Prevention of Medical Device Infections via Multi-action Nitric Oxide and Chlorhexidine Diacetate Releasing Medical Grade Silicone Biointerfaces

Abstract

The presence of bacteria and biofilm on medical device surfaces has been linked to serious infections, increased health care costs, and failure of medical devices. Therefore, antimicrobial biointerfaces and medical devices that can thwart microbial attachment and biofilm formation are urgently needed. Both nitric oxide (NO) and chlorhexidine diacetate (CHXD) are possess broad-spectrum antibacterial properties. In the past, individual polymer release systems of CHXD and NO donor S-nitroso-N-acetylpenicillamine (SNAP) incorporated polymer platforms have attracted considerable attention for biomedical/therapeutic applications. However, the combination of the two surfaces has not yet been explored. Herein, the synergy of NO and CHXD was evaluated to create an antimicrobial medical-grade silicone rubber (SR). The 10 wt% SNAP films were fabricated using solvent casting with a topcoat of CHXD (1, 3, and 5 wt%) to generate a dual-active antibacterial interface. Chemiluminescence studies confirmed the NO release from SNAP-CHXD films at physiologically relevant levels (0.5 – 4 × 10⁻¹⁰ mol min⁻¹ cm⁻²) for at least 3 weeks and CHXD release for at least 7 d. Further characterization of the films via SEM-EDS confirmed uniform distribution of SNAP and presence of CHXD within the polymer films without substantial morphological changes, as confirmed by contact angle hysteresis. Moreover, the dual-active SNAP-CHXD films were able to significantly reduce E. coli and S. aureus bacteria (> 3-log reduction) compared to controls with no explicit toxicity towards mouse fibroblast cells. The synergy between the two potent antimicrobial agents will help combat bacterial contamination on biointerfaces and enhance the longevity of medical devices.

Graphical Abstract

Synergistic effect of S-nitroso-N-acetyl-penicillamine (SNAP) and Chlorhexidine diacetate (CHXD) to reduce bacterial contamination and enhance the durability of medical devices. The multi-action of the two bactericidal compounds leads to >3-log reduction in viable bacterial colonization on surface.

Introduction

Indwelling medical devices, like intravascular and urinary catheters, are the major source of catheter-associated bloodstream or urinary tract infections. Biomedical devices provide a suitable surface for bacterial adhesion which can eventually lead to biofilm formation. Devices that succumb to biofilm buildup on the surfaces often tend to have compromised functionality with reduced durability that can extend hospital stays and increase the financial burden on patients. In the US alone, a total of 250,000 cases of device-associated infections are reported every year.¹ When bacteria from the surrounding environment encounter a medical device, the first step of the multifaceted process is attachment to the surface. The attachment of bacteria is followed by uninhibited proliferation on the surface which then leads to the production of the extracellular polymeric substances (EPS) comprising of eDNA, genes, polysaccharides, lipids, enzymes, proteins, etc. The protective EPS matrix not only provides nutrients to growing bacteria but also protects embedded bacteria from the action of traditional antibiotics that fail to penetrate the defensive layer. Over time, bacteria protected within the biofilm can shift from sessile state to planktonic and use these indwelling medical devices as a port of entry into the body, leading to many chronic, nosocomial, and medical device-related infections.^2–6

When it comes to bacterial pathogenesis, both Gram-positive and Gram-negative bacterial species have been seen to form biofilms on medical device surfaces. Despite keeping the surroundings clean and sterilization of medical devices before use, many medical implants and prosthetic devices can succumb to bacterial contamination in both short- and long-term applications. This situation has become worse due to the emergence of antibiotic resistance in bacteria. It is estimated that >1000 times dosage of antibiotic is required to eradicate bacteria encapsulated within the biofilm as opposed to free-floating bacteria.^{7, 8} Due to the shift in efforts of managing cases, the COVID-19 emergency has diverted the focus of conventional infection control tactics which has resulted in a >50% increase in central line-associated bloodstream infections (CLABSI) rates.^{9, 10} Therefore, there is a dire need for broad-spectrum antimicrobial approaches that can act upon the pathogenesis of microorganisms.

Infections arising from medical devices can be modulated by developing infection-controlling strategies that can inhibit catheter-related infections and radically lower the instances of morbidity, mortality, and associated medical care costs. Previous studies have reported bactericidal or bacteria resistant approaches to reduce the microbial burden on medical devices. These strategies include impregnation of antimicrobial agents such as silver, zinc^{11, 12} in silicone-based polymers^{13, 14}, anti-fouling mechanisms (e.g., SLIPs, zwitterions)^14–17, antibiotic and antimicrobial coated/impregnated catheters (chlorhexidine (CHXD), silver sulfadiazine, rifampicin, auranofin, etc.)^18–21, and nitric oxide (NO)-releasing therapeutic approaches.^{22, 23}

Chlorhexidine is an extensively used antimicrobial agent in hospital-based settings for skin disinfection and as well as prevention of bacterial contamination on common surfaces.²⁴ The positively charged CHXD binds to the negatively charged bacterial cell wall. The strong binding of moieties leads to loss of membrane integrity and malfunctioning of proteins and enzymes that causes cellular damage and cytoplasmic leakage resulting in microbial death.^25–28 The broad-spectrum antibacterial properties of CHXD have been widely studied for long-term antibacterial applications. Several researchers have reported sustained release from CHXD impregnated, incorporated, and coated surfaces for the use of medical devices such as dental implants, vascular catheters, and antimicrobial dressings.^29–33 Some of these techniques have also made it to pre-clinical stages and are currently being used to treat catheter-associated infections in patients.¹⁹ For example, CHXD and silver sulfadiazine impregnated catheters are commercially available. However, infections on medical devices persist and are continuing to rise.³⁴ One solution to avoid using higher concentrations of CHXD that are toxic to mammalian cells is to combine CHXD with other antibacterial agents that can augment antibacterial properties and as well as enhance the biocompatibility of medical devices.

