Wearable nitric oxide-releasing antibacterial insert for preventing device-associated infections

Abstract

Medical device-associated infections are a pervasive global healthcare concern, often leading to severe complications. Bacterial biofilms that form on indwelling medical devices, such as catheters, are significant contributors to infections like bloodstream and urinary tract infections. This study addresses the challenge of biofilms on medical devices by introducing a portable antimicrobial catheter insert (PACI) designed to be efficient, biocompatible, and anti-infective. The PACI utilizes nitric oxide (NO), known for its potent antimicrobial properties, to deter bacterial adhesion and biofilm formation. To achieve this, a photoinitiated NO donor, S-nitroso-N-acetylpenicillamine (SNAP), is covalently linked to a polydimethylsiloxane (PDMS) polymer. This design allows for higher NO loading for long-term impact and prevents premature donor leaching, a common challenge with SNAP-blended polymers. The SNAP-PDMS material was applied to a side-glowing fiber optic and connected to a wearable light module emitting 450 nm light, creating a functional antimicrobial insert. Activation of the fiber optic, accomplished with a one-click mechanism, enables real-time NO release, maintaining controlled NO levels for a minimum of 24 hours. The therapeutic levels of NO released via photocatalysis from the PACI demonstrated remarkable efficacy, with >90 % reduction in bacterial viability against S. aureus S. epidermidis, and P. mirabilis without any cytotoxic impact on mammalian cells. This study underscores the potential of the NO-releasing insert in clinical settings, providing a portable and adaptable solution for preventing catheter-associated infections.

1. Introduction

Catheter-associated infections are a significant concern in healthcare settings, particularly among patients requiring long-term catheterization. Catheters are crucial for various medical treatments, monitoring, and fluid administration, but they also provide a direct pathway for microorganisms, increasing infection risks. In the United States alone, catheter-related bloodstream infections (CRBSIs) exceed 250,000 cases annually, with a mortality rate of around 35 %. The cost per infection ranges from $34,000 to $56,000, contributing to approximately $2.3 billion in annual medical expenses [1,2]. The rise in catheter-related infections is primarily due to biofilm formation. Over 1 million hospital-acquired infections are reported annually, with 60–70 % attributed to bacterial contamination and biofilm formation on medical devices [3,4]. When catheters are inserted into the body, they create a stream that can serve as a potential entry point for bacteria and other pathogens [5,6]. Once inside, these microorganisms can adhere to the catheter’s surface and form a biofilm composed of extracellular polymeric substances (EPS) that protect bacteria from antibiotics and immune responses, leading to complications such as localized or systemic infections, catheter-associated urinary tract infections (CAUTIs), and surgical implant infections.

Preventive measures and proper care are essential, especially for individuals with long-term catheters in home settings. Failure to follow recommended maintenance procedures can significantly increase infection risk. Understanding the underlying risk factors and implementing proactive strategies can help reduce infections associated with medical devices. Current strategies for combating biofilms on medical devices include using antibiotics, antimicrobial peptides, and silver coatings [7–11]. Additionally, antiseptic barrier products such as chlorhexidine-impregnated caps and alcohol-soaked swabs aim to reduce microbial contamination and prevent catheter-associated infections [12]. However, these methods have notable limitations, including the potential for antibiotic resistance, narrow activity spectra, decreased effectiveness over time, incomplete biofilm eradication, and possible long-term safety concerns [13,14]. Moreover, while effective against bacteria within the catheter, these techniques may fall short in addressing bacterial contamination on the external surfaces of the catheter. Consequently, there is an urgent need for innovative, broad-spectrum antimicrobial strategies that can be seamlessly integrated with medical devices throughout their lifespan. These solutions should minimize reliance on antibiotics, reduce the risk of resistance, and address both internal and external bacterial contamination.

Nitric oxide (NO) has emerged as a versatile antimicrobial agent due to its non-toxic nature and potent antimicrobial properties, making it an attractive option for combating infections and enhancing biomedical device safety [15,16]. This small, gaseous molecule plays a crucial role in the body’s immune response and various physiological processes (wound healing, vasodilation etc.). It interacts with other molecules to form reactive nitrogen species, such as peroxynitrite (ONOO−), which exhibit strong antimicrobial effects by damaging bacterial DNA, proteins, and lipid membranes [17–20]. Unlike conventional antimicrobial agents, NO-releasing materials demonstrate superior biocompatibility and long-term efficacy, making them suitable for applications in medical devices such as catheters (intravascular, urinary, etc.), implants, endotracheal tubes, and wound dressings [21,22]. This property makes it suitable for biomedical device applications, where the goal is to eliminate pathogens without harming healthy tissues.

However, the short half-life and gaseous nature of NO present challenges for its direct use in long-term biomedical applications. To address this, various NO donors have been developed and integrated into polymers to achieve controlled and sustained NO release [23,24]. Notable advancements include the use of S-nitrosothiols (RSNOs) like S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (GSNO), which release NO in response to environmental triggers like heat, light, metal ions or pH changes [25]. Photocatalysis, particularly at around 340 nm and 520–590 nm wavelengths, corresponding to the S-nitrosothiol bond’s electronic transition offers a promising strategy for precise NO release, enhancing the effectiveness of NO-releasing systems [25–27]. Researchers have explored various photoactive compounds, including L-arginine, modified-RSNO, N-diazeniumdiaolates, or other nitrosated compounds for creating materials with photodynamic effects for ROS propagation [28]. These molecules can be modified or integrated into materials such as cationic polymers, chitosan-based NO conjugates, and layer-by-layer assembled coatings, which provide sustained antimicrobial activity and reduce bacterial resistance [29,30]. Despite these advancements, practical implementation and integration of NO-releasing surfaces into existing medical devices remain underexplored, highlighting the need for continued research in this field.

Inspired by the potential of nitric oxide (NO) and light-driven antimicrobial techniques, this study introduces a groundbreaking application of a NO-releasing polymer in the shape of a Portable Antimicrobial Catheter Insert (PACI). Building on our previous smartphone-based Disposable Catheter Disinfection Insert, the PACI features a fully self-contained, battery-powered system [31]. It utilizes a photoresponsive NO donor, S-nitroso-N-acetylpenicillamine (SNAP), covalently attached to the backbone of PDMS polymer to overcome leaching issues and enable high NO loading. The SNAP-PDMS polymer solution was coated onto the side-glowing fiber optic, which was then connected to a Versalume^™ wearable blue light module, illuminating the entire length of the PACI (Fig. 1A). To trigger NO release and combat pathogenic bacteria, the PACI can be inserted into medical devices and powered by a battery-operated light source (Fig. 1B). Under LED irradiation with a wavelength of 450–650 nm, SNAP degradation generates NO. The light and luminous intensity were assessed using spectroscopy and light meter measurements. The surface of the PACI was characterized using Scanning Electron Microscopy (SEM) equipped with energy-dispersive X-ray spectroscopy (EDS). The NO release kinetics from the PACI and NO donor leaching were assessed using chemiluminescence detection and UV–vis spectroscopy, respectively. Nitrite generation and cytocompatibility with mouse fibroblast cells were also investigated in vitro to determine the biocompatibility of the insert. Additionally, the antibacterial efficacy of the PACI was studied through a 24 h bacterial adhesion assay against three common bacterial strains associated with CRBSIs and CAUTIs (S. aureus, S. epidermidis, and P. mirabilis). The compact PACI is designed to fit within the catheter lumen cap, extending NO release for the entire lifespan of the indwelling device, effectively preventing medical device-associated infections. Its wireless design enables patients with long-term catheters, even those in remote facilities like their homes, to benefit from this technology. The PACI is user-friendly, cost-effective, and highly efficient in addressing current medical device-associated infections. The adaptable nature of fiber optic combined with the NO-releasing coating allows it to be customized for various types of medical device tubing, ensuring a sterile environment for optimal device performance.

