Liquid-Infused Nitric-Oxide-Releasing Silicone Foley Urinary Catheters for Prevention of Catheter-Associated Urinary Tract Infections

Abstract

Urinary catheterization is one of the most common medical procedures that makes a patient susceptible to infection due to biofilm formation on the urinary catheter. Catheter associated urinary tract infections (CAUTIs) are responsible for over 1 million cases in the United States alone and cost the healthcare industry more than $350 million every year. This work presents a liquid-infused nitric-oxide-releasing (LINORel) urinary catheter fabricated by incorporating the nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP) and silicone oil into commercial silicone Foley catheters through a two-stage swelling process. This synergistic combination improves NO-releasing materials by providing minimal SNAP leaching and a more controlled release of NO while incorporating the nonfouling characteristics of liquid-infused materials. The LINORel urinary catheter was successful in sustaining a controlled NO release over a 60 day period under physiological conditions with minimal SNAP leaching during the initial 24 h period, 0.49 ± 0.0061%. The LINORel-UC proved successful in reducing bacterial adhesion and biofilm formation for Gram positive Staphylococcus aureus (98.49 ± 2.06%) over a 7 day period in a drip flow bioreactor environment. Overall, this study presents a desirable combination that incorporates the antifouling advantages of liquid-infused materials with the active release of a bactericidal agent, an uncharted territory in aiding to prevent the risk of CAUTIs.

Graphical Abstract

INTRODUCTION

In healthy individuals, the urinary tract is a sterile environment, and implanting a urinary catheter creates an ideal milieu for bacteria to remain in the urinary tract, making it one of the most susceptible medical devices for infection.^1,2 However, urinary catheterization has become a common medical procedure to enable the drainage and removal of urine for various medical purposes, such as during and post surgeries, or for urinary incontinence. The two main types of urinary catheters are short-term use and long-term use catheters. Short-term use refers to when the catheter is only used for a few weeks and long-term use catheters are typically used for multiple months.³ Of patients undergoing short-term catheterization, catheter associated urinary tract infections (CAUTIs) occur in 10–15% of patients while virtually all patients undergoing long-term catheterization will become infected, making prolonged catheterization an imperative risk factor.^1,3–5 Extended catheterization can lead to biofilm formation, wherein the attached bacteria produce extracellular polymeric substances that colonize the surface, and allows the biofilm community to develop, gaining nutrients from the gentle flow of warm urine through the catheter.^3,6 The most common bacteria associated with CAUTIs are Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Enterococcus faecalis, and Klebsiella pneumoniae.^3,7 Once a biofilm is formed on the surface, the infection becomes harder to treat due to the formation of the extracellular polymeric substance that protects the bacteria cells, inhibiting antibiotics and other bactericidal agents from penetrating the substance and eradicating the biofilm.³ This allows free-floating (planktonic) bacteria and pathogens within the urine to adhere to the urinary catheter as a vehicle for colonization, making the patient more susceptible to infections.^2,3 Thus, CAUTIs are one of the most common hospital acquired infections, accounting for 80% of all nosocomial infections worldwide with more than 1 million cases in the United States per year.^3,7,8 These alarming statistics illustrate the necessity to develop effective means to eradicate the potential for CAUTIs.

Typically, catheters are routinely replaced to prevent the formation of infection, or the patient is given antibiotics; both undesirable solutions as to not promote antibiotic resistance and cause further discomfort to the patient.^3,6 If CATUIs are left untreated, the severity of the situation increases to the potential of causing infection in the kidneys and bloodstream.⁹ Currently, silver eluting urinary catheters are used as a treatment for eradicating CAUTIs without antibiotics.^1,10–13 Silver has been vastly researched and commercialized to be an effective antimicrobial agent. However, it is still yet to be confirmed if silver eluting urinary catheters significantly reduce CAUTIs clinically.^14,15