Additionally, NO is an endogenous gas molecule responsible for several regulatory functions such as vasodilation and inhibition of platelet activation. Immune cells such as macrophages and neutrophils utilize NO to fight the invading pathogen by exhibiting antimicrobial and anti-inflammatory properties.³⁵ To exogenously mimic the innate functions of NO, several NO donors have been synthesized and integrated with medical-grade polymers to develop antimicrobial, hemocompatible, and biocompatible surfaces. Synthetic and endogenous S-nitrosothiols, such as S-nitroso-N-acetyl-penicillamine (SNAP) and S-nitrosoglutathione (GSNO), NO donors have demonstrated improved stability when incorporated within polymer matrices for the use of medical device application.³⁶ These polymers have also been shown to release NO at physiologically relevant levels by the means of catalyst (heat, light, or metal ion).³⁷ Nitric oxide released from the polymer systems can emulate NO release from endothelial cells at a surface flux of 0.5 – 4 × 10⁻¹⁰ mol cm⁻² min⁻¹, to prevent platelet activation and adhesion.³⁸ SNAP incorporated polymers utilizing medical-grade polyurethane-based silicone elastomers (e.g., CarboSil, ChronoSil, silicone rubber) have been extensively studied to generate NO-releasing medical devices with prolonged and regulated NO release.^39–42 However, the levels of NO emitted from these matrices can eventually decrease as the NO payload is depleted, which might restrict their ability to fully eradicate the bacteria in long-term applications. Therefore, combining NO-releasing materials with other broad-spectrum antibacterial agents, such as CHXD, that can elevate long-term applications is one possible solution to bacterial colonization on biomaterials.

Previously, both SNAP and CHXD have been individually incorporated/impregnated within polymer systems. However, the combination of these two compounds as an infection control strategy has not been studied to date. This study aims to combine SNAP and CHXD in a single polymer matrix to achieve amplified antibacterial properties for the use of potential biomedical applications. This material design can also be applied to other polymer-based medical device interfaces such as endotracheal tubes, insulin cannulas, and hemodialysis catheters that face the challenges of bacterial contamination. In this study, a commercial-grade silicone rubber (SR) polymer was used to fabricate SR films with dual antibacterial properties comprising of NO donor SNAP and CHXD using the solvent evaporation and dip-coating method. Varying concentrations of CHXD were optimized on the SNAP incorporated SR and tested for cytocompatibility towards NIH 3T3 mouse fibroblast films. Examination of the surface characteristics of films was carried out via contact angle, scanning electron microscopy, and elemental mapping of modified and unmodified films. The NO release from the samples was tested over 4 weeks using a chemiluminescence method and leaching of NO donor and CHXD in the soaking buffer was assessed using the UV-vis spectroscopy technique. Finally, the antibacterial potential of the films was investigated in a 24 h bacterial adhesion assay against E. coli (Gram-negative) and S. aureus (Gram-positive) bacteria, two major bacterial strains frequently linked to infections arising from medical devices.

2. Materials and Methods

2.1. Materials

Ethylenediaminetetraacetic acid (EDTA), tetrahydrofuran (THF), and sterile phosphate buffer saline powder with 0.01 M, pH 7.4, containing 0.138 M NaCl, 2.7 mM KCl, were purchased from Sigma Aldrich (St. Louis, MO). S-nitroso-N-acetylpenicillamine was purchased from PharmaBlock (Hatfield, PA). Chlorhexidine diacetate hydrate and glutaraldehyde solution (50%/Certified/BioReagent) were obtained from Fisher Scientific (Hampton, NH). All aqueous solutions were prepared using deionized water. Phosphate buffer saline (PBS) 0.01M with 100 μM EDTA was used for all material characterization and NO analyzer studies. Dulbecco’s modified Eagle’s medium (DMEM) and trypsin-EDTA were purchased from Corning (Manassas, VA20109). The Cell Counting Kit-8 (CCK-8) was purchased from Sigma-Aldrich (St. Louis, MO). Antibiotics penicillin-streptomycin (Pen-Strep) and fetal bovine serum (FBS) were obtained from Gibco-Life Technologies (Grand Island, NY). The bacterial strain S. aureus (ATCC 6538), E. coli (ATCC 25922), and 3T3 mouse fibroblast cells (ATCC 1658) for cytotoxicity studies were obtained from American Type Culture Collection (ATCC). All the buffers and media used in microbial and tissue culture were sterilized in an autoclave at 121 °C, 100 kPa (15 psi) for 30 minutes prior to use.

2.2. Preparation of SR-SNAP-CHXD polymer films

DOWSIL^™ 3–1944 RTV silicone rubber with or without 10 wt% of SNAP was prepared by a solvent evaporation method. Films were prepared to fabricate four test groups: SR, SR-CHXD, SR-SNAP, and SR-SNAP-CHXD (Figure 1). To prepare the polymer solution for the base films, 270 mg of SR was weighed out and dissolved in 1 mL of THF and 30 mg of SNAP was added to achieve 10wt% (w/w) as the final concentration of SNAP. The solution was vortexed until all the SNAP crystals were completely dissolved and then cast into the circular Teflon molds of 2.5 cm diameter. The polymer films in the mold were cured overnight in a fume hood initially and then 24 h in the vacuum desiccator enabling the solvent to evaporate completely. For the corresponding controls without SNAP, 300 mg of SR was dissolved in 1 mL of THF and cast resulting in blank SR films. Once all the films were cured, smaller disks of diameter 0.65 cm were cut from the parent base film. Table 1 describes all the sample types fabricated for this study and highlights the methodology followed to fabricate films. All the base films (SR and SR-SNAP) were first top-coated with 3 topcoats of a plain SR topcoat solution (1 mL SR-THF solution at 300 mg mL⁻¹ concentration) at a 15 min interval. The films were allowed to dry for 2 h at room temperature. To prepare the polymer solution for the CHXD topcoat, 297, 291, and 285 mg of SR was weighed out and dissolved in 1 mL of THF with 3, 9, and 15 mg of CHXD to achieve 1, 3, and 5 wt% (w/w) as the final CHXD concentration, respectively. To generate the 1, 3, and 5 wt% CHXD coated films, the SR and SR-SNAP films were top coated with 2 topcoats of the CHXD solution at 15 min intervals, resulting in SR-CHXD and SR-SNAP-CHXD films. For corresponding SR and SR-SNAP controls without CHXD, films were top coated with 1 mL of SR dissolved in THF (2 topcoats at 15 min interval) at 300 mg mL⁻¹ concentration to result in SR and SR-SNAP films. The small disks were dried overnight and then dried under vacuum for an additional 24 hours. This was done to remove any residual THF and ensure the polymers were cured. The weights and thickness of the final films were recorded using a Mettler Toledo analytical balance and a Mitutoyo micrometer. The prepared polymer samples were kept in the freezer (−20 °C) in the dark to retain their NO-releasing properties before further analysis.

Figure 1.

A) Methodology to fabricate SR, SR-SNAP, SR-CHXD and SR-SNAP-CHXD films. B) Schematic representation of SR-SNAP-CHXD films to combat medical device infections. SR-SNAP films are top coated with SR-CHXD to generate dual-active antimicrobial surfaces.

Table 1.