Fig. 1.

(A) Development of a battery-powered Portable Antimicrobial Catheter Insert (PACI). NO-releasing covalently conjugated SNAP-PDMS polymer is applied to a side-glow fiber optic and connected to a portable LED light source to create the PACI device. (B) Application scenario of the PACI: The light-insert can be seamlessly integrated into commonly used medical catheters and tubing. Therapeutic NO levels can be easily activated using a one-click mechanism through SNAP-PDMS photocatalysis. The PACI offers a solution to combat bacterial infections associated with medical devices, prolonging device lifespan and reducing treatment costs.

2. Materials and methods

2.1. Materials

Acetic anhydride, pyridine, hydrochloric acid, magnesium sulfate anhydrous, N-acetyl-D-penicillamine (NAP), polysiloxane (−OH terminated, 2,973 cSt), (3-aminopropyl)trimethoxysilane (APTMS), dibutyltin dilaurate (DBTDL), dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA), modified Griess reagent (GR), sodium nitrite (NaNO₂), toluene (Tol), 1,4,8,11-tetraazacyclotetradecane (Cyclam) and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO USA). T-butyl nitrite (TBN, 90 % pure) was purchased from Fisher Scientific (Waltham, MA USA). All reagents were > 95 % purity and used as-is unless otherwise specified. Versalume^™ Single Color Wearable Module Blue color, LC connectorized Corning^® Fibrance^® Light-Diffusing Fiber were purchased from Versalume^™™ (Versalume^™, CA). Bacterial strains S. aureus (ATCC 6538), S. epidermidis (ATCC 14990), P. mirabilis (ATCC 29906), mouse fibroblasts (3 T3, ATCC CRL-1658) were cultured from stocks originally sourced from American Type Culture Collection (Manassas, VA USA). Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), trypsin, and penicillin-streptomycin (P/S, 5,000 U mL⁻¹) were purchased from VWR (Atlanta, GA USA). Deionized water was used to prepare all aqueous solutions. For material characterization and NO analyzer studies, a 0.01 M phosphate buffer saline (PBS) with 100 μM EDTA was utilized (pH 7.4). Before conducting bacteria and biocompatibility experiments, all buffers and media were sterilized in an autoclave at 121 °C, 100 kPa (15 psi) above atmospheric pressure for a duration of 30 min.

2.2. Fabrication of the NO donor-modified polydimethylsiloxane (SNAP-PDMS) coating

Synthesis of the covalently-immobilized NO donor conjugate of polysiloxane for NO-releasing coatings (SNAP-PDMS) began with the preparation of the thiolactone derivative (NAPTH) of NAP following our previously reported method [32]. Subsequently, SNAP-PDMS was prepared following our previously reported method with minor deviation (Fig. 2A) [33]. Polysiloxane (3.2 g) was dissolved in Tol (80 mg mL⁻¹) for 10 min, then APTMS (600 mg) and DBTDL (14.4 mg) were added. The polysiloxane was stirred for 12 h at room temperature, afterwards, NAPTH (600 mg) was added. The casting solution was stirred for an additional 48 h, with TBN (washed 3× times with an equal volume of 25 mM Cyclam) added (6 mL). The casting solution was stirred for an additional 30 min with protection from light, and then used for dip-coating.

Fig. 2.

(A) Reaction scheme for covalently binding the NO donor S-nitroso-N-acetylpenicillamine (SNAP) to hydroxy terminated polydimethylsiloxane (PDMS) polymer via a sequential reaction with a thiolactone derivative of N-acetyl-penicillamine and nitrosation. (B) FTIR characterization of SNAP-PDMS polymer supported the presence of the RSNO group through characteristic bond vibration (1497 cm⁻¹) as well as other characteristic peaks consistent with the NO donor precursor NAP. (C) Design of battery-operated Portable Antimicrobial Catheter Insert (PACI). NO-releasing PDMS is coated on a side glow fiber optic and connected to a portable LED light source to develop the PACI device. (D) PACI device innovation involves covalently linking light-responsive NO donor SNAP with PDMS. LED illumination of the fiber optic, activated with a one-click mechanism, controls real-time NO release. The therapeutic levels of NO released via photocatalysis from PACI can help addressing bacterial infections linked to medical device usage, thereby substantially enhancing device longevity, and leading to lowered treatment expenses.

2.3. Fourier transform infrared spectroscopy (FTIR) analysis

FTIR studies of the as-prepared SNAP-PDMS polymer and precursor components were performed using a universal attenuated total reflectance accessory with a Spectrum 3 infrared spectrometer from Perkin-Elmer (Waltham, Massachusetts). Samples were analyzed over a total of 64 scans collected at a resolution of 4 cm⁻¹ along the detection range of 4000 to 650 cm⁻¹.

2.4. Formulation of portable antimicrobial catheter insert (PACI)

All fiber optics used in this study were obtained from Versalume^™ LLC (Santa Clara, California). The fiber optics were cut into ~10 cm length and ~ 4 cm of the fiber was coated with either PDMS or SNAP-PDMS polymer solution. PDMS coating solution was prepared by mixing 10 parts Sylgard 184 with 1 part curing agent followed by the addition of 6.2 mL Toluene. The solution was allowed to stir for 30 min. For the fabrication of PDMS-coated fiber optics, the fibers were dip-coated 6 times with the PDMS solution resulting in a control PDMS group. Similarly, SNAP-PDMS coated fiber optics were prepared by dip coating the fibers in SNAP-PDMS solution 5 times with a final coating of PDMS, namely SNAP-PDMS group. Two minutes of drying time was allowed between each coat for both the control and test groups. Samples were dried in a hot air oven for 1 h at 60 °C followed by overnight drying at room temperature and 24 h drying in a vacuum desiccator to ensure all the solvent from coating was evaporated. Then, coated fiber optic samples were connected to a Versalume^™ single-color wearable module with the ability to emit blue light leading to PDMS-Light and SNAP-PDMS-Light (PACI) for further experiments.

2.5. Characterization of PACI

2.5.1. Surface characterization using scanning electron microscopy (SEM) equipped with energy dispersive X-ray spectroscopy (EDS)

The surface morphology of uncoated (bare) and SNAP-coated fiber optics (SNAP-PDMS) was assessed by Field-emission scanning electron microscopy (FESEM, FEI Teneo, FEI Co.). The samples were mounted onto aluminum stubs using double-sided carbon tape. These mounted samples were sputter coated with 10 nm Au–Pd by a Leica EM ACE200 sputter coater (Buffalo Grove, IL). The sputter-coated samples were then subjected to an accelerating voltage of 10.00 kV and imaged at different magnifications. Additionally, energy-dispersive X-ray spectroscopy (EDS, Oxford Instruments) was used to detect the presence of elements on the surface of the fiber optics.

