the tested dose.⁵⁹ In conclusion, Cu GSNO composite is not toxic to mouse fibroblast cells, providing strong evidence about its biocompatible nature. This combined with the antibacterial and antithrombic effects makes the material highly translational and supports its testing in clinical models in future.

3.5. In Vivo 4 h Extracorporeal Hemocompatibility in Rabbit Model

The ability for materials to prevent thrombus formation and platelet consumption is critical for blood contacting medical devices. Evaluation of novel materials in vivo is critical as in vitro assays are commonly required to include various anticoagulants to decrease the thrombotic nature of the blood. Similarly, the activity and viability of cells in the blood decrease as it is removed from the body, where high variability can be seen between freshly drawn and stored blood. Therefore, the ECC model is valuable in its ability to maintain fresh blood in vivo without the use of anticoagulants. Both platelet concentration (% of baseline) and overall thrombus formation were used as metrics to determine the overall hemocompatibility of the various polymer coatings.

Platelet counts were monitored over the 4 h ECC experiments, corrected for hemodilution from the IV fluids, and are summarized in Figure 7A. During the entire 4 h procedure, Cu GSNO loops maintained higher platelet counts compared to other test loops. At the end of 4 h duration, Cu GSNO loops maintained 89.3% ± 8.5% of the baseline platelet count, while GSNO, Cu, and CarboSil loops dropped to 56.71% ± 9.09%, 76.7%, and 67.6 ± 20.4%, respectively, indicating that the higher NO release exhibited by the Cu GSNO loops assisted in maintaining the platelet counts. While the platelet counts for the Cu and CarboSil loops appear to be higher than the GSNO loop, both non-NO-releasing material combinations had loops that showed complete occlusion of the ECC circuit (< 5 mL min⁻¹) prior to the 4 h end point. CarboSil control loops showed N=1 loop occluding at 2 h after loop placement, while Cu loops showed N=2 occluding at 2.5 h and 3 h (Figure 7B). Average platelet count for non-surviving loops could not be accounted for beyond point of occlusion. Because one of the CarboSil and two of the Cu loops clotted prior to the end of the experiment, no statistical significance could be calculated with these two groups. On the other hand, for both NO-releasing loops (GSNO and Cu GSNO), all 3 rabbits survived the 4 h procedure. However, though all GSNO loops survived the 4 hr duration, GSNO loops showed a lower platelet count compared to Cu GSNO loops. The higher NO release from the Cu GSNO combination showed significantly higher platelet counts compared to the GSNO loops alone (p = 0.02). This indicates that the higher levels of NO released during the 4 h period significantly increases the hemocompatibility of the device. As established in previous literature, higher levels of NO release from the surface are needed to prevent platelet activation and clot formation.³⁴ Similar elevated NO flux from ECC loops has showed to preserve >80% of the platelet count compared to control loops.³⁵ Moreover, Cu NPs combined with SNAP have shown to preserve >90% of the platelet count compared to control loops, while ECC loops with only Cu NPs or with only SNAP did not preserve platelet counts as well (~25% platelet consumption).³⁷ This can further be observed in the images of the thrombo-chambers post-procedure (Figure 8). These serve as a visual representation of the clot formation that occurred in the control loops in comparison to the lack of occlusion in the Cu GSNO samples, which can be attributed to the higher-level nitric oxide release present in the Cu GSNO loops. The CarboSil control, Cu, and GSNO loops all showed thrombus formation, while only one Cu GSNO loop showed any clot formation at the end of the 4 h procedure. The lack of thrombus formation found only with Cu GSNO samples suggests that higher levels of nitric oxide release are necessary to create a surface environment that reduces thrombus formation and continues to preserve platelet count. Similar findings have been made with on NO donors at similar higher levels of NO release are better able to reduce clot formation and prevent platelet count reduction, which improves the hemocompatibility of the device.^{37, 41} Overall, Cu GSNO loops showed the greatest percentage of platelet count and least amount of thrombus formation, indicating this combination creates the greatest hemocompatibility of the tested devices.

Figure 7 –

Analysis of the platelet count (A) based of the baseline count and rabbit survival (B) (n=3). Cu GSNO loops maintained the highest platelet count throughout the 4 h application period. Both NO-releasing groups showed 100% survival. The data are means ± SD. Statistical significance (*) was found using a standard two-tailed student t-test between the Cu GSNO and GSNO groups at 2 h and 4 h (P < 0.05). Because some CarboSil and Cu loops did not survive, t-tests could not be performed for these groups beyond 2 h and 3 h, respectively.

Figure 8 –

Representative photos of thrombo-chambers after 4 h ECC. (A) CarboSil control, (B) Cu, and (C) GSNO showed more clot formation when compared to (D) Cu GSNO.

4. CONCLUSION

In this work, Cu NPs were layered with the NO donor GSNO to create a catalyzed NO-releasing medical grade polymer to improve the hemocompatibility and reduce chances of infection of medical devices. The addition of Cu NPs and GSNO had no significantly negative effect on the surface morphology of the coated ECC loops in comparison to the bare PVC and CarboSil controls. Both the GSNO (indicated by the sulfur present in the GSNO molecule) and Cu NPs were deposited evenly throughout their respective layers and the weight percentage of Cu NPs found (~2.5 wt. %) was close to the actual amount of Cu NPs originally used (3 wt. %). The incorporation of Cu NPs increased the NO release from a maximum of 2.3 ± 0.2 ×10⁻¹⁰ mol cm⁻² min⁻¹ to a maximum of 10.3 ± 0.8 ×10⁻¹⁰ mol cm⁻² min⁻¹ during the 4 h application period, demonstrating a NO flux that is approximately five times higher than the GSNO loop. After exposing each sample type to a Gram-positive (S. aureus) and Gram-negative (P. aeruginosa) bacterial strain, the Cu GSNO showed the greatest reduction in viable bacterial adhesion, reducing the count of viable bacteria on the polymer surface by 99.94% ± 0.03% and 96.7% ± 0.5% for S. aureus and P. aeruginosa, respectively (up to 3-log reduction). The both the individual controls as well as the combination of Cu NPs and GSNO were found to be noncytotoxic towards mammalian cells as demonstrated by the CCK-8 assay performed on 3T3 mouse fibroblast. The Cu GSNO combination was able to significantly reduce overall thrombus mass and decrease platelet consumption (10.7 ± 8.5% of baseline) after 4 h exposure to blood in a rabbit ECC model. Overall, we found that the Cu GSNO combination can increase the nitric oxide released from a polymer for relevant window of time for extracorporeal blood circulation periods, resulting in a reduction in viable adhered bacteria and platelet adhesion, which suggests that this combination can improve biocompatibility while preventing infection for medical applications.