Nitric-oxide-releasing (NORel) materials have been readily implemented in various research areas since NO was discovered as an endogenous gas released from the endothelium and proven to be a strong bactericidal agent.^16–22 The endogenous production can be mimicked and NO can be produced exogenously by incorporating various NO donors into a polymeric material via physically blending the NO donor within the polymer,^16,21 swelling the polymer with a swelling solution,^19,23 or immobilization onto the polymer itself.^24,25 Common donors include N-diazeniumdiolates^20,25,26 and S-nitrosothiols (RSNOs), with S-nitroso-N-acetylpenicillamine (SNAP) being one of the most commonly used RSNO (Figure 1).^16,17 Nitric oxide donors allow for several advantages, including storage of NO within the polymer until the initiation of release, ability to take advantage of the tunable concentration, and adjusting the desired release kinetics of localized delivery for varying applications.¹⁷ For RSNOs, NO release initiation commences upon exposure to heat, catalyst (e.g., copper ions, Cu²⁺), visible light, and often interactions with moisture (Figure 1).^27,28 The efficacy of NO-releasing materials has demonstrated bactericidal properties in vitro using a number of materials, such as Elast-eon E2As, Tygon, and silicone rubber.^{16–19,22,28} While NO-releasing materials have proven to significantly reduce the presence of bacteria in vivo,^20,21 decreasing the risk of biofilm formation is very desirable to prevent the chance of infection from the beginning. NO-releasing materials only inhibit the proliferation of bacteria, lacking the ability to prevent bacterial adhesion. With this, the development of an antibiofouling NO-releasing material can provide vast improvements to the bactericidal properties of silicone Foley urinary catheters.

Figure 1.

(A) RSNO donor, SNAP, molecular structure. (B) NO release and disulfide bond formation mechanism from RSNOs.

Liquid-infused materials have been gaining momentum on the research front due to the fact that they create a low-adhesion interface between the material and the contacting liquid. The low-adhesion interface is created by the infiltration of the infused liquid into the polymer network where the liquid takes advantage of the capillary forces and the chemical affinity present between the infused liquid and the underlying polymer.²⁹ The slippery liquid-infused porous surfaces (SLIPS) idea originates from the thick liquid mucus lining of the gastrointestinal tract.²⁹ The mucus layer guards the vulnerable tissue in the tract from colonization of bacteria. Similarly, the Nepenthes pitcher plant utilizes a liquid water layer to create a low friction surface to prevent the attachment of insects.^30,31 These two ideas pave the way for a biocompatible, antibiofouling surface coating. SLIPS has been proven effective when a biocompatible lubricating liquid was infiltrated into a polytetrafluoroethylene (PTFE)-based system that was able to prevent up to 96–99% of common bacterial biofilms from attaching over a 7 day period solely on the mobility of the slippery interface.³² However, there is still the question on the ability of bacteria, or other microorganisms, to breach the lubricating layer, colonizing on the underlying polymeric material, and establishing “beach-heads”.³³ As SLIPS cannot influence the behaviors of planktonic microorganisms, this method only prevents the adhesion.³³ This approach could be further optimized with an antimicrobial agent.

Herein, we have combined the bactericidal properties of NO-releasing polymers and SLIPS as a synergistic approach to not only prevent the adhesion of pathogenic microorganisms and the formation of biofilms, but also prevent the growth and proliferation of those organisms in the surrounding environment. The approach of incorporating NO release with the low fouling capabilities of liquid-infused materials has been previously proven to effectively prevent biofilm formation.²² Therefore, this method is incorporated here to be further optimized in commercial urinary catheters. We use a dual swelling method, first swelling the silicone Foley catheter with solvent containing the NO donor SNAP, followed by swelling the NORel urinary catheter with silicone oil. The fabricated catheters provide the desired traits of liquid-infused materials and NO-releasing materials, while controlling the undesired burst release kinetics typical of NO-releasing materials. We demonstrate that liquid-infused nitric oxide-releasing (LINORel) urinary catheters effectively prevent the biofilm formation of pathogens commonly associated with CAUTIs over a 24 h and 7 day period. The proposed urinary catheter successfully combines the advantages of liquid-infused materials with the active release of an antibacterial agent, a novel method in aiding to prevent the risk of CATUIs that has become one of the major hospital acquired infections.

MATERIALS AND METHODS

Materials.

N-Acetyl-d-penicillamine (NAP), sodium chloride, potassium chloride, sodium phosphate dibasic, potassium phosphate monobasic, ethylenediaminetetraacetic acid (EDTA), tetrahydrofuran (THF), phosphate buffered saline (PBS), and sulfuric acid were purchased from Sigma-Aldrich (St. Louis, MO). Methanol, hydrochloric acid, silicone oil, and sulfuric acid were obtained from Fisher Scientific (Pittsburgh, PA). Silicone Foley urinary catheters (UC), size 18 Fr, were purchased from Medline Industries (Sauget, IL). Phosphate buffered saline (PBS), pH 7.4, containing 138 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate, 100 μM EDTA was used for all experiments. Staphylococcus aureus (ATCC 6538) and Pseudomonas aeruginosa (ATCC 27853) were originally obtained from American Type Culture Collection (Manassas, VA, ATCC). Luria broth (LB) and Luria Agar (LA) were purchased from Fischer BioReagents (Fair Lawn, NJ).