2.5.2. Light emission using spectroscopy analysis

In order to ascertain the wavelength of the LED light emitted by the light module, a wireless spectrophotometer (PS-2600, PASCO Scientific) with a detection range spanning 380–950 nm was utilized. The fiber optic samples, connected to the blue color light module, were firmly secured with a clamp, and subjected to the light detector to measure the wavelength of the emitted light. To ensure accurate characterization of the intended light sources, light studies were conducted in an environment devoid of ambient light. This precautionary measure was taken to ensure that only the desired lights were being analyzed and recorded.

2.5.3. Determination of luminous intensity of the light insert

In determining the luminous intensity of the fiber optic linked to the Versalume^™’s blue light module, a digital light meter (MT-912, URCERI) was employed. The fiber optic samples, connected to the blue light module, were securely positioned using a clamp, and the light meter was employed to quantify the emitted light’s intensity. For precise evaluation of the targeted light sources, the light assessments were conducted in a controlled environment, eliminating any ambient light interference.

2.6. Photoinitiated NO release from NO-releasing PACI

The quantification of NO release from both light-exposed (SNAP-PDMS-Light) and non-light-exposed samples (SNAP-PDMS) was performed using the gold standard Zysense chemiluminescence Nitric Oxide Analyzer (NOA) 280i (Frederick, CO). This analysis was conducted under a nitrogen atmosphere and physiologically relevant conditions. The NO release from the inserts is normalized to the surface area and presented as moles min⁻¹ cm⁻².

2.6.1. Dynamic regulation of NO release in real time

The SNAP-PDMS-Light samples were carefully placed inside an amber NOA sample cell with 7 mL of PBS-EDTA solution at a physiological temperature (37 °C) while ensuring protection from ambient light. To enable real-time control over the release of NO, a wireless, battery-operated blue light module was connected to the fiber optic. The NO flux from the samples was recorded while the light source was intermittently switched on and off using the wearable module. The experimental procedure began with obtaining the baseline measurements without the light insert. Subsequently, the insert with the light on was introduced, and the light source was alternately switched on and off to demonstrate the dynamic control and monitoring of NO release in real-time.

2.6.2. 24-Hour NO release kinetics

The SNAP-PDMS (no light) and SNAP-PDMS-Light samples were placed in an amber NOA sample cell containing 7 mL of PBS with 100 μM EDTA (pH 7.4) at the physiological temperature of 37 °C. For the SNAP-PDMS-Light samples, the Versalume^™ blue light module was activated and the photoinitiated NO release was recorded in the static light mode. The NO release profiles in the absence and presence of light were recorded at 0, 4, 8, 12 and 24 h time points until the release reached a plateau. Samples were incubated at 37 °C with PBS-EDTA in between the testing intervals.

2.7. Investigation of SNAP dissemination in soaking buffer

The UV–vis spectrophotometric method was employed to determine the extent of SNAP diffusion from SNAP-PDMS coated fiber optics in the presence and absence of light. The PACI samples with and without light (SNAP-PDMS-Light, SNAP-PDMS) were immersed in 10 mM PBS with 100 μM of EDTA at pH 7.4 and incubated at 37 °C for 48 h. The soaking buffer was analyzed at various time intervals (2, 4, 6, 8, 24, and 48 h) to assess the concentration of SNAP in the buffer. The molar absorptivity of SNAP in 10 mM PBS-EDTA at pH 7.4 was determined to be 996 M⁻¹ cm⁻¹ at a wavelength of 340 nm. The samples were maintained at 37 °C in PBS throughout the experimental duration, and the results were evaluated by determining the SNAP concentration in each sample, normalized by the surface area of the samples.

2.8. Accumulation of NO metabolite

Studies of NO metabolite accumulation (i.e., nitrites) in buffers and media were performed using a modified Griess assay following our previously reported method with minor deviation [34]. Extracts collected in the vehicle were processed following incubation with coated fiber optics for 24 h (surface area-to-volume ratio of ~0.55 cm² mL⁻¹) by diluting 1:10 in Griess reagent (final concentration of 20 mg mL⁻¹) and reacting for 20 min at ambient conditions. Afterward, the samples were read for absorbance at 540 nm. A separate calibration curve was prepared via serial dilution of NaNO₂ (20 μg mL⁻¹) in solution. For cell culture studies, DMEM without phenol red supplement was used. The results are reported as the mean equivalent of nitrite (μg) ± SD (N = 9 repeats).

2.9. Biocompatibility screening of antimicrobial insert

Biocompatibility screening of the coated and uncoated inserts with and without light was performed per the International Organization for Standardization’s (ISO) standards for the biological evaluation of medical devices via tests for in vitro cytotoxicity (10993–5) [35]. Standards were followed with some minor deviations in the protocol.

3T3 cells were revived from cryopreserved stocks and subcultured in DMEM supplemented with FBS (10 % v/v) and P/S (1 % v/v) under a humidified atmosphere with 5 % CO₂ at 37 °C. Cells were grown to ~70 % sub-confluency, afterwards, cells were enzymatically detached (0.05 % v/v trypsin with 5 mM EDTA) and pelleted via centrifugation (200 rcf, 5 min). Cells were resuspended in clean media, with counts determined via trypan blue staining using an EVE^™ automated cell counter from NanoEnTek (Waltham, MA USA). For cytotoxicity screening, cells were seeded onto 96-well polystyrene plates (5,000 cells well⁻¹) and incubated for 24 h. Concurrently, extracts were prepared from inserts by incubating fiber optic segments in complete media (surface area-to-volume ratio of ~0.55 cm² mL⁻¹) for 24 h. Afterward, fiber optic segments were removed, with extracts used for testing. Each sample extract (treatment) was plated (100 μL well⁻¹) in triplicate. Negative (vehicle) control samples were prepared by treating cells with complete media pre-incubated for 24 h at 37 °C. Extract-treated plates were grown for an additional 24 h, afterwards, media was aspirated off and replaced with MTT (0.5 mg mL⁻¹ in PBS (1×)). Cells were incubated with MTT for an additional 3 h, then the MTT solution was aspirated off, with the remaining formazan precipitate dissolved in DMSO (200 μL well⁻¹). Well-plates were read for absorbance at 570 nm with a further reference reading at 630 nm. The relative percent cell viability was then calculated according to Eq. (1). Final data are reported as the mean percent cellular viability ± SD (N = 3 repeats).

$$\text{Relative Percent Cell Viability}=\frac{{\left(\mathrm{O}{\mathrm{D}}_{570}-\mathrm{O}{\mathrm{D}}_{630}\right)}_{\text{treatment}}}{{\left(\mathrm{O}{\mathrm{D}}_{570}-\mathrm{O}{\mathrm{D}}_{630}\right)}_{\text{vehicle}}}\times 100\mathrm{\%}$$ (1)

2.10. In vitro antibacterial evaluation using 24 h bacterial adhesion assay

The antibacterial effectiveness of light-triggered NO-releasing fiber optics was evaluated against S. epidermidis, S. aureus, and P. mirabilis strains using a 24-h bacterial adhesion assay. To conduct the assay, a single colony of either S. epidermidis or S. aureus was cultured in LB media at 37 °C with shaking at 150 rpm overnight. Similarly, P. mirabilis was cultured in CLED media under the same conditions. Once the bacteria reached the logarithmic growth phase, the bacterial suspension was centrifuged to obtain a bacterial pellet, which was then washed with PBS buffer and resuspended in sterile PBS buffer to achieve a concentration of approximately 10⁸ CFU mL⁻¹.