Composition for each sample type used in the study

Sample type	Base film	Topcoat
SR	300 mg mL−1 SR	5 topcoats of 300 mg mL−1 SR
SR-SNAP	300 mg mL−1 SR with 10 wt% SNAP	5 topcoats of 300 mg mL−1 SR
SR-CHXD	300 mg mL−1 SR	3 topcoats of 300 mg mL−1 SR, 2 topcoats of 300 mg mL−1 SR with 1,3,5 wt% CHXD
SR-SNAP-CHXD	300 mg mL−1 SR with 10 wt% SNAP	3 topcoats of 300 mg mL−1 SR, 2 topcoats of 300 mg/mL SR with 1,3,5 wt% CHXD

2.1. Characterization of SNAP-CHXD films for optimized NO-releasing polymers

2.2.1. NO-release measurements

NO release kinetics from the films was evaluated using Zysense chemiluminescence Nitric Oxide Analyzer (NOA) 280i (Zysense, Frederick, CO). For this, samples were submerged in NOA samples cell with Phosphate Buffer Saline (PBS) substituted with 100 μM EDTA (pH 7.4). EDTA was added to the buffer to quench the metal ion activity from water that can non-specifically catalyze NO from SNAP. The temperature of the system was maintained at 37 °C to emulate the physiological conditions. The NO released from the polymer films in the NOA cell was constantly carried into the reaction chamber via highly purified nitrogen gas at a continuous flow rate of 200 mL min⁻¹. The cell pressure of the NOA cell ranged from 8.9 to 9.7 torr with a supply pressure between 6.4–6.6 psi. All experiments were performed in triplicates protected from ambient light.

To quantitate the real-time NO release under physiological conditions, SR-SNAP and SR-SNAP-CHXD films (n=3) were each placed in 2 mL of PBS-EDTA in an NOA sample cell. The samples were tested until the steady-state of NO release was attained. The films were incubated in PBS-EDTA at 37 °C and the NO release from the films was recorded at various time points. The amount of NO released from the samples (ppb/ppm) was normalized with the surface areas of the samples to obtain the NO flux levels with mol cm⁻² min⁻¹ units. For evaluating the effect on the presence of CHXD, films were tested in a short-term experiment up to 24 h with all different CHXD concentrations (1, 3, and 5 wt% CHXD topcoat). To understand the full potential of films, optimized concentrations of SR-SNAP-CHXD (5 wt%) were compared to SR-SNAP films in a long-term NO release study up to 4 weeks. For the long-term study, the soaking buffer was replaced on each testing day during the incubation to prevent the saturation of SNAP leached in the soaking buffer.

2.2.1. Effect of sterilization on NO release

To test the sterilization compatibility of SR-SNAP-CHXD films, samples were exposed to the EO sterilization process under AN 74i Anprolene EO gas sterilizer (Anderson Sterilizers). For this, SR-SNAP-CHXD films were packaged in a peel pouch and sterilized under EO gas under 24 h cycle followed by 2 h of air purging. To maintain a minimum of 35% humidity, a Humidichip was added to the sterilization bag and the whole process was done at room temperature. To test the influence of UV-light on the SNAP stability and corresponding NO release, SR-SNAP-CHXD films were exposed to UV-light under biosafety cabinet administered by REDISHIP Purifier^® Logic^®+ Class II A2 Biosafety Cabinets, Labconco^® for 30 min. NO release from the sterilized samples was measured using the same methodology as Section 2.1.1. Samples were tested for their NO release on day 0 and day 1 and compared to freshly prepared samples that were not exposed to any sterilization process.

2.2. Evaluation of SNAP and CHXD release from polymer films

The amount of SNAP and CHXD released from the polymer films into the soaking buffer was measured by recording the absorbance of buffer solutions for up to 1 week. Three samples for each type of film were prepared and weighed before initiating the study. These films were then soaked in PBS (with EDTA) at 37 °C. A UV-vis spectrophotometer (Cary 360, Agilent Technologies) was used to measure the absorbance of the buffer solutions every day. The absorbance of solutions was measured using Quartz cuvette at 340 and 255 nm which is the maxima in the UV-Vis absorbance spectra for SNAP and CHXD, respectively.⁴³ The calibration graph of SNAP and CHXD in PBS with EDTA was used to interpolate the absorbance measurements recorded from the study and convert them to concentrations of the compound in the measured sample. Care was taken to make sure that the buffer solution amount for each sample was maintained at the same amount throughout the experiment to avoid any inconsistent readings and three replicates were used for each measurement.

2.3. Characterization of films

2.3.1. Static contact angle measurement

Polymer films that were compatible with the goniometer instrument were prepared by spin coating enabling the reproducible evaluation of any effects of SNAP or CHXD on the surface wettability of the samples. To measure the static water contact angle, thin films were prepared by spin coating SR, SR-SNAP, and SR-CHXD solutions on 15 mm Deckgläser Cover Glasses using a programmable MTI VTC-100A spin coater. The static contact angle measurement for the SR, SR-SNAP, SR-CHXD, and SR-SNAP-CHXD polymer matrixes were conducted using Ossila Contact Angle Goniometer with sessile drop method with deionized water. A 10 μL of deionized water was dropped on different locations on film and the droplet was allowed to sit for 10 seconds before recording the values. Four replicates were used for each measurement, and the average of left and right contact angles was measured via the Ossila software.

2.3.2. Scanning electron microscopy equipped with elemental mapping

Surface morphology and composition of SR, SR-SNAP, SR-CHXD, and SR-SNAP-CHXD were evaluated using a scanning electron microscope and elemental mapping techniques, respectively. All the films were sputter-coated with a 10 nm gold-palladium mixture using a sputter coater (Leica Microsystems). Field emission scanning electron microscopy (FESEM) instrument (FEI Teneo, FEI, Inc., Hillsboro, OR USA) with an accelerating voltage of 5 kV at a working distance of 10 mm was utilized to obtain surface morphology images. Energy-dispersive X-ray spectroscopy (EDS –Oxford instruments) with an accelerating voltage of 20 kV, was used to evaluate the dispersion of chlorine (Cl) on the surface of CHXD coated films and sulfur (S) in the cross-section of SNAP incorporated samples as the characteristic element of SNAP composition.