SNAP Synthesis Procedure.

SNAP was synthesized using a previously described method.³⁴ In short, an equimolar ratio of NAP and sodium nitrite was dissolved in a 1:1 mixture of water and methanol containing 2 M HCl and 2 M H₂SO₄. After dissolving, the reaction vessel was cooled in an ice bath in order to precipitate the green SNAP crystals. The crystals were collected via vacuum filtration, rinsed with DI water, and dried under ambient conditions. The reaction mixture and the subsequent crystals were protected from light during the entirety of the experiment.

Preparation of the SNAP Impregnated Urinary Catheters and the Liquid-Infused NO-Releasing Urinary Catheters.

The SNAP swelling solution was prepared by dissolving SNAP in THF at a concentration of 125 mg mL⁻¹, previously found to be the highest concentration of SNAP possible, due to solubility in THF.¹⁹ The silicone Foley catheter was cut into 1 cm long segments and submerged in the SNAP swelling solution for 24 h. The catheter segment was removed from the solution and dried for 24 h, allowing ample time for the THF to evaporate. Upon the completion of drying, the samples were placed in a 20 mL vial with DI water and sonicated for 5 min in a Fisher Scientific 1.9 L sonicating bath in order to remove any residual SNAP crystals adhered to the surface of the catheter segment. During the entire preparation period, the samples were kept in the dark. Using the method described above, the NORel-UC samples were prepared. The liquid-infused urinary catheters (LI-UC) and LINORel-UC were prepared by taking a 1 cm segment of the silicone Foley catheter and the NORel-UC and immersing each in silicone oil for 72 h.

Silicone Oil Swelling Characterization.

The swelling and deswelling characteristics of the LI-UC and LINORel-UC were examined. For swelling, 1 cm segments of the silicone Foley catheter and the SNAP-impregnated catheter were massed and submerged in silicone oil for a total of 72 h. These characteristics were defined by the swelling ratio, SR (eq 1).

$$\text{SR=}\frac{M_i}{M_o}$$ (1)

The SR is defined as the ratio of the mass of the infused urinary catheter containing both the mass of the oil and of the polymer (M_o) and the initial mass of the polymer (M_i), prior to swelling. At various time points, the mass of each segment was measured to determine the respective SR. The samples were kept at room temperature during swelling. The deswelling of the oil was observed by incubating the LI-UC and LINORel-UC in PBS with EDTA at 37 °C and measuring the SR at various time points for a total of 14 days.

Nitric Oxide Release Measurements from NORel-UC and LINORel-UC.

The nitric oxide release from the prepared urinary catheters was measured using a Sievers chemiluminescence Nitric Oxide Analyzer (NOA), model 280i (Boulder, CO). For the measurements, 1 cm sections of the NORel-UC or LINORel-UC (n = 3) was placed in 4 mL of PBS buffer with 100 μM EDTA in an amber glass vial at 37 °C. Upon placing the sample in the amber vial, the nitric oxide release from the surface of the catheter was purged from the surrounding buffer by a constant flow of bubbled nitrogen which was then measured by the chemiluminescence detection chamber. After each measurement, the buffer was refreshed so that it did not become saturated with neither SNAP nor silicone oil. In between NO release measurements, the NORel-UC and LINORel-UC segments were kept in 4 mL of PBS with EDTA in an incubator at 37 °C in the dark in order to mimic physiological conditions.

SNAP Leaching and Quantification from NORel-UC and LINORel-UC.

The total amount of SNAP leached from the prepared catheters during the first 24 h was measured. Segments (1 cm, n = 3) of the NORel-UC and LINORel-UC were submerged in 2 mL of PBS with EDTA and kept in an incubator at 37 °C. At each time point, 1 mL of the buffer was measured for the concentration of SNAP using a Thermo Scientific Genesys 10S UV–vis spectrophotometer. The buffer was returned back to the original sample container after each measurement to maintain the total incubation volume throughout the experiment. Absorbance for SNAP molecule has local maxima at 340 and 590 nm. Throughout the duration of this experiment, 340 nm was used.^16,35,36 The absorbance of the solution was recorded, and the concentration of SNAP in the solution was obtained from a predetermined calibration curve. Pure PBS with EDTA was used as the blank for the entire experiment. The samples were protected from light for the duration of the study.