All four sample types (PDMS, PDMS-Light, SNAP-PDMS, SNAP-PDMS-Light) were sterilized under UV light for 30 min before being immersed in microcentrifuge tubes containing the bacterial suspension. For the light-exposed samples (PDMS-Light and SNAP-PDMS-Light), fiber optics were connected to a Versalume^™ single-color blue light module (450 nm wavelength, ~20 mW fiber coupled power), providing continuous exposure throughout the 24-h incubation. The SNAP-PDMS coating was applied directly to the fiber optic surface, maintaining a zero distance between the light source and the NO-releasing coating to ensure optimal light delivery and maximize NO release. All samples were incubated at 37 °C for 24 h. Afterward, the portion of fiber optics submerged in the bacterial solution was removed, rinsed with PBS, and homogenized using an Omni-TH homogenizer (Omni, Kennesaw, GA) at 25,000 rpm for 1 min. The bacterial solution was also vortexed for an additional 1 min. Subsequently, the solution was diluted, and for S. epidermidis and S. aureus, the diluted solution was plated on LB agar plates, while for P. mirabilis, it was plated on CLED agar plates using a spiral plater (Eddy Jet 2, IUL Instruments). The agar plates were incubated at 37 °C for 24 h, and the number of colonies formed on each plate was quantified using an automated colony counter (Sphere Flash, IUL Instruments). The percent and log₁₀ reduction in bacterial viability were calculated relative to the control group (PDMS-coated fiber optics) and normalized to the surface area. The following equation was used to determine the percent reduction in adhered bacteria, where C represents the concentration of viable bacteria in CFU cm⁻² (Eq. (2))

$$\mathrm{\%}reduction=\frac{C\left(PDMS\right)-C\left(\mathit{\text{Test}}\right)}{C\left(PDMS\right)}\mathrm{*}100\mathrm{\%}$$ (2)

3. Statistical analysis

All results in the study are based on a minimum sample size of n ≥ 3. Data are presented as either mean ± standard deviation (SD) or standard error of mean (SEM). To assess statistical significance between different sample types, a student’s t-test was employed. A significance level of p < 0.05 was considered to evaluate the statistical differences between the test groups (SNAP-PDMS, PDMS-Light, SNAP-PDMS-Light) and the control group (PDMS).

4. Results and discussion

4.1. Fourier-transform infrared spectroscopy (FTIR) analysis

To address the issue of bacterial contamination on medical devices, this study introduces a portable antimicrobial catheter insert (PACI) that is efficient, biocompatible, and anti-infective. The PACI leverages the potent antimicrobial properties of blue light and nitric oxide (NO) to prevent bacterial adhesion and biofilm formation on medical device surfaces. To achieve this, S-nitroso-N-acetylpenicillamine (SNAP), a photoinitiated NO donor, is covalently bonded to a polydimethylsiloxane (PDMS) polymer using a nitrosation method (Fig. 2A). This approach allows for higher NO loading for sustained effectiveness and prevents early loss of the NO donor, a frequent problem with SNAP-blended polymers. To verify the successful conjugation of SNAP to the PDMS polymer, FTIR analysis was conducted on the prepared SNAP-PDMS. The analysis confirmed successful crosslinking of the polysiloxane with the aminosilane crosslinker, evidenced by the 3293 cm⁻¹ N-H stretching. Further functionalization was achieved via aminolysis with the thiolactone of NAP, introducing a terminal tertiary thiol pendant group (Fig. 2B). Nitrosation of the SNAP-PDMS casting solution with TBN and subsequent thermosetting confirmed the presence of tertiary S-nitrosothiol groups (1497 cm⁻¹, Fig. 2B), consistent with our previous reports [33,36].

4.2. Development and characterization of PACI

4.2.1. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS)

The Versalume^™ wireless wearable module offers a user-friendly and highly portable approach to easily integrate PACI into a daily routine. With a single click, the light source can be powered on and off, and it can be conveniently charged using a micro-USB type-B connector (Fig. 2C). To imbue long-term NO-releasing properties, a well-established technique involving dip coating of a polymer solution onto a substrate was employed (Fig. 2D).

This approach was used to apply the SNAP-PDMS solution formulated earlier onto the fiber optic, creating a portable antimicrobial light insert in conjunction with the Versalume^™ wearable light module. The thickness of the fiber optics after dip coating with PDMS or SNAP-PDMS polymer solution was measured with precision using a micrometer. The outer diameter of the fiber optics used in the study was 0.931 ± 0.002 mm. After dip coating, the thickness of the inserts increased, with the PDMS-coated inserts measuring 0.95 ± 0.01 mm and the SNAP-PDMS-coated inserts measuring 1.12 ± 0.01 mm.

To ensure consistency and avoid introducing any variability in surface topography, a comparative analysis was conducted between uncoated and coated surfaces. Scanning electron microscopy (SEM) was employed to investigate the surface and cross-sectional morphology of both uncoated and SNAP-PDMS-coated surfaces, revealing no apparent differences in surface topography when comparing these two groups (Fig. 3A–C). Furthermore, cross-sectional imaging of SNAP-PDMS coated fiber optics clearly showed distinct coated parts with uniform thickness around the outer jacket of the fiber optics (Fig. 3D). A representative cross-sectional schematic is shown in Fig. 3E to illustrate this. Additionally, elemental mapping of the surface of the bare and SNAP-PDMS coated fibers showed the presence of Si, O, and C groups for both surfaces. In contrast, sulfur groups were only detected in SNAP-PDMS coated samples (Fig. 3F–N), which confirmed the successful incorporation of SNAP. These findings are further quantified in Tables 1 and 2, which provide detailed elemental compositions obtained from EDS analysis.

Fig. 3.

Surface characterization of NO-releasing Portable Antimicrobial Catheter Insert (PACI) using Scanning Electron Microscopy (SEM). SEM images were captured for bare fiber optic with (A) Top view and (B) cross-section and as well as for SNAP-PDMS coated fiber optics (C) Top view and (D) cross-section. The thickness of the SNAP-PDMS coating is identified with the red arrow, which clearly distinguishes it from the fiber optics jacket. (E) Schematic representation of PACI components with SNAP-PDMS coating on top and fiber optic contents inside. Energy Dispersive X-ray Spectroscopy analysis of (F–I) bare and (J-N) SNAP-PDMS coated fiber optics. The presence of sulfur in the SNAP-PDMS coating indicates the presence of SNAP via the identification of sulfur in the chemical structure. Scale bar represents 25 μM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 1.

