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Drug Delivery

Drug Delivery - Poster Session A

Poster I20 - Antibacterial Assessment of a Nitric Oxide-Generating Anticoagulant in Solution and Characterization of a Nitric Oxide-Releasing Substrate

Thursday, October 24, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Internal blood-contacting medical devices often encounter significant complications, including thrombosis and bacterial infections, which compromise their hemocompatibility [3]. Current clinical approaches aim to mitigate thrombus formation but can lead to excessive blood loss and patient death [2]. Literature highlights three essential properties for achieving optimal hemocompatibility: immobilization of anticoagulants, controlled release of antithrombotics, and surface chemistry modifications mimicking endothelial properties [1]. This project aims to incorporate all three properties by creating an aqueous-soluble anticoagulant molecule modified with a nitric-oxide NO-generating (NOgen) moiety. This design is expected to exhibit antibacterial capabilities by preventing bacterial proliferation and biofilm formation. Additionally, the NOgen anticoagulant was applied onto an NO-releasing (NOrel) substrate to characterize its surface properties and assess its potential in overcoming challenges with indwelling devices.

Materials and

Methods:  The molecule was fabricated by facilitating a bond between the carboxylic acid of the anticoagulant and the amine of the NOgen moiety over a 24- hour period, and it was dissolved in PBS . In vitro bacterial studies were performed on the base anticoagulant as well as the newly synthesized molecule to assess bacterial killing because of the covalent addition. Various clinical concentrations of both solutions were exposed to Gram-positive Staphylococcus aureus (S. aureus) bacteria over a 24-hour incubation period under physiological conditions (150 rpm, 37°C). Subsequently, bacterial cultures were plated on Luria-Bertani (LB) agar plates to count colony forming units (CFU). Absorbances of S. aureus and Gram-negative Escherichia coli (E. Coli) solutions in 96-well plates were measured to identify the concentration where 50% reduction occurred for each strain compared to bacteria controls, which is the MIC50 value. An NO donor was incorporated into 8 mm commercial-grade polymer films, which were subsequently plasma treated and submerged in concentrated solution with the doped anticoagulant molecule to facilitate surface adhesion . Anticoagulant adhesion was quantified via toluidine blue assay, during which toluidine blue dye binds to a specific functional group in the NOgen anticoagulant molecule when present.

Results, Conclusions, and Discussions: Overall, NOgen-A demonstrated superior antibacterial capabilities compared to the control anticoagulant. In Figure 1A, significant bacterial eradication of S. aureus was observed at high concentrations of NOgen-A, while the control showed no effect. At lower concentrations, an increase in growth was noted with the control anticoagulant (A-C4), which suggests a potential proliferative effect on S. aureus that was eliminated with the covalent addition. Figure 1B displays agar plate images illustrating S. aureus CFU counts for each concentration group. Additionally, as shown in Figure 2, NOgen-A had an MIC50 value of approximately 440 µg/ml when exposed to S. aureus. The E. coli MIC50 assay indicated similar efficacy, with NOgen-A showing highest antimicrobial capacities but at a lower concentration of 110 µg/ml. With both strains, control A showed no considerable evidence of bactericidal activity. Optimization of NOgen-A attachment to a NOrel surface revealed that higher concentrations led to increased adhesion of NOgen-A to the film, both before and after incorporating a PBS washing step to the protocol to mitigate the risk of inpatient leaching. Over a 14-day observation period where films were submerged in PBS at 37°C to mimic physiological conditions, 1.17±1.24 µg of NOgen-A leached off into solution after day 14. The newly synthesized molecule NOgen-A is hypothesized to incorporate essential components to achieve endothelium-mimicking functionality, which proves beneficial both in solution and adsorbed onto an NOrel polymer. Initial tests have demonstrated that NOgen-A has effective antibacterial properties in vitro. Further studies will explore its antimicrobial effects on additional bacterial strains, evaluate its antithrombotic potential, and assess its cytotoxicity levels in combination with NO.

Acknowledgements (Optional):  This material is based upon work supported by a National Science Foundation Research Experiences for Undergraduates (REU) site program under Grant No. 1950581.

References:
1. Ashcraft, M., et al. (2021). "Combination strategies for antithrombotic biomaterials: an emerging trend towards hemocompatibility." Biomaterials Science 9(7): 2413-2423
2. Gbyli, R., et al. (2018). "Achieving Totally Local Anticoagulation on Blood Contacting Devices." Advanced Materials Interfaces 5(4): 1700954.
3. Pavithra, D. and M. Doble (2008). "Biofilm formation, bacterial adhesion and host response on polymeric implants—issues and prevention." Biomedical Materials 3(3): 034003.