The total amount of SNAP leached during the oil swelling study was also examined to confirm that significant amounts of SNAP were not lost during the 72 h swelling period. SNAP impregnated urinary catheter segments, 1 cm, were massed and submerged in 5 mL of silicone oil. The absorbance spectrum of the silicone oil was measured at various time points over the 72 h swelling period. Due to the fact that pure silicone oil had an absorbance of 0.0 when compared to PBS with EDTA buffer as the blank at 340 nm, the same calibration curve was used to measure the amount of SNAP within the silicone oil swelling solution.

The total wt % of SNAP impregnated into the urinary catheters was calculated. Segments (1 cm, n = 3) of the NORel-UC and LINORel-UC were submerged in 5 mL of THF and stirred for 24 h to ensure complete extraction of the SNAP molecule impregnated during the swelling period. During the SNAP extraction period, the samples were protected from light. After the 24 h period, 1 mL of the THF was measured for the concentration of SNAP using a UV–vis spectrophotometer. As described above, 340 nm was used to record the absorbance of the solution. The concentration of SNAP in the THF was calculated from a predetermined calibration curve. Pure THF was used as the blank for the entire experiment.

Bacteria Adhesion Analysis: 24 h and 7 day Exposure.

Inhibition of bacteria adhesion on the fabricated catheters was studied using models for both 24 h and 7 days. To obtain the required pathogenic cultures, S. aureus and P. aeruginosa were grown overnight in LB media to a CFU mL⁻¹ of 10⁶–10⁸. This culture was then washed twice in PBS by centrifuging at 4400 rpm for 7.5 m. This was done to get rid of waste and unused media from the overnight culture. The bacteria pellet was then resuspended with PBS to obtain the suspension culture used for the bacteria adhesion study.

For the 24 h study, UC samples (UC, LI-UC, NORel-UC, and LINORel-UC; n = 3) were incubated with the bacteria suspension culture (~10⁸ CFU mL⁻¹) in a shaker incubator (200 rpm, 37 °C). This high number was used initially to start the study as it is a short span and a preliminary test to examine if the antimicrobial efficacy is sufficient to kill a high number in a short span of time. Unlike the bioreactor study for 7 days, there was no continuous flow of nutrient media into the incubating well and no output of waste. At the end of 24 h, the samples were washed with sterile PBS to get rid of any loose bacteria and then homogenized using a homogenizer. The samples were homogenized for 1 min each to remove the attached bacteria into a consistent volume of sterile PBS for each sample. This process also helped in homogenizing any bacterial biofilm formed in the 24 h study. The bacterial solutions obtained were then serially diluted and plated on LA media plates. The plates were incubated in 37 °C for 24 h and CFU cm⁻² measurements were done from them.

For the 7 day study, a drip flow bioreactor model (Biosurface Technologies, DFR) was used to study bacteria biofilm inhibition. A modified form of the ASTM E2647–13 protocol was used for the experiment. An overnight culture of the bacteria was grown up to ~10⁶ CFU mL⁻¹. A lower initial CFU mL⁻¹ was used for the bioreactor study since infection in physiological environments start at lower counts of bacteria. Exposure to nutrients from the environment would cause an increase in number of bacteria that was expected to be seen in control UCs while antimicrobial containing UCs would prevent an increase. This culture was further processed like mentioned previously and resuspended in sterile PBS. The samples to be tested were placed in the chambers of the sterile DFR and incubated with the bacteria solution for 1 h. This 1 h incubation was done to allow for the bacteria to settle on the catheters. After an hour of incubation, nutrient media was allowed to flow through the chambers at a rate of 0.8 mL min⁻¹. This flow rate was used to mimic the conditions of urine flow in a urinary catheter and allow for low shear conditions. At the end of 7 d, catheters were homogenized and the obtained bacteria was serially diluted. The serial dilutions were plated and counted after 18 h of incubation in 37 °C.

Statistical Analysis.

Data for the 24 h and 7 day bacteria adhesion analysis is expressed as mean ± standard deviation (SD) and for all other experiments is expressed as mean ± standard error of the mean (SEM). The results between the data for the UC, LI-UC, NORel-UC, and LINORel-UC were analyzed by comparison of means using Student’s t-test. Values of p < 0.05 were considered statistically significant for all tests.

