Poster N17 - Antibacterial and Hemocompatible Lock Solution Applying S-Nitrosoglutathione (GSNO) Catalyzed by Copper Nanoparticle Coated Catheter for Antibacterial and Antithrombotic Applications

Introduction: The development of medical devices that implement the incorporation of NO donor molecules has displayed that combination strategies for surface modifications have further enhanced the results observed from these donor molecules. Specifically, metal ion catalysis using metals such as copper nanoparticles to interact with these donor molecules has shown promising preliminary data that exhibits an elevation in NO release to maximize the antimicrobial and hemocompatible properties of the surface they are implemented into while still maintaining cytocompatibility1. This catalysis works through a reduction chemistry mechanism in which the metal ion interacts with disulfide bonds present in the biological system and NO donor to reduce Cu2+ to Cu1+ 2. This reduced copper ion then reacts with the RSNO molecule and releases NO. A key feature these metal ion interactions possess includes the conversion back to Cu2+ during the reduction reaction which leads to re-initiation of the cycle which in turn also leads to endogenous NO generation/catalysis and interactions with the biomedical surface. The combination of multiple surface modifications to address surface-related complications associated with catheter lock solutions remains to be further characterized3. Herein, a combination of a GSNO lock solution implemented into a copper-coated catheter will be assessed to promote antibacterial and antifouling nature. These characterizations provide a pathway to the implementation of therapies that prevent these complications from proliferating.

Materials and

Methods: Experimental catheters for this study were fabricated using a dip-coating method on stainless steel mandrels with a clinically relevant silicone rubber substrate. For implementation of metal nanoparticles, 40-60nm, >99% purity Cu nanoparticles and added to the surface via dip coating using a polymer solution at specific concentrations to control the catalysis of NO release in addition to NO generation properties. GSNO synthesis was done through a facile nitrosation of the precursor molecule glutathione (GSH) and precipitated to yield a solid, soluble product. In order to quantify the nitric oxide release kinetics of the lock solution through the polymer interface, a gold-standard chemiluminescence method of measuring nitric oxide release via a Nitric Oxide Analyzer (NOA) was used for these characterization studies. Bacterial adhesion studies were conducted using a 48 h biofilm model and 24 h adhesion studies to analyze both early and onset infection of the interior and exterior interface of catheter samples. Both gram positive and gram-negative representative strains were analyzed using S. aureus (ATCC 6538) and E.coli (ATCC 25922) respectively. Cytocompatibility studies were conducted with a 24 h leachate study using NIH 3T3 mouse fibroblast cells and Human umbilical vein endothelial cells (HUVECs) following ISO-10993-5 standards. All blood compatibility and platelet adhesion studies were conducted using porcine blood and LDH assay for quantification.

Results, Conclusions, and Discussions: Nitric oxide release kinetics display the role of Cu coatings on the fabricated catheters by elevating the catalysis of NO through the polymer. At lower concentrations (5 mg mL-1) of GSNO-GSH lock solution with Cu coated catheters, the highest elevation of NO release was observed with an average NO flux of 12.89 x 10-10 mol min-1 cm-1 compared to control catheters with 6.45 x 10-10 mol min-1 cm-1 in 24 h (Figure 1). These findings suggest that at lower cytocompatible concentrations of GNSO-GSH, reaction efficiency is enhanced by allowing the NO donor to react with more Cu2+ active sites present on the interior catheter surface. In order to assess the antimicrobial efficacy of the proposed materials, a 48 h biofilm eradication study was conducted. Cu coated catheters with 5 mg mL-1 GSNO GSH lock solution displayed >2 log reduction (>99%) vs. control catheters when exposed to E.coli 25922 and S.aureus 6538 (Figure 2). In addition, a 24 h bacterial adhesion study exploring exterior lumen
bacterial eradication showed similar trends with over 1.5 log reduction in both gram positive and negative strains (Figure 3). Apart from the bactericidal properties of the materials, different concentrations of GSNO-GSH lock solutions did not illicit cytotoxic effects against mouse fibroblast and HUVEC cells with viability above 70% for all test samples in an MTT
assay. Blood compatibility studies also displayed significant anti-platelet effects by reducing platelet activation on the external lumen of the catheter by 72.09% compared to controls.

NO releasing lock solution therapies have displayed viable antibacterial and hemocompatible results compared to other antibiotic and anticoagulant loaded solutions however, the incorporation of a metal catalyst such as copper nanoparticles provide for the potential for enhancement of these effects both within the catheter and on the external polymer interface. This project combined and NO releasing lock solution in conjunction with a copper nanoparticle coated catheter to combat bacterial infection and achieve hemocompatibility on both interior and exterior interfaces of catheter applications.

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References:
(1) Douglass, M. E.; Goudie, M. J.; Pant, J.; Singha, P.; Hopkins, S.; Devine, R.; Schmiedt, C. W.; Handa, H. Catalyzed Nitric Oxide Release via Cu Nanoparticles Leads to an Increase in Antimicrobial Effects and Hemocompatibility for Short-Term Extracorporeal Circulation. ACS Applied Bio Materials 2019, 2 (6), 2539-2548. DOI: 10.1021/acsabm.9b00237.
(2) Pant, J.; Goudie, M. J.; Hopkins, S. P.; Brisbois, E. J.; Handa, H. Tunable Nitric Oxide Release from S-Nitroso-N-acetylpenicillamine via Catalytic Copper Nanoparticles for Biomedical Applications. ACS Applied Materials & Interfaces 2017, 9 (18), 15254-15264. DOI: 10.1021/acsami.7b01408.
(3) Ashcraft, M.; Douglass, M.; Garren, M.; Mondal, A.; Bright, L. E.; Wu, Y.; Handa, H. Nitric Oxide-Releasing Lock Solution for the Prevention of Catheter-Related Infection and Thrombosis. ACS Applied Bio Materials 2022, 5 (4), 1519-1527. DOI: 10.1021/acsabm.1c01272.