Marcus J. Goudie

Antimicrobial and Physicochemical Characterization of Biodegradable, Nitric Oxide-Releasing Nanocellulose–Chitosan Packaging Membranes

Biodegradable composite membranes with antimicrobial properties consisting of nanocellulose fibrils (CNFs), chitosan, and S-nitroso-N-acetyl-d-penicillamine (SNAP) were developed and tested for food packaging applications. As a nitric oxide donor, SNAP was encapsulated into completely dispersed chitosan in 100 mL of 0.1 N acetic acid and was thoroughly mixed with CNFs to produce a composite membrane. The fabricated membranes had a uniform dispersion of chitosan and SNAP within the CNFs, which was confirmed through scanning electron microscopy (SEM) micrographs and a chemiluminescence nitric oxide analyzer. The membranes prepared without SNAP showed lower water vapor permeability than that of the membranes with SNAP. The addition of SNAP resulted in a decrease in Youngs modulus for both two- and three-layer membrane configurations. Antimicrobial property evaluation of SNAP-incorporated membranes showed an effective zone of inhibition against bacterial strains of Enterococcus faecalis