RESULTS AND DISCUSSION

Liquid-Infused Characterization of the Urinary Catheter.

In order to evaluate whether the impregnation of SNAP into the silicone Foley catheter alters the liquid-infused properties when the LINORel-UC is swelled with oil, the swelling and deswelling ratios were observed before and after the incorporation of SNAP into the urinary catheter. The immersion of the silicone Foley catheter in silicone oil expands and swells the polymer due to the polymer chains extending to maximize polymer–solvent interactions.²⁹ The swelling ratio was observed over 72 h. The presence of SNAP within the tubing increased the swelling capacity from 1.44 ± 0.003 for the LI-UC, to 1.54 ± 0.005 for the LINORel-UC, confirming that SNAP does not negatively affect the swelling capabilities of the silicone Foley catheter (Figure 2). The larger swelling ratio for the LINORel-UC is hypothesized to be due to unfavorable interactions between the polymer matrix and the crystalline SNAP that is present after the solvent swelling process.²² These interactions allow for further infusion of oil into the NORel-UC rather than what occurs within the commercial UC. The theory that the amount of infused SNAP increases the swelling ratio is also demonstrated by Goudie et al. where the presence of SNAP at a concentration of 25 mg mL⁻¹ increased the swelling ratio for silicone rubber (SR) from 1.53 ± 0.003 for the LI-SR, to 1.59 ± 0.005 LINORel-SR.²² The presented data herein supports the theory that increasing the amount of infused SNAP increases the differences in the swelling ratio between the control UC and NORel-UC especially since the difference in swelling ratios for the higher concentration of SNAP reported here is larger than the difference reported by Goudie et al. Both LI-UC and LINORel-UC catheters were able to maintain these swelling ratios for over 14 days at 37 °C. When compared to previous studies, the swelling ratio is lower with an increase in maximum swelling time.^22,29 This increase in swelling time corresponds to the decrease in the diffusion of oil from the polymer matrix, resulting in a lower swelling ratio for the urinary catheters. Because of this, the lubricating surface of the LI-UC and LINORel-UC may be affected.

Figure 2.

Swelling characteristics over 72-h period and deswelling characteristics over the remaining 14-day period of the urinary catheter in silicone oil for the LI-UC and LINORel-UC (n = 3). Error bars are excluded since they are on the order of data point size.

The sliding angle was not observed for the prepared urinary catheters as previous reports have shown that the sliding angle for liquid-infused materials was significantly reduced, with a sliding angle of 2.1°, compared to the control material that had a sliding angle of 40.1°.²⁹ Additionally, the incorporation of SNAP into the polymer has been shown to have negligible effects on the sliding angle as the LINORel material had a similar sliding angle when compared to the LI material.²² Therefore, we have substantial evidence that the liquid-infused properties stand in the presence of SNAP.

Quantification of Leaching of SNAP from NO-Releasing Urinary Catheters.

Leaching of the NO donor from the polymer can have disadvantageous effects on the longevity of the release characteristics and could result in a nonlocalized release. After solvent impregnation, the wt % of SNAP within the catheter was observed to be 11.45%. Previous work has reported SNAP to be marginally hydrophobic, meaning it would prefer to stay within the polymer, although some SNAP is likely to diffuse into the surrounding solution.^28,37 Low amounts of SNAP leaching has been found to be noncytotoxic in vivo for a variety of NO-releasing materials including silicon,²³ and Elast-eon.²¹ In addition to SNAP diffusing into the surrounding solution, NAP and NAP-dimers products form after the release of NO from the SNAP molecule and have been reported to have similar leaching patterns as SNAP.²⁸ NAP is often used to treat heavy metal poisoning and does not induce cytotoxicity within the body; therefore, small amounts of NAP and/or NAP disulfide leached from the urinary catheter into the urine or surrounding tissues would unlikely induce a toxic response in clinical applications.^28,38 However, the complete understanding on how NAP and NAP-dimer leaching impacts urethral tissues requires further investigation. Thus, in order to ensure that a minimal amount of SNAP was leached from the catheter surface, the absorbance of the PBS with EDTA buffer and the silicone oil swelling solution containing the SNAP impregnated urinary catheter was measured. Using UV–vis spectroscopy, the amount of SNAP leached from the prepared urinary catheter samples during both the first 24 h submerged in PBS and the 24 h oil swelling was determined (Figure 3). Within the first 24 h, 0.80 ± 0.077% of SNAP was shown to leach out of the NORel-UC and 0.49 ± 0.0061% of SNAP leached from the LINORel-UC, meaning that there is a high amount of SNAP retention within the polymer of the fabricated urinary catheters. The total amount of SNAP leached from the LINORel-UC was reduced by ca. 38% than that of the NORel-UC from PBS incubation and reduced by ca. 88% from oil incubation. The presence of the silicone oil in the NORel-UC successfully inhibited the additional leaching of SNAP since the lubricating layer hinders the surrounding fluid from reaching the surface. During the 72 h oil swelling period, the amount of SNAP leached did not significantly increase after 9.5 h (p > 0.05), demonstrating that swelling the UC segments in oil does not have an effect on the amount of SNAP present in the UC sample during the required swelling period. The solubility of SNAP in oil if found to be 0.4 μg mL⁻¹.²² Due to the low solubility of SNAP in silicone oil and minimal SNAP leaching during the oil swelling stage, 11.45%, was taken as the amount of SNAP loaded for both NORel-UC and LINORel-UC.