2.4. Antibacterial efficacy of dual-active SNAP-CHXD polymer films

2.4.1. 24 h bacterial adhesion assay

To assess the antibacterial efficacy of the polymer films, a 24 h antibacterial adhesion assay was used. All the samples SR, SR-SNAP, SR-CHXD, and SR-SNAP-CHXD were tested against two strains of bacteria E. coli and S. aureus. Bacterial cultures were prepared by inoculating single isolated colonies in LB media that were incubated and grown for 5 h at 120 rpm at 37 °C. Bacteria from the stock were extracted at the mid-log phase, washed in PBS at 3500 rpm, 7 min to remove the dead cells and debris. Samples were exposed to a bacterial solution with adjusted optical density (O.D) of 0.5 measured at 600 nm wavelength corresponding to a final concentration of bacteria ~ 10⁷ colony forming units (CFU) mL⁻¹. Polymer films with bacteria were incubated at physiological conditions (pH 7.4, 37 °C) for 24 h at 120 rpm. To evaluate the viable colonies adhered on the surface of films, polymer films were transferred into fresh PBS buffer, and homogenized and vortexed for 60 sec each. Diluted samples were then plated on the L.B agar media using a bacteria Spiral Plater (Eddy Jet 2W, IUL Instruments) (Log mode 100 uL). Results were recorded based on the growth of viable colonies on the agar plate after overnight incubation at 37 °C utilizing an automated bacteria colony counter (Sphere Flash, IUL Instruments). The results obtained from the study were normalized by the surface area of the films and represented as the percent reduction in the adherence of viable bacteria on the test surface (SR-CHXD, SR-SNAP, and SR-SNAP-CHXD) with respect to the control surface (SR) determined by Equation 1.

$$\%\mathit{\text{bacterial}}\mathit{\text{reduction}}=\frac{(SR\mathit{\text{control}})-(\mathit{\text{Test}})\times 100}{(SR\mathit{\text{control}})}$$ (1)

2.4.2. Bacteria morphological analysis

S. aureus and E. coli adhered on the surface were visualized using SEM to analyze the effect of CHXD and SNAP on morphological changes of Gram–positive and –negative bacteria strains. The SEM analysis was carried out using the previously reported method with slight modifications.⁴⁴ Bacteria cultured in LB media overnight were diluted to 10⁷ CFU mL⁻¹ concentrations and samples cut into disks with 0.65 cm diameter were placed in 2 mL microcentrifuge tubes. Each sample (SR, SR-CHXD, SR-SNAP, SR-SNAP-CHXD) was exposed to 1 mL bacteria culture in a shaker incubator at 37 °C and 120 rpm. After 24 h of incubation, samples were gently taken out and rinsed with fresh sterilized PBS to detach the loosely adhered bacteria from the surface and immersed in 3% glutaraldehyde in 0.1 M PBS solution overnight to fix the samples. Samples were dehydrated with an increasing amount of ethanol with 20 min intervals (50, 60, 70, 80, 90, and 100% v/v in DI water). Finally, samples were left at ambient temperature to dry completely in the fume hood, protected from light. Samples were mounted on double-sided carbon tape on an SEM specimen mount and sputter-coated with a 10 nm thick gold-palladium mixture using Leica EM ACE600 coater (Leica Microsystems, Wetzlar Germany). Microscope images of bacteria on the surface of different samples were obtained using a FESEM instrument (FEI Teneo, FEI, Inc., Hillsboro, OR, USA) with an accelerating voltage of 5 kV at a working distance of 10 mm.

2.5. In vitro cytocompatibility study

The compatibility of films with mammalian cells was assessed by following the ISO standards (ISO10993–5:2009 Test for evaluating compatibility of medical devices in vitro). This was done as per the previously reported protocol with slight modification.⁴⁵ All test and control group SR, SR-SNAP, SR-CHXD, and SR-SNAP-CHXD samples (n = 3) each were cleansed with 70% ethanol and sterilized under UV-light in biosafety cabinet for 15 mins on each side. Each sample was submerged in complete DMEM media supplemented with 10% FBS and 1% Penicillin-streptomycin mixture (2 mL) and incubated for 24 h at 37 °C, 5% CO₂. The leachate of the samples was then used for exposure to the cells and further analysis.

NIH 3T3 mouse fibroblast cell line was used to test the cytocompatibility of the films. Cells at 5000 cells/ well were seeded into a cell-culture treated 96-well plate and incubated for 24 h at 37 °C, 5% CO₂ in humidified conditions. After 24 h, the old media from the wells was aspirated out and substituted with an equivalent volume of leachates prepared in Section 2.8.1 and exposed to the confluent cells for each specific sample type. Cells exposed to leachates were then incubated for an additional 24 h to allow the leachates to act out on cells. All experiments were done in triplicates. After 24 h, CCK-8 was added to the cells and incubated for 2 h (10 μL). The absorbance of the cells was measured at 450 nm wavelength using a micro well-plate reader (Cytation 5 imaging multi-mode reader, BioTek). Results from the study are represented as cell viability of test group (SR, SR-SNAP, SR-CHXD 1,3, and 5 wt%, and SR-SNAP-CHXD 1,3, and 5 wt%) relative to control 3T3 fibroblast cells in media alone that received no treatment.

$$\mathit{\text{Relative}}\mathit{\text{cell}}\mathit{\text{viability}}(\text{\%})=\frac{\mathit{\text{absorbance}}\mathit{\text{treatment}}\mathit{\text{group}}}{ \mathit{\text{absorbance}}\mathit{\text{cell}}sin\mathit{\text{media}}\mathit{\text{without}}\mathit{\text{treatment}}}\times 100$$

2.6. Statistical Analysis

All experimentations in this study were conducted with a sample size n ≥ 3. Data obtained from the study are presented as mean ± standard error of the mean (SEM). To ascertain the statistical significance between the test (SR-SNAP-CHXD) and control group (SR, SR-SNAP, SR-CHXD), an unpaired Student’s t-test was used with an assumption of unequal variance. p values of < 0.05 were considered statistically significant.

3. Results

4.1. NO release kinetics

The physical attributes of the sample weight and dimensions of the polymer films were recorded using an analytical balance (Mettler Toledo^™ XS105DU, Columbus, OH) and a micrometer. The weights of the films before and after top coating were found to be 9.38 ± 0.53 and 19.55 ± 1.21 mg, respectively. All the films synthesized in the study (SR, SR-SNAP, SR-CHXD, and SR-SNAP-CHXD) were of uniform weight and thickness. The addition of five topcoats on the base films resulted in a corresponding increase in the thickness of the disks before and after topcoats from 0.36 ± 0.01 to 0.85 ± 0.08 mm, respectively. To understand if the addition of CHXD on the SNAP loaded polymer affects the NO release, the SR-SNAP and SR-SNAP-CHXD films were tested for the NO release kinetics using the chemiluminescence method under physiological conditions (Figure 2A). While the SR-SNAP control films exhibited an initial flux of 5.46 × 10⁻¹⁰ mol min⁻¹ cm⁻², SR-SNAP-CHXD with 1, 3, and 5 wt% CHXD exhibited 6.54, 6.06, and 5.67 × 10⁻¹⁰ mol min⁻¹ cm⁻², respectively (Figure 2B). The NO release from the study demonstrated no significant differences in the release kinetics with the presence of CHXD on the surface irrespective of CHXD concentration. In a longer-term study, the SR-SNAP and SR-SNAP-CHXD with 5 wt% CHXD were tested over 4 weeks (Figure 2C). Results from the long-term study demonstrated similar NO release levels for samples with and without CHXD present in the topcoat. The initial and final NO release levels from samples are reported in Table 2.