Elemental Composition of Unmodified Fiber Optic Analyzed via Energy Dispersive Spectroscopy (EDS) and Scanning Electron Microscopy (SEM).

Element	wt%	σ
C	75.1	0.6
O	24.8	0.6
Si	0.1	0.1

Table 2.

Elemental Composition of SNAP-PDMS Coated Fiber Optic Analyzed via Energy Dispersive Spectroscopy (EDS) and Scanning Electron Microscopy (SEM).

Element	wt%	σ
C	49.0	0.6
O	21.7	0.4
Si	28.5	0.4
S	0.8	0.1

Earlier investigations that involved the dip-coating of SNAP-doped polymer solutions onto substrates yielded similar findings regarding the absence of any noticeable changes in the surface topography of manufactured catheters when SNAP was introduced [37].

4.2.2. Analysis of the light emission from portable antimicrobial catheter insert (PACI)

The PASCO wireless spectrometer’s optical probe was utilized to validate the wavelength of emitted light from the wireless light source. The specific wavelength of the light was determined by connecting the fiber optic to the illuminated light source in static mode. The light source can be activated with a single click, instantaneously illuminating the full length of the fiber optic (Fig. 4A–B). The inherent flexibility of the fiber optic allows seamless integration with various existing indwelling devices, such as intravenous, urinary, hemodialysis catheters, endotracheal tubes, etc. currently available in the market (Fig. 4C–D). The small and pliable nature of the light inserts makes it a universally adaptable technology for diverse catheters of varying dimensions.

Fig. 4.

(A) Fiber optic connected to the light source in the absence of light (B) Light from the fiber optic connected to a portable light source can be activated instantaneously with a simple click (C) Illustration of the extensive adaptability of the portable light insert to medical devices like endotracheal tubes, indwelling catheter (intravascular, urinary, etc.) to decontaminate in between clinical applications. (D) The PACI insert is fabricated with commercially available side glowing fiber optics (which are inserted within the catheter lumen and are replaceable) and a wireless light source controller/battery unit that can be secured to the patient’s skin using standard adhesive IV catheter securement dressings. (E) Validation of the emitted light wavelength from the Versalume^™ wearable blue light module (F) Calculation of luminous intensity of fiber optic presented as mean ± SD (G) Spectral analysis of the blue light module emitting light within the visible range, specifically between 435 and 450 nm wavelength. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

The length, thickness, and width of the insert can be easily customized to match catheter specifications. This adaptability positions the insert as a versatile technological solution to meet contemporary biomedical requirements. The study’s results conclusively demonstrated that the Versalume^™ blue light wearable module emits light falling within the 440–450 nm range (Fig. 4E). The intensity of the light emitted by the fiber optic connected to the Versalume^™ blue color wearable light module was evaluated using a digital light meter (Fig. 4F). The study’s outcomes demonstrated a luminous intensity of 295.93 ± 7.70 Lux units, signifying the brightness level of the emitted light. This measurement provides valuable insight into the practical illumination capability of the fiber optic module in real-world scenarios.

Spectral analysis of the blue light module emitting light within the visible range, specifically between 435 and 450 nm wavelength (Fig. 4G). Fiber optics with the ability to emit blue light within the 435–500 nm wavelength range, are utilized in numerous medical settings for effectively sanitizing medical equipment surfaces. The emitted light has been validated for compatibility with endoscopes, respiratory tools, catheters (such as endotracheal tubes and urinary catheters), and wound-healing bandages [38]. This specific wavelength possesses robust antimicrobial characteristics, capable of DNA disruption in bacteria and pathogens without leading to resistance [39]. When applied to medical surfaces and devices, blue light can systematically eliminate harmful microorganisms, contributing to infection prevention and establishing a safer healthcare environment.

4.3. Quantification and control of real-time NO release

4.3.1. Real-time modulation of NO release

The NO donor SNAP has the capacity to catalytically release NO when exposed to heat, light, or metal ions (Fig. 5A). This research introduced a small side glow fiber optic system, engineered for potential applications in medical device sterilization and designed to be universally compatible with a wide range of clinical devices, including endoscopes, respiratory equipment, catheters (including endotracheal and urinary catheters) (Fig. 5B). The adaptability of fiber optics enables smooth integration with diverse medical devices, streamlining minimally invasive procedures. Notably, unlike other antibacterial methods involving heat or electrical currents, fiber optics do not generate heat or electric currents and are made from biocompatible materials, thereby reducing the risk of tissue damage during biomedical interventions [40]. Leveraging these exceptional qualities, this study successfully combined fiber optics with a NO-releasing surface, resulting in the development of an exceptionally functional and efficient anti-bacterial insert. In clinical practice, the PACI device would be attached to the catheter lumen cap and inserted within the indwelling medical device. To evaluate its performance, real-time NO release from the insert was measured using a chemiluminescence-based nitric oxide analyzer (NOA). Impressively, the insert could be instantly activated with a single click, underscoring its efficacy in clinical applications (Fig. 5C).

Fig. 5.

(A) Chemical structure of the NO donor S-nitroso-N-acetylpenicillamine (SNAP). RSNOs like SNAP can be triggered by the stimulus of heat, light, or metal ions to cleave the S–N bond and release NO. (B) Picture of the Portable Antimicrobial Catheter Insert (PACI) device comprised of SNAP conjugated to PDMS polymer coated on a side glow fiber optic. (C) Representative graph of steady-state NO release from SNAP-PDMS with and without 450 nm blue light at physiological conditions (37 °C) (D) Representative example of tunable NO release via alternating on/off cycles of light irradiation. (E) Quantification of real time NO release from SNAP-PDMS with and without light measured using chemiluminescence measured with the trigger of blue light at 37 °C in PBS with 100 μM EDTA up to 24 h (n ≥ 3). (F) Quantitation of amount of SNAP present in the PBS-EDTA (soaking buffer) from SNAP-PDMS and SNAP-PDMS-Light samples at 37 °C in dark and presence of blue light, respectively. Data normalized to surface area of the polymer. (G) Quantification of nitrite generation in solution. (H) Cytocompatibility of PACI and controls measured towards 3 T3 mouse fibroblast cells using a 24 h indirect leachate exposure study. All data are reported as mean ± standard deviation (n ≥ 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Although NO-releasing materials possess immense clinical potential, they often lack dynamic NO control, a crucial feature for adaptable dosing in real-time. Photoactivated NO donors offer rapid, precise, and adjustable NO delivery, ensuring therapeutic levels without over- or under-dosing. This is vital in acute medical situations, where optimal NO concentration is critical. For example, catheter implantation may require higher initial NO levels to deter bacterial colonization, followed by lower maintenance levels. Contaminated surfaces may necessitate increased NO doses for disinfection. Achieving modulatory NO levels is feasible with photosensitive NO donors responding to light. Previous studies have combined RSNO donors with metal-based compounds to control NO release [41]. However, metal-based compounds can leach into the bloodstream, triggering cytotoxic immune responses and posing risks to healthy tissues. In contrast, light-based photodynamic therapy is non-invasive and safer, reducing the risk of unintended systemic effects or harm to healthy tissues.