Figure 3.

Leaching characteristics of SNAP from NORel-UC and LINORel-UC during first the 24 h period of soaking in PBS at 37 °C and the 24 h leaching characteristics of SNAP from LINORel-UC during the 72 h silicone oil swelling period under ambient conditions using UV–vis spectroscopy (n = 3). Samples were protected from light throughout the study. Data represent mean ± SEM.

Nitric Oxide Release Measurements in Vitro.

Nitric oxide release was examined for both the NORel-UC and LINORel-UC using a Sievers Chemiluminescence Nitric Oxide Analyzer (Figure 4A). Over a 60 day period, it was observed that the initial NO release for the NORel-UC was 3.59 ± 0.13 × 10⁻¹⁰ mol cm⁻² min⁻¹ with a final NO release of 0.10 ± 0.04 × 10⁻¹⁰ mol cm⁻² min⁻¹, while the LINORel-UC initially released 0.4 ± 0.04 × 10⁻¹⁰ and 0.41 ± 0.05 × 10⁻¹⁰ mol cm⁻² min⁻¹ at the end of the 60 day period. The cumulative release of NO from the both the NORel-UC and LINORel-UC over the 60 day NO release period as a percentage of total loading is shown in Figure 4B. The amount of NO released is attributed to both the leaching of the SNAP molecule and the degradation of SNAP to NAP that occurs over the release period. After the 60 day period, only 21.20% of the total NO loaded was released for the NORel-UC, and 32.04% for the LINORel-UC. These low percentages indicate that there is still the capacity for the fabricated urinary catheters to release NO. Majority of the NO released from the NORel-UC is attributed to the burst release that occurs at the beginning of the testing period, while the LINORel-UC cumulative NO release is more linear and gradually increases. The NORel-UC showed an initial burst release of NO, resulting in a typical release profile consistent with other previously reported materials.^16,19,22,23 Many NO-releasing polymers exhibit this large burst release, which can affect the cytotoxicity levels and the overall efficacy of the material, so limiting this burst release phenomenon is desirable. The incorporation of the silicone oil works to prevent the large initial burst release of NO when the material first comes into contact with increased temperature. The presence of silicone oil also contributes to a more uniform and constant NO release over the anticipated release period, as shown in Figure 4A. Both of these desirable characteristics can be attributed to the fact that the slippery surface created from the infusion of silicone oil prevents total hydration of the urinary catheter due to a slow uptake up water, allowing for a more even and controlled release.

Figure 4.

(A) Average nitric oxide release measurements from NORel-UC and LINORel-UC over a 60 day period (n = 3). NO release measured from catheter samples submerged in PBS at 37 °C using a Sievers Chemiluminescence Nitric Oxide Analyzer. Data represent mean ± SEM. (B) Cumulative release of NO from NORel-UC and LINORel-UC over a 60 day period under physiological conditions resulting from the leaching and degradation of the SNAP molecule.

Biofilm Formation Inhibition in a 24 h and 7 day Drip Flow Bioreactor Model.