Figure 2.

(A) The chemical structure of NO donor S-nitroso-N-acetylpenicillamine (SNAP). The RSNOs, such as SNAP, have the capacity to be triggered by heat, light, or metal ions to cleave the S-N bond and release NO. (B) The NO release levels from SR-SNAP and SR-SNAP-CHXD with 1, 3, and 5 wt% CHXD top coated films tested using Nitric oxide analyzer for up to 24 h. (C) Long-term NO release quantification from SR-SNAP and SR-SNAP-CHXD(5wt%). (D) Effect of ethylene oxide and UV-sterilization process on NO releasing SR-SNAP and SR-SNAP-CHXD 5wt% films. All NO release studies were performed at physiological conditions of pH 7.4 and 37 °C. Data represents mean ± SEM (n ≥ 3).

Table 2.

Initial and final NO release kinetics from SR-SNAP and SR-SNAP-CHXD (5wt%)

	NO release (x 10−10 mol min−1 cm−2)
Sample type	Initial (Day 0)	Final (Day 29)
SR-SNAP	5.46 ± 0.64	0.28 ± 0.03
SR-SNAP-CHXD (5 wt%)	5.67 ± 0.31	0.41 ± 0.15

4.2. Ethylene oxide (EO) and Ultraviolet (UV) light sterilization

To determine the stability of SR-SNAP-CHXD (5 wt%) films to withstand clinically relevant sterilization processes, in this study ethylene oxide and ultraviolet light sterilization were utilized to determine the suitable sterilization method for the SNAP and CHXD containing polymer films. Samples were first synthesized using the dip-coating method followed by EO and UV light sterilization for 24 h and 30 min, respectively. After exposure, the NO release from the samples was quantified and compared to NO release from fresh samples (Figure 2D). While the freshly prepared samples released NO at 5.67 ± 0.31 × 10⁻¹⁰ mol min⁻¹ cm⁻² NO flux, both EO and UV-light exposed samples exhibited 4.87 ± 0.41 and 4.90 ± 0.33 × 10⁻¹⁰ mol min⁻¹ cm⁻² NO flux, respectively on day 0. There was a slight reduction observed in the level of NO release from sterilized samples compared to freshly prepared samples on day 0. However, the difference between the NO release of fresh and sterilized samples was not statistically significant (p > 0.05) and the levels of NO continued to be at physiologically relevant levels post-sterilization for at least 24 h following the same trend as freshly prepared samples

4.3. In vitro analysis of SNAP and CHXD release in soaking buffer

Diffusion of SNAP and CHXD from polymer films into the PBS buffer was analyzed over a 7 d period using UV-vis spectroscopy. For this, SR-SNAP and SR-CHXD films were soaked in PBS-EDTA at 37 °C. At each time point, the soaking buffer was collected and analyzed to examine the amount of CHXD and SNAP that diffused out of the polymer films. To quantify the amount of SNAP and CHXD leached into the solution, a standard curve of both SNAP and CHXD was prepared in PBS-EDTA and plotted. The molar extinction coefficient of CHXD and SNAP in PBS-EDTA at room temperature was determined to be 2670 M⁻¹ cm⁻¹ and 999 M⁻¹ cm⁻¹ at 255 and 340 nm, respectively (Figure 3A–D). After 24 h, 0.27 ± 0.01 μg CHXD and 0.48 ± 0.03 μg of SNAP mg⁻¹ of total film mass was detected in the buffer. In both cases, CHXD and SNAP diffusion was observed to be higher on the initial days due to the water-rich polymer layer that enables more rapid diffusion of these species into the buffer. However, the diffusion of the compounds stabilized with each testing day (Figure 3E). The results indicated that a total of 0.47 ± 0.02 μg CHXD mg⁻¹ and 2.06 ± 0.08 μg SNAP mg⁻¹ polymer diffused out of the polymer films after 7 d of soaking.

Figure 3.

UV-vis calibration curve of (A) CHXD and (B) SNAP in PBS-EDTA buffer. The characteristic peak of (C) CHXD was observed at 255 nm and (D) SNAP at 340 nm wavelength. (E) Using the standard curve at 255 and 340 nm wavelength, the amount of CHXD and SNAP released from the polymer films was assessed for 7d at physiological conditions of pH 7.4 and 37 °C in PBS buffer. Data represents mean ± SEM (n ≥ 3).

4.4. Surface characterization

4.4.1. Characterization of polymer surface via contact angle

Static contact angle measurements provide insights into the surface wettability of the polymers by determining the hydrophobicity or hydrophilicity of the surface, where materials at or above 90° are classified as hydrophobic. To analyze how the modification of the surface through the incorporation of SNAP or top coating with CHXD altered the surface wettability, the static contact angle of water on the surface of the films was evaluated (Figure 4). The unmodified surface (SR) showed an initial water contact angle of 92.47 ± 2.14°. Both SR-SNAP and SR-CHXD exhibited a water contact angle of 96.16 ± 2.69° and 90.66 ± 1.80°, respectively, indicating that the surface hydrophobicity was not significantly affected by the inclusion of the SNAP and CHXD. Similarly, the results from SR-SNAP-CHXD films showed 94.59 ± 4.09°. When comparing the SR-CHXD film to the SR control, a slightly lower average contact angle was observed in SR-SR-CHXD (90.67 ± 1.80°). This result is expected as an increase in CHXD concentration will increase the surface hydrophilicity.⁴⁶ Overall, all the films exhibited hydrophobicity and remained statistically insignificant after modification. In summary, film preparation through the incorporation of CHXD and SNAP did not alter the surface hydrophobicity significantly compared to the unmodified SR surface and no statistical differences in wettability were observed between the sample types.

Figure 4.

Surface wettability of polymer surfaces determined by static water contact angle. No significant difference was observed between the sample groups. Data represents mean ± SEM (n ≥ 3).