To assess PACI’s real-time modulatory capacity, the SNAP-PDMS insert was connected to a Versalume^™ wearable light module emitting blue light (Fig. 5D). The study used alternative cycles of light on/off irradiation to measure NO release from SNAP-PDMS-coated PACI. Results showed that blue light instantly initiates higher NO levels in real-time. Across six cycles, photoinitiated NO release exhibited consistent trends, indicating PACI’s photosensitive properties and potential for real-time NO control. This precision and portability will allow patients to use antibacterial inserts in various settings while maintaining mobility. Light acts as a catalyst, enabling precise NO level modulation for antibacterial purposes. Real-time NO control enhances precision, safety, and versatility, making it valuable in healthcare, research, and industry.

4.3.2. 24-h NO release kinetics

The NO release from SNAP-PDMS (Light off) and SNAP-PDMS-Light (Light on) was measured for a duration of 24 h under physiological conditions (37 °C in PBS buffer) using chemiluminescence method (Fig. 5E). This study was conducted for 24 h, with the idea that the PACI device could be replaced daily in clinical applications. At 0 h time point, it was observed that NO release from SNAP-PDMS (Light off) and SNAP-PDMS-Light (Light on) measured at 1.38 and 27.40 × 10⁻¹⁰ mol cm⁻² min⁻¹, respectively. This finding underscores the profound catalytic effect of light, which significantly enhances the rate of NO release from the insert. Over the course of 24 h, NO release was monitored at various time points (0, 4, 8, 12 and 24 h). The levels of NO released at various time points are presented in Table 3.

Table 3.

NO release levels measured from SNAP-PDMS and SNAP-PDMS-Light (PACI) at different time points at 37 °C using a chemiluminescence nitric oxide analyzer. Data represents mean ± SD (n ≥ 3).

Time (h)	NO Release (SNAP-PDMS) (× 10−10 mol cm−2 min−1)	NO Release (SNAP-PDMS-Light) (× 10−10 mol cm−2 min−1)
0	1.38 ± 0.42	27.40 ± 8.35
4	1.28 ± 0.58	12.34 ± 0.85
8	1.63 ± 0.26	10.06 ± 1.96
12	1.46 ± 0.16	7.41 ± 2.58
24	0.59 ± 0.21	3.15 ± 0.98

One key benefit of the PACI design is the ability to regulate NO release by introducing light via fiber optics. These findings indicate that light can effectively be used as a trigger to modulate NO levels, precisely allowing for targeted antibacterial effects. Remarkably, SNAP-PDMS-Light continued to exhibit a substantially higher rate of NO release even at the 24 h mark, releasing 3.15 × 10⁻¹⁰ mol cm⁻² min⁻¹ of NO flux in contrast to the 0.59 × 10⁻¹⁰ mol cm⁻² min⁻¹ of NO from released by the Light off samples.

The introduction of blue light consistently elevates the rate at which NO is depleted from the samples, maintaining above physiologically relevant levels even at the 24 h time point. Overall, both PACI samples, with and without light exposure, adeptly emulate the NO release levels observed from endothelium (0.5–4 × 10⁻¹⁰ mol cm⁻² min⁻¹) for at least 24 h [42]. Previous studies on NO-releasing materials have successfully demonstrated their capacity to reduce inflammation, prevent fibrosis, eliminate various microbial strains (including antibiotic-resistant ones), obstruct, disrupt, and disperse microbial biofilm formation, inhibit platelet activation, and reduce clotting and thrombosis when maintaining NO flux within these ranges [16,43]. Once NO release from the PACI falls below physiological levels, a new PACI device can be readily replaced within the medical device. This approach ensures the sustained delivery of NO at biologically relevant concentrations is always maintained, making it a promising strategy for a range of clinical applications.

The antibacterial catheter insert offers exceptional convenience and versatility, making it well-suited for diverse healthcare settings. Its portable design allows easy use in hospitals, clinics, and home care environments, addressing infection prevention needs across various patient populations. The insert can be seamlessly integrated into different catheter types, including vascular, urinary, and hemodialysis catheters, ensuring its applicability across various medical scenarios. Delivering NO directly to the catheter’s surface provides targeted anti-bacterial action without impacting surrounding tissues or organs. Moreover, the controlled release mechanism guarantees a consistent supply of NO over an extended period, effectively inhibiting bacterial growth. This approach reduces the reliance on systemic antibiotics, mitigating the risk of antibiotic resistance and potential side effects. Optimal coating thickness in this study was chosen to balance NO release rates, antibacterial effectiveness, and durability, ensuring that NO levels remain near physiological levels without causing cytotoxic effects. However, this thickness can be tailored by adjusting the number of SNAP-PDMS topcoats, depending on the specific application (e.g., urinary or vascular catheters), the type of test subjects (e.g., small or large animals, humans), and the desired NO release levels and duration. The primary objective is to ensure the insert fits securely within the catheter lumen. Overall, the antibacterial catheter insert is a cost-effective solution that can be easily integrated into existing commercially available catheter designs, enhancing infection prevention and patient care.

4.4. Detection of NO donor in soaking buffer

Leaching can impact the biocompatibility of material by introducing foreign substances. If the leachates are toxic or trigger an immune response, they can lead to adverse reactions, inflammation, or tissue damage. Moreover, the leaching of compounds can disrupt the intended controlled drug delivery system, compromising patient safety and treatment outcomes. Therefore, managing and preventing drug leaching is crucial for maintaining the desired therapeutic effect and ensuring reliable and effective medical interventions. Parent molecule of SNAP is N-acetyl-D-penicillamine (NAP) which is an FDA-approved drug utilized for the treatment of heavy metal poisoning [44,45]. The degradation pathway ensures that the leaching of SNAP (if any) remains safe and non-toxic while maintaining its antibacterial properties. It is well understood that the covalent conjugation of drugs onto the surface can prevent the premature loss of compounds into biological systems and physiological fluids. Conventional non-covalently attached drugs usually face the issue of leaching which can significantly impact the functionality of the biomaterials. Such unintended release can lead to inadequate therapeutic effects, reduced efficacy, or adverse reactions. In order to evaluate the efficiency of the covalent conjugation of SNAP to polymer, the diffusion of SNAP from the SNAP-PDMS coated inserts was measured in the presence and absence of light (Fig. 5F). Both SNAP-PDMS and SNAP-PDMS-Light (PACI) were incubated in PBS at 37 °C, and the amount of SNAP released into the soaking buffer was quantified at various time points using UV–vis spectroscopy at a wavelength of 340 nm. After 24 h, it was found that 9.5 μg cm⁻² and 14.5 μg cm⁻² of SNAP had diffused out from SNAP-PDMS and SNAP-PDMS-Light, respectively. The covalent conjugation of the NO donor radically reduced the amount of SNAP that diffused into the soaking buffer. The examination of leachate levels in SNAP-PDMS films revealed a slight increase in the initial few hours of measurement. However, beyond this period, cumulative leachate levels remained almost constant throughout the study. This behavior can be attributed to the chemical reactions between NAP-thiolactone and the aminosilane cross-linker in PDMS, which limit nitrosation to covalently bound thiol groups. Any unreacted NAP-thiolactone retains its ring structure and does not form unbound SNAP. These findings align with prior research in which the covalent bonding of SNAP to polymers such as PDMS and polyvinyl chloride (PVC) exhibited comparable levels of donor leaching, signifying a substantial reduction in donor release compared to its impregnation within the polymer. [46,47].