Biofilms are a key hindrance faced by urinary catheter during both short- and long-term operations. Free-floating (or planktonic) bacteria can come across a surface submerged in the urine and within minutes become attached. These free-floating bacteria are widely present in the microflora of the patient’s skin or urinary tract and find an easy way to the surface of the urinary catheters.^3,39 The attached bacteria then produce slimy, extracellular polymeric substances (EPS) that cover the catheter and form the conditioning film for the stationary, attached bacteria. Extracellular polymeric substance production allows the emerging biofilm community to develop a complex, three-dimensional structure that is influenced by a variety of environmental factors. This structure, now called the biofilm, protects the bacteria from antibiotics, and hence, have become a major hurdle for healthcare-associated infections.⁴⁰

In our study, we examined the fabricated catheters’ ability to inhibit bacterial infection by testing them in 24 h and 7 day models with the NO-releasing urinary catheters. It is important to note here that even though NO-releasing materials do kill bacteria within the biofilm, they are not effective in preventing biofilm formation (which contain both exopolysaccharides and bacteria themselves). Hence, through the antimicrobial analysis we expect to see a greater decrease in biofilm formation and bacteria adhesion in the LINORel-UCs due to the presence of infused silicone oil in addition to NO. The bacteria used are commonly found uropathogens: S. aureus and P. aeruginosa.⁴¹ S. aureus is a Gram-positive bacteria, and P. aeruginosa is a Gram-negative bacteria. Both have been commonly studied for antimicrobial resistance purpose. Previously, antibacterial analyses of NO-releasing urinary catheters have been mainly done with E. coli, S. epidermidis, and P. mirabilis.^42–45

For the 24 h studies, a shaking environment was established, wherein temperature was maintained at 37 °C but no nutrient media was supplied or waste discarded continuously (as seen in the 7 day bioreactor model). The total viable P. aeruginosa adhered on the catheter samples’ surface was determined after 24 h. Plate count for 24 h P. aeruginosa biofilms showed that the viable bacteria attached on the surface of the LINORel-UC samples was 98.678 ± 0.214% less than that on UC controls (Figure 5, Table 1; n = 3). This corresponded to a ca. 2 log reduction in viable bacteria growth. There was also a reduction of 72.12 ± 4.51% and 64.09 ± 5.81% on LINORel-UC when compared to NORel and LI-UC, respectively. Similarly, total viable S. aureus adhered on the catheter samples’ surface was determined after 24 h exposure at 37 °C. Plate count for 24 h S. aureus biofilms showed that the viable bacteria attached on the surface of LINORel-UC samples was 99.958 ± 0.004 less than on UC controls (Figure 5, Table 2; n = 3). This corresponded to a ca. 3.5 log reduction in viable bacteria adhesion. In addition to the reduction compared to UC control samples, the LINORel samples also displayed a 94.08 ± 0.60% and 71.43 ± 2.86% reduction in S. aureus adhesion when compared to LI-UC and NORel-UC, respectively (Figure 5; n = 3). From both the bacteria 24 h results, it was proven that LINORel-UCs provided good antimicrobial efficacy compared to the other UCs at high microbial content for 24 h exposure. Even though LI-UCs and NORel-UCs were able to provide antimicrobial efficacy, each had drawbacks which led them to have lower efficacy compared to the LINORel-UCs. With this in mind, a longer study of a 7 day bioreactor model was carried out next.

Figure 5.

S. aureus and P. aeruginosa bacteria adhesion per cm² for control commercial urinary catheter, NORel-UC, LI-UC, and LINORel-UC over 24 h period (n = 3). Data represent mean ± SD.

Table 1.

Bacteria Adhesion per cm² at 24 h for P. aeruginosa

	control-UC	LI-UC	NORel-UC	LINORel-UC
avg CFU cm−2	1.22 × 109	4.49 × 107	5.79 × 107	1.61 × 106
% reduction compared to control-UC		96.32 ± 0.83	95.26 ± 0.30	98.68 ± 0.21
p value vs control-UC		0.025	0.026	0.024
p value vs LINORel-UC	0.024	0.041	8.79 × 10−5

Table 2.