4.4.2. Surface morphology and elemental mapping

Surface morphology and elemental mapping analysis were carried out to evaluate the surface of the samples and the dispersion of CHXD and SNAP in the structure (Figure 5). The surface of each sample observed by SEM imaging exhibited a relatively smooth surface, and no significant changes were observed on the surface compared to unmodified SR. The cross-sectional view of films clearly showed the presence of SNAP crystals which was confirmed by the EDS mapping of the sulfur element present in the S-NO bond of SNAP moiety (Figure 5A–B). Furthermore, the SEM-EDS showed dispersed CHXD within the topcoat layer via mapping of the Chlorine element in the CHXD structure confirming its presence on films (Figure 5C–D).

Figure 5.

Surface SEM-EDS images of SR-SNAP-CHXD films. (A) SEM of cross-section of SR-SNAP-CHXD films. (B) Elemental mapping of SNAP to evaluate distribution in polymer films via mapping of sulfur group (depicted by red color) as the representative element of SNAP. (C) SEM image of films (top view). (D) Elemental mapping of chlorine (depicted with green color) dispersion on the surface of samples representing characteristic element of CHXD. Scale bar represents 100 μM.

4.5. Antibacterial efficacy of SR-SNAP-CHXD films

4.5.1. In vitro antibacterial adhesion assay

The antibacterial activity of the multi-active films was evaluated using 24 h bacterial adhesion assay against S. aureus and E. coli bacteria. Both SR-SNAP and SR-CHXD (5 wt%) resulted in 0.74 and 2.75 log reductions in E. coli bacteria viability, respectively (p < 0.05). However, the combinational effect of SNAP and CHXD into SR-SNAP-CHXD(5wt%) films resulted in the highest E. coli bacterial inhibition with 3.41 log bacterial reduction compared to SR control (p < 0.05) (Figure 6A). The synergy of SNAP and CHXD in SR-SNAP-CHXD also reduced the viable CFU of S. aureus bacteria on the film surface by 3.79-log reduction compared to SR control (p < 0.05), demonstrating the broad-spectrum antibacterial activity. While SR-SNAP and SR-CHXD also individually reduced the S. aureus adhesion by 0.91 and 2.76 log reductions, respectively (p < 0.05) (Figure 6B), the synergy of both SNAP-CHXD exhibited maximum reduction.

Figure 6.

Antibacterial activity of polymer films calculated as a log of the colony forming units (CFU) cm⁻² of surface area against (A) S. aureus (B) E. coli using a 24 h bacterial adhesion assay. Data represents mean ± standard error of mean (n≥3), * represents p < 0.05, ** p < 0.01 SR vs. SR-SNAP, SR-CHXD and SR-SNAP-CHXD.

4.5.2. Bacteria morphological analysis

The FESEM analysis of bacteria on the surface of films revealed the effect of individual CHXD and SNAP and as well as their synergy through SR-SNAP-CHXD in bacterial eradication (Figure 7). Bacterial clusters with intact membranes on SR surface were evident in SEM images (Figure 7A and E) that showed smooth outer membranes confirming the viability of adhered bacteria after 24 h. However, the SR-SNAP, SR-CHXD, and SR-SNAP-CHXD (Figure 7B–D, and F–H) exhibited a significant reduction in bacteria colonization due to their inherent antibacterial properties. The bactericidal effect was more enhanced with the synergy of SNAP-CHXD. The SEM analysis showed disrupted, wrinkled, and indented bacterial cell walls with blisters indicating the antimicrobial action of CHXD.⁴⁷ Cellular debris from dead cells was also evident on samples with bactericidal capacities.

Figure 7.

Representative images of S. aureus (A-D), and E. coli (E-H) adhesion on SR (A and E), SR-CHXD (B and F), SR-SNAP (C and G), and SR-SNAP-CHXD (D and H) samples after 24 h of incubation at 37 °C. Bacteria cell morphology was depicted in magnified insets within each image. Blisters appeared on bacteria cell walls on surface containing CHXD (B, D, F, and H). Bacteria cell wall destruction was also observed on the surface of the NO releasing samples (C, D, G, and H). The extended white scale bar represents 10 μm and the scale bar in the insets represent 2 μm.

4.6. Cytocompatibility of films towards mammalian cells

To assess the compatibility of SR-SNAP-CHXD films, mammalian NIH 3T3 fibroblast cells were exposed to leachates obtained from films over 24 h and tested for viability using CCK-8 cell viability kit. The results from this study demonstrated that all the samples SR, SR-SNAP, SR-CHXD, and SR-SNAP-CHXD at 1, 3, and 5 wt% CHXD exhibited more than 70% cell viability threshold (Figure 8). There was a slight variation in all the tested groups due to certain experimental variations; however, none of the results were significantly different. Polymer samples top-coated with concentrations greater than 5 wt% CHXD resulted in a reduction of cell viability (data not shown). Therefore, 5 wt% CHXD top-coated samples were utilized in all experiments.

Figure 8.

Cytocompatibility of polymer films tested against NIH 3T3 mouse fibroblast cells using a 24 h leachate exposure study. All samples exhibited >70% cell viability threshold (indicated by dashed line). Results are calculated as relative cell viability normalized to control cells that received no treatment. Data represents mean ± SEM (n ≥ 3).

5. Discussion

Medical devices coated or impregnated with CHXD are currently clinically used to combat catheter-related bloodstream and other infections. However, despite these efforts, the rate of infections is continuing to rise. To overcome the challenges associated with bacterial contamination, various surface strategies have been developed in the literature. The SNAP-incorporated NO-releasing materials have been widely studied for both antimicrobial and blood-contacting biomedical device applications.^{17, 48, 49} These polymer systems provide excellent antimicrobial properties that can prolong the lifetime and augment the biocompatibility of medical devices. However, over time the levels of NO may decrease due to the depletion of the NO reservoir within the material, which limits the ability of these materials to eradicate all bacteria. Although NO holds great potential in conquering bacteria and associated issues, NO-releasing materials have not been commercialized yet. Therefore, combining NO-releasing materials with other clinically available broad-spectrum antibacterial agents, such as CHXD, can be one possible solution to bacterial colonization on biomaterials and provide multiple mechanisms at the medical device interfaces to ensure microbial eradication. Since the initial timepoint during the implantation or insertion of a medical device is considered extremely critical in determining the fate of the device, the synergy of two antimicrobial interfaces is expected to lower the degree of bacterial adhesion at this potential early onslaught of infection. The subsequent physiological levels of NO from the samples can then provide prolonged antibacterial action against pathogens and continue to inhibit biofilm formation.