In addition to covalent bonding, past research has demonstrated that using a polymer topcoat can reduce or control the leaching of NO donors as a preventive strategy [46]. However, this approach merely provides a short delay in the release of blended components, which restricts the potential applications of materials requiring sustained and long-lasting NO release. A noteworthy characteristic of covalently bound SNAP to PDMS polymer is its ability to eliminate the need for a topcoat to prevent leaching in aqueous environments while enabling localized release of NO at the desired material interface and location. Interestingly, in this study, the covalent conjugation of SNAP to PDMS resulted in a reduction of SNAP leaching by over 89 % when compared to impregnated SNAP in the PDMS polymer reported previously [31]. Covalent conjugation of the NO donor SNAP to hydrophobic polymers like PDMS has been previously reported to substantially reduce leaching, thereby extending the NO release from the polymer [48,49]. This reduction in leaching is attributed to the intramolecular hydrogen bonding between SNAP molecules and the low water uptake of hydrophobic polymers, effectively containing the dissolution and dissemination of SNAP out of the polymer matrix [37,50].

4.5. NO metabolite accumulation (Griess assay)

In addition to NO detection using chemiluminescence, the quantification of residual nitrite resulting from NO’s reaction with superoxide was also tested using colorimetric Griess assay (Fig. 5G). This assay indirectly measures NO levels by assessing nitrite (NO₂⁻) production through NO’s autooxidation in solution [51]. The NO₂⁻ group then reacts with sulfanilamide to form a diazonium salt intermediate which reacts with N-1-naphthyl ethylenediamine to form an azo dye. This azo dye is detectable at 540 nm spectrophotometrically [52]. For this study, the suspensions of PDMS, PDMS-Light, SNAP-PDMS, and SNAP-PDMS-Light were collected in buffers and media, incubated with Griess reagent, and data was collected by reading the samples for absorbance. Results from the study showed that the samples without NO donors (i.e., PDMS and PDMS-Light) exhibited no nitrite presence. Conversely, NO-releasing SNAP-PDMS and SNAP-PDMS-Light samples released 21.82 ± 1.07 and 86.01 ± 11.76 μg cm⁻² of nitrite into the solution, consistent with the chemiluminescence data from the nitric oxide analyzer. In all cases, the introduction of light to SNAP-PDMS resulted in increased NO/nitrite accumulation compared to light-off samples, attributed to the photocatalytic effect of light on SNAP.

4.6. Biocompatibility screening

Studies of the biocompatibility of the NO-releasing PACI and controls were performed to evaluate suitability for further antimicrobial screening. Testing of the fiber optics demonstrated retention of biocompatibility compared to SR control surfaces (Fig. 5H), showing that the light source and potential accumulation of NO metabolites did not lead to any significant cytotoxic effect. These observations were consistent with our prior study of NO-releasing catheter inserts [53], supporting the biocompatibility of the present technology. Unlike prior NO-releasing technologies based on the controlled release of NO by donor embedment into polymers, this strategy with SNAP-PDMS leads to very minimal donor/byproduct leaching, thereby enabling a more robust system for direct correlation of cytotoxic effects based on the total amount of NO metabolite accumulation. Notably, the practical application of this technology in catheters could be paired with traditional saline lock solutions, whereby lock solution leakage into the surrounding interstitial fluid could lead to further metabolite dilution [43]. In any eventuality, photoexcitation of the covalently integrated NO donor in the fiber optic coating enables a robust, cytocompatible technology for the prevention of catheter-associated infection.

4.7. Antibacterial activity of PACI using 24 h bacterial adhesion assay

4.7.1. Evaluating the antibacterial efficacy of PACI

Medical devices are susceptible to bacterial adhesion due to their surface characteristics and challenges in maintaining sterile conditions during medical procedures. Bacterial colonization and biofilm formation are major contributors to infections associated with the usage of catheters, such as catheter-related bloodstream infections and catheter-associated urinary tract infections. These infections not only shorten the device’s lifespan but also necessitate replacement before they become life-threatening, leading to patient suffering and increased healthcare costs. Addressing bacterial colonization on biomedical devices is crucial to reducing the incidence of infections and improving patient outcomes.

RSNOs possess photosensitivity, offering the potential for non-invasive localized antimicrobial strategies to prevent medical complications associated with medical devices. When exposed to light, particularly in the presence of specific wavelengths or with the aid of photosensitizing agents, RSNOs can undergo photodissociation, where the nitroso group breaks away from the thiol group, resulting in the release of NO. [31] This process can be harnessed for various applications, including in the field of medicine, where the release of NO can be used for its antimicrobial properties or as a signaling molecule with various physiological effects. The photoresponsive property of RSNO can be combined with blue light with an ability to photodynamically deactivate bacteria. The combination of the RSNOs with antibacterial light can not only catalyze the NO release from the material but also help in eradicating bacteria synergistically.

The blue wavelengths within the visible light spectrum (435–500 nm) have intrinsic antimicrobial properties and can effectively inactivate a wide range of bacteria, including both Gram-positive and Gram-negative species. In this study, a 450 nm wavelength blue light-emitting fiber optic was integrated with the photosensitive NO donor SNAP to develop a battery-operated, portable, NO-releasing antibacterial catheter insert. The antibacterial efficiency of the developed PACI device was evaluated against three bacterial strains: S. aureus, S. epidermidis, and P. mirabilis using a 24 h bacterial adhesion assay. The bacterial cells adhered to the surface of the PACI were quantified and normalized to the insert’s surface area to determine viable CFU cm⁻². The study’s outcomes on SNAP-PDMS-Light synergy unveiled a 95.90 % (1.44 log) reduction in viable S. aureus adhesion compared to the control PDMS group (Fig. 6A–B). Independently, PDMS-Light and SNAP-PDMS exhibited reductions of 72.17 % (0.52 log) and 75.82 % (0.59 log), respectively, due to their individual antibacterial effects. Consequently, the combined effect of SNAP-PDMS and Light resulted in the most substantial reduction. These results were similarly reflected with S. epidermidis and P. mirabilis bacteria (Fig. 6C–F). In both instances, SNAP-PDMS-Light achieved the highest bacterial inhibition, translating to a 99.76 % (2.68 log) and 93.51 % (1.19 log) reduction, respectively. In contrast, SNAP-PDMS control samples achieved 76.37 % (0.68 log) and 88.99 % (1.01 log) reduction against S. epidermidis and P. mirabilis, respectively. PDMS-Light samples, however, demonstrated a mere 0.53 % (0.03 log) reduction against S. epidermidis and a 40.19 % (0.24 log) reduction against P. mirabilis. The inadequacy of PDMS-Light against S. epidermidis can be attributed to the unique characteristics of S. epidermidis bacteria, biofilm formation, antioxidant defenses, cell wall composition, efflux pumps, genetics, pigmentation, and adaptive responses that collectively protect the bacteria from oxidative stress-induced damage, rendering it comparatively less susceptible to blue light in contrast to other bacteria [54]. Blue light, with a wavelength of 450 nm, chiefly impacts bacteria through photochemical damage to their DNA and cellular structures [39]. However, bacterial responses to light can differ based on factors like cell wall composition, pigmentation, and defense mechanisms. Prior investigations involving NO and light have demonstrated bacterial susceptibility to exposure to blue light [55]. This mechanism involves the photosensitizer absorbing blue light, generating reactive oxygen species (ROS) like singlet oxygen and free radicals. These ROS exhibit strong reactivity, capable of harming bacterial cells by disrupting cell membranes and vital biomolecules, ultimately causing bacterial death. Blue light-induced bacterial deactivation has garnered interest as a potential substitute for conventional antibiotics, particularly against antibiotic-resistant strains. This approach presents various benefits, including safety, diminished risk of antibiotic resistance, and the possibility of precise and localized treatment. The NO similarly exhibits antibacterial effects at high concentrations, prompting biofilm dispersal across various bacterial strains, signifying its therapeutic relevance against biofilm-related infections.

Fig. 6.

Antimicrobial activity of the NO-releasing Portable Antimicrobial Catheter Insert calculated as a log of the colony forming units (CFU) cm⁻¹ against: (A) S. aureus (B) S. epidermidis and (C) P. mirabilis. Corresponding log reduction of (D) S. aureus, (E) S. epidermidis and (F) P. mirabilis calculated with respect to PDMS control for 24 h. All data are represented as mean ± SEM (* p < 0.05, ** p < 0.01, *** p < 0.001, ****p < 0.0001, n ≥ 3). (G) Antibacterial mechanisms of NO and its byproducts.

NO is a reactive gas, with a transient nature and the capability to spontaneously permeate cell membranes. Its antibacterial mechanism involves the formation of reactive species such as peroxynitrite (ONOO⁻), nitrogen dioxide (NO₂), and dinitrogen trioxide (N₂O₃) through reactions with oxygen and reactive oxygen intermediates (Fig. 6G). In addition to NO and superoxide ions, reactive nitrogen species also hinder the synthesis of extracellular polysaccharides, which are crucial for bacterial adhesion on surfaces. These reactive species can interact with proteins, metalloenzymes, DNA, and cell membranes of bacteria, leading to multiple damages and ultimately causing cell death.

Activation of NO release from SNAP and other photosensitive NO-releasing composite materials has been documented in prior studies utilizing visible and near-infrared LED lights [56,57,58]. The ability of RSNOs to respond to light can be utilized in controlling the release of NO from polymers. The on-demand release of NO from the polymer matrix upon light exposure highlights the responsiveness of RSNOs to photolytic stimulation. While previous research has shown the benefits of precise control over NO release levels, [47,59] this study focuses on the practical application of adjustable photo-release characteristics of RSNOs in clinical settings using the PACI device. Using photo-triggered NO release from PACI offers a powerful and localized antimicrobial solution. It is characterized by portability, biocompatibility, affordability, and easy application. These qualities make it an optimal choice for integration with medical devices for efficient functioning. The disposable nature of the PACI device makes it highly suitable for sustained applications of NO on catheter surfaces. This application efficiently prevents the attachment of microorganisms, thus inhibiting the formation of biofilms on surfaces and effectively reducing the incidence of CRBSIs and medical device-associated infections. Consequently, the development of PACI technology is an important step towards averting microbial contamination in both short- and long-term indwelling medical devices that inherently face higher infection risks, including endotracheal tubes, intravenous, urinary, insulin, peritoneal dialysis, and hemodialysis catheters. Implementing this technology can significantly decrease mortality rates and associated healthcare expenses. Additionally, PACI can be combined with traditional antibiotics and photodynamic therapies. Research indicates that NO enhances antibiotic effectiveness by disrupting biofilms and increasing bacterial susceptibility, which is especially beneficial for resistant infections [60]. Combining PACI with photodynamic agents, which produce ROS when exposed to light, can offer a more comprehensive approach to controlling biofilm-forming bacteria and improving infection management in complex medical scenarios.

Previous studies have shown that incorporating RSNOs and releasing NO from this polymer in solvent-casted films or as a device coating reduces bacterial adhesion and biofilm formation in both in vitro and in vivo models. Based on prior evidence that SNAP-loaded polymers significantly reduce bacterial growth and are safe in vivo, we anticipate similar outcomes with the PACI device [36]. However, further evaluation using in vivo models is crucial to fully realize this technology’s clinical potential. These studies are currently underway and essential for assessing the PACI device’s long-term efficacy, safety, and biocompatibility in clinical settings. The continuation of this research will be key to demonstrating the viability of this approach for a wide range of medical applications, ultimately advancing the development of next-generation antibacterial solutions for medical devices.

5. Conclusions

In healthcare settings, bacterial biofilms are commonly associated with chronic and nosocomial infections, often linked to the usage of medical devices. These infections, which can lead to severe complications like bloodstream infections, urinary tract infections, and localized catheter-related infections, pose significant health risks to patients and result in elevated healthcare costs and extended hospital stays. To combat bacterial colonization on catheters and reduce infection risks, this study explores the potential of nitric oxide (NO) as an antimicrobial agent. NO is renowned for its broad-spectrum antimicrobial properties, yet its effective delivery to catheter surfaces is challenged by its gaseous nature and short half-life. This research introduces an innovative solution for addressing medical device-related infections through the use of the battery-operated Portable Antimicrobial Catheter Insert (PACI). The PACI is designed to prevent bacterial adhesion and biofilm formation on medical devices. It achieves this by utilizing S-nitroso-N-acetylpenicillamine (SNAP), a photoresponsive NO-releasing compound covalently conjugated to polydimethylsiloxane (PDMS) polymer, ensuring secure NO storage and controlled release. In the PACI, SNAP-PDMS is coated onto a fiber optic and connected to a Versalume^™ wearable light module, emitting 450 nm wavelength light. A simple button click instantly activates NO release, offering sustained and on-demand NO release in a wireless setup. Chemiluminescence studies confirmed the successful and real-time modulation of NO release from the PACI, maintaining physiologically relevant NO levels for at least 24 h. In vitro bactericidal evaluations demonstrated the broad-spectrum antibacterial activity of PACI against S. aureus, S. epidermidis, and P. mirabilis pathogens responsible for catheter-related bloodstream and urinary tract infections. The PACI effectively eliminated over 90 % of viable bacterial cells, preventing their adherence, and biofilm formation on surfaces. Biocompatibility assessments confirmed the safety of the light insert through leachate exposure studies with mouse fibroblast cells, indicating its suitability for clinical applications. Overall, this research demonstrates practical applications of NO-releasing materials, particularly in the context of medical device-related infections. The PACI’s portability, user-friendly, and universally adaptable design make it a promising tool for improving infection prevention and extending the lifespan of numerous medical devices like catheters across various clinical and home-based settings.

Data availability

Data will be made available upon a reasonable request.