Bacteria Adhesion per cm² at 24 h for S. aureus

	control-UC	LI-UC	NORel-UC	LINORel-UC
avg CFU cm−2	4.75 × 107	3.38 × 105	7.00 × 104	2.00 × 104
% reduction compared to control-UC		99.29 ± 0.84	99.85 ± 0.10	99.96 ± 0.00
p value vs control-UC		0.179	0.177	0.177
p value vs LINORel-UC	0.177	0.303	0.206

For the 7 day study, we employed a drip flow bioreactor model to analyze the efficacy of the catheters against S. aureus. Drip flow bioreactor models have been used previously to study biofilm formation for developing antimicrobial materials that release nitric oxide.^46,47 However, in these studies, NO was released by the material only at the end of the drip flow biofilm growth by electrochemical mechanisms and not over the entirety of the experiment. Considering the ability of biofilms to grow well in a drip flow system, compared to a CDC high-shear bioreactor,⁴⁸ the antimicrobial activity of NO release would have to be continuously significant for the entirety of the study. As seen in Figure 6 (Table 3; n = 3), total viable bacteria count adhered to NORel-UC and LI-UC was reduced by 93.52 ± 1.48% and 76.38 ± 14.15% when compared to UC controls. This is due to the antimicrobial function of NO and hydrophobic nature of the LI-UCs. However, this reduction was increased for LINORel-UC at 98.49 ± 2.06%. As hypothesized earlier, this increase in reduction of adhered bacteria is possibly due to the synergistic combination of silicone oil with NO-releasing surfaces. While the oil acts as a passive method to prevent any stagnant formation of bacteria colonies, NO is the active bactericidal agent. It is important to note here that a reduction of 93.62 ± 8.72% was seen on LINORel-UC compared to NORel-UC. As mentioned in the previous section, the continuous release of NO overtime also contributes to an even release and prevents bacterial colonization even with a 7-day period of exposure to common nosocomial pathogens in physiological implantation conditions.

Figure 6.

Bacteria adhesion for control commercial urinary catheter, NORel-UC, LI-UC, and LINORel-UC after 7 days of exposure to S. aureus in a drip flow bioreactor study (n = 3). Data represent mean ± SD.

Table 3.

Bacteria Adhesion per cm² at 7 days for S. aureus

	control-UC	LI-UC	NORel-UC	LINORel-UC
avg CFU cm−2	2.06 × 107	4.87 × 106	1.33 × 106	3.11 × 105
% reduction compared to control-UC		76.38 ± 14.15	93.52 ± 1.48	98.49 ± 2.06
p value vs control-UC		0.137	0.092	0.085
p value vs LINORel-UC	0.085	0.116	0.028

CONCLUSION

Here, the antifouling advantages of liquid-infused materials was incorporated with the active release of an antibacterial agent in a silicone Foley catheter, through the two stage swelling of silicone oil and the NO donor SNAP. The presence of SNAP in the silicone Foley catheter proved to have no significant effects on the slippery properties of the surface. However, to our advantage, the implementation of silicone oil successfully aided in controlling not only the initial NO burst release, typical of NO-releasing materials, but provided an overall controlled and consistent release over a 60 day period, due to the lubricating layer hindering the total hydration of the urinary catheter surface. There was a high retention of SNAP within the LINORel-UC, only leaching 0.031 ± 0.0004 mg SNAP mg⁻¹ tubing, while the NORel-UC leached 0.050 ± 0.0049 mg SNAP mg⁻¹ tubing. The NORel-UC exhibited an initial NO-release of 3.59 ± 0.13 × 10⁻¹⁰ mol cm⁻² min⁻¹ and a final release of 0.10 ± 0.04 × 10⁻¹⁰ mol cm⁻² min⁻¹ over the 60 day period, while the LINORel-UC NO-release had a more consistent release between 0.4 ± 0.04 × 10⁻¹⁰ mol cm⁻² min⁻¹ and 0.41 ± 0.05 × 10⁻¹⁰ mol cm⁻² min⁻¹ over the 60 day period. The LINORel-UC exhibited a steady NO release, prolonging the desired properties of the liquid-infused nitric oxide releasing urinary catheter. Bacterial adhesion and biofilm formation were examined over a 24 h well model and a 7 day drip flow bioreactor model. In the 24 h model, 98.68 ± 0.21% and 99.96 ± 0.00% reduction was seen in P. aeruginosa and S. aureus viable bacteria adhesion, respectively. This reduction was higher than those seen in LI-UCs and NORel-UCs. Similarly, a 98.49 ± 2.06% reduction was noted for Gram positive S. aureus for the LINORel-UCs in the 7 day drip flow bioreactor model. Both of the positive results seen from different time spans in different models of bacteria exposure confirm that LINORel-UCs do in fact have a lasting antimicrobial function and may be tested further for long-term applications. Overall, the results suggest that the synergistic combination of the active release of NO and the passive release of silicone oil to prevent biofilm formation on silicone Foley catheters potentially provides a promising application in reducing the risk of CAUTIs.