To combine the NO-releasing SR polymer with CHXD, a facile and easy method is proposed in this study. Medical grade SR polymer was incorporated with 10 wt% of SNAP and top-coated with CHXD to combine two antibacterial strategies into one system for potential biomedical device application. Previous studies using SNAP have shown that polymeric materials retain their mechanical properties up to 10 wt% SNAP.⁴¹ Beyond 10 wt% materials tend to demonstrate a sharp decline in ultimate tensile strength.⁴¹ Therefore, a 10 wt% SNAP was chosen for this study. To optimize the concentration of CHXD, three different wt% of CHXD in SR-THF solution (1, 3, and 5 wt%) were dip-coated on the 10 wt% SNAP incorporated SR films (Figure 1). The NO release kinetics from varying amounts of CHXD on SR-SNAP samples were measured using a NO analyzer. The levels of NO released from polymer films were observed to be above physiologically relevant levels (0.5 – 4 × 10⁻¹⁰ mol min⁻¹ cm⁻²) up to 24 h (Figure 2B). Since results from the study showed no significant difference between the release levels, the highest concentration of CHXD top-coated films (5 wt%) was chosen for further evaluation to maximize the antimicrobial potential of these materials. To analyze the long-term release levels of NO from SR-SNAP-CHXD as compared to SR-SNAP samples, the films were tested over 4 weeks at physiological conditions (Figure 2C). The data revealed similar levels of NO and duration from both SR-SNAP and SR-SNAP-CHXD films with initial levels of NO 5.46 ± 0.64 and 5.67 ± 0.31 × 10⁻¹⁰ mol min⁻¹ cm⁻² on day 0. The final NO release values tested from films on day 29 were 0.28 ± 0.03 and 0.41 ± 0.15 × 10⁻¹⁰ mol min⁻¹ cm⁻² for SR-SNAP and SR-SNAP-CHXD films, respectively (Table 2). Values obtained in this study align with previously reported NO flux from SNAP incorporated RTV polymer at same initial SNAP loading (10 wt%) that showed 5.34 ± 0.91 × 10⁻¹⁰ mol cm⁻² min⁻¹ of NO release on day 0.³⁹

To evaluate the translational properties of these surfaces for medical device applications, the SR-SNAP-CHXD films were also tested for their compatibility with conventional ethylene oxide and UV sterilization methods used in hospital settings. Bioactive materials that contain NO donor compounds can lose their functions during sterilization due to their thermal and moisture sensitivity. However, the results obtained in this study confirmed that the NO levels from the sterilized samples continued to stay above physiological levels similar to that of freshly fabricated films (Figure 2D). Similar results have been reported about both SNAP and CHXD compatibility with various sterilization methods.^{41, 50} These studies have presented the ability of SNAP incorporated films (10 wt%) in retaining more than 90% of initial SNAP loaded into Elast-eon E2As (a copolymer of combined soft portions of polydimethylsiloxane and poly(hexamethylene oxide)) polymer matrix after UV and EO sterilization.⁴¹ Similarly, Sherertz et al. have reported the compatibility of CHXD coated triple lumen polyurethane catheters with an EO sterilization method which were also able to preserve their antibacterial efficacies against S. aureus pathogen in an in vivo rabbit model.⁵⁰ Overall, the results obtained from this study confirm that both ethylene oxide and UV-light sterilization methods are viable options with the combinational films highlighting the potential of this material for clinical translation.

The amount of SNAP and CHXD released from polymer films into the PBS buffer under physiological conditions was also analyzed over 7-d using UV-vis spectroscopy. The data were extrapolated using the standard curve and characteristic peak of SNAP and CHXD compounds at 255 and 340 nm wavelength, respectively (Figure 3A–D). After 24 h, 0.27 ± 0.01 μg CHXD and 0.48 ± 0.03 μg of SNAP mg⁻¹ of total film mass was detected in the buffer which translates to 53.98% and 4.80% of the initial SNAP and CHXD loaded in the polymer, respectively. In both cases, CHXD and SNAP diffusion was observed to be higher on the initial days. However, the diffusion of the compounds stabilized with each testing day (Figure 3E). It was found that after 7 d of testing, 95.04% and 20.63% of CHXD and SNAP leached into the soaking buffer, respectively. The higher amount of CHXD leaching from the polymer samples can be beneficial at the onset of infection in the initial days. The corresponding slow NO release from polymer would then help in the prevention of biofilm development on medical device surfaces over relatively longer durations. The cross-linked nature of silicone polymer with very low water uptake properties makes it a suitable choice for long-term antibacterial applications since it slows the release of both SNAP and CHXD from the polymer matrix.

This data is consistent with NO release flux from SNAP films that showed higher levels of initial NO flux during the 24 h incubation (Figure 2B). The high levels of SNAP leaching and NO flux can be attributed to the SNAP present in the water-rich layer of the polymer matrix. Leaching of SNAP and its byproducts is not a concern as SNAP is synthesized from its parent thiol, N-acetyl-D-penicillamine (NAP), which is an FDA-approved drug used to treat heavy metal poisoning.⁵¹ In fact, several studies have been reported in the past using rabbit and sheep animal models that confirmed the safety of SNAP incorporated materials during in vivo testing of SR-SNAP.^{52, 53} Overall, results obtained from NO release and CHXD diffusion confirm the longevity of the polymer films.

The hydrophobicity of the drug and polymer both can dictate the drug release kinetics. Therefore, the release behavior of drugs from polymers can be altered by changing the parameters such as initial drug loading, polymer substrate (hydrophobic vs. hydrophilic), and thickness of topcoat (diffusion) based on type and duration of application. The surface wettability of polymers can play a critical role in regulating the NO release from polymers by prolonging or preventing moisture from reaching the embedded SNAP molecule in the polymer matrix to stimulate NO release. Therefore, SNAP was integrated with silicone rubber polymer which is known to exhibit hydrophobic characteristics. These properties of silicone rubber make it an excellent choice for catheter-related applications. In order to analyze the effect of modification on SR polymer, surface characterization of polymer films was carried out using contact angle measurement (Figure 4). Contact angle measurements on the samples revealed no significant difference between unmodified SR and modified surfaces (SR-SNAP, SR-CHXD, and SR-SNAP-CHXD) and all the samples remained hydrophobic with contact angle > 90° (Figure 4). Results from this study corroborate with previously published reports that showed no significant difference in water contact angles of thermoplastic polyurethane polymers with and without SNAP incorporation (10 wt%) assuming that interface between blood proteins and film surface will stay unaltered with the addition of SNAP.^{39, 41} In addition, using scanning electron microscopy and EDS mapping results confirmed homogenous distribution of SNAP and CHXD in the polymers (Figure 5). These results demonstrate that SNAP incorporation through solvent evaporation and dip coating CHXD on the top of films is a facile and reliable technique to generate these dual-active antimicrobial surfaces.

Bacterial colonization on biomedical devices leads to ca. 90% of all hospital-acquired infections.⁵⁴ Bacteria like S. aureus and E. coli are a part of the group of ESKAPE pathogens that are the leading cause of nosocomial infections around the globe. Overuse of antibiotics and their abuse has triggered antimicrobial resistance in these opportunistic pathogens making them even harder to eradicate. Moreover, the altered gene expression of bacteria encapsulated within biofilm guards them against host immune response. In these scenarios, it will be beneficial to have strategies that can help potentially thwart the progression of bacterial colonization on medical devices. Both NO and CHXD are known to exhibit broad-spectrum antimicrobial effects. Previous studies have reported the use of NO and CHXD in medical device applications such as dental implants, sutures, vascular catheters, antimicrobial dressings, antibiotic lock solutions, etc. to combat bacterial infections^{26, 55–58}. However, to date, no study has been reported that combined NO and CHXD within polymer materials for developing dual-active antibacterial interfaces. The antibacterial activity of the films was analyzed against S. aureus (Gram-positive) and E. coli (Gram-negative) bacteria using a 24 h antibacterial adhesion assay and imaged using FESEM. Results from the study showed the highest bacterial killing with SR-SNAP-CHXD against both S. aureus and E. coli bacteria with >3-log reduction in just 24 h (Figure 6). Although individual SR-SNAP and SR-CHXD films did exhibit antibacterial properties, the method of combining the two agents is more potent. The results from SR-SNAP films are equivalent to previous studies that utilized polyurethane-based polymers to incorporate or impregnate SNAP that released similar NO levels over a 24 h period.^{39, 59, 60} The NO released can damage bacterial cell membrane by the means of lipid peroxidation that disrupts the outer membrane of cells.^61–63 Notably, much of antibacterial effects were found to be through antiseptic CHXD in addition to SNAP in both S. aureus and E. coli 24 h antibacterial studies. It was also observed that SR-SNAP-CHXD was more potent against S. aureus bacteria which is a Gram-positive strain as opposed to E. coli. These results are analogous to previous reports that showed differential actions of CHXD on Gram-positive vs. Gram-negative bacteria.⁶⁴ This can be explained by the high binding efficiency of CHXD to the Gram-positive bacteria cell wall. To visualize the morphology of bacteria on the films scanning electron microscopy was utilized (Figure 7). The reduction of bacteria on the bactericidal surface was very evident and it was observed that CHXD coated surfaces caused blisters on S. aureus (Figure 7B and D) and wrinkles on E. coli bacteria (Figure 7F and H) which aligns with previously studied effects of CHXD on bacteria.^{64, 65} The cationic nature of CHXD structure attracts the negatively charged bacteria membrane with strong adsorption to phosphate-containing groups. This binding results in loss of integrity in the cell membrane and malfunctioning of proteins and enzymes that lead to cell damage and intracellular compound leakage.^25–28 Similar to the results from the plate counting method, SEM imaging validated the highest reduction of bacteria on SR-SNAP-CHXD films.

While the films demonstrated excellent antibacterial properties, it was also important to determine the safety of films towards mammalian cells. NO-releasing materials have been observed to carry enhanced biocompatibility in vitro due to the inherent endogenous regulatory functions of NO. The NO donor SNAP is easy to synthesize and a well-characterized NO donor with enhanced biocompatibility. For this reason, SNAP-loaded materials are extensively studied both in vitro and in vivo. Previous studies have shown polymeric medical devices with NO-releasing capacity for the use of endotracheal tubes, insulin cannula, catheters, extracorporeal circulation, etc.^{17, 49, 52} Results obtained in this study align with previously published articles that emphasize the compatibility of SNAP loaded polymers for biomedical applications.^{59, 66} Cell compatibility studies confirmed >70% cell survival rate with all samples (Figure 8). Results from the cytotoxicity study were in alignment with previously published reports where higher concentrations of CHXD were seen to trend towards cytotoxicity (data not shown). For example, higher concentrations of CHXD have previously been reported to damage skin cells.^67–69 Therefore, 5 wt% samples were selected for this study which showed excellent compatibility towards mouse fibroblast cells. The approach of combining both SNAP and CHXD can moderate CHXD mediated toxicity and add potential benefits of SNAP to medical devices. The combination of the two compounds can amplify the antibacterial efficacy of medical devices without causing adverse effects to native cells and tissues in the body.

The results from the bacteria studies highlight the success of combining the two bioactive approaches. Both SNAP and CHXD have previously been used individually to incorporate the bactericidal properties into various other polymeric medical devices such as polyurethane, E2As, PVC, etc.^{10, 17, 22, 56} To reduce the cytotoxicity induced by higher concentrations of CHXD, CHXD materials can be combined with NO (second antibacterial agent). Because NO possess several other regulatory functions such as wound healing, anti-inflammatory, and anti-thrombotic properties, the combination of a dual-functional surface can be one collective solution to several medical device-related issues.⁷⁰ Moreover, due to the short half-life of NO, there have been no reported cases of resistance in microorganisms which makes it a superior therapeutic as opposed to conventional antibiotic therapy for overcoming antimicrobial resistance.^{71, 72} Together the results from cytocompatibility and antibacterial studies underscore the necessity for bringing two antimicrobial surface strategies into one polymer system to effectively tackle bacteria and device-associated infections.

6. Conclusions

To utilize the antibacterial effects of both NO and CHXD, a CHXD salt and the NO donor SNAP were incorporated in medical-grade silicone rubber (SR) polymer for biomedical and therapeutic applications. The 10 wt% SNAP incorporated films were fabricated using a solvent-evaporation process with a topcoat of CHXD (1, 3, and 5 wt%) to generate a dual-active antibacterial interface. Chemiluminescence studies confirmed the NO release from SR-SNAP-CHXD films at physiologically relevant levels (0.5 – 4 × 10⁻¹⁰ mol min⁻¹ cm⁻²) of NO for at least 4 weeks with a minimal amount of leaching. Further characterization of the films via SEM-EDS confirmed uniform distribution of SNAP and presence of CHXD within the polymer films without substantial morphological changes confirmed by contact angle hysteresis. Moreover, the dual-active SNAP-CHXD films were able to significantly reduce E. coli and S. aureus bacteria (> 3-log reduction) compared to plain SR and individual SNAP and CHXD controls with no explicit toxicity towards NIH 3T3 mouse fibroblast cells. The synergy between the two systems can reduce bacterial contamination on the surface and enhance the durability of medical devices with a potential application for a wide range of biointerfaces (e.g., urinary catheters, blood catheters, insulin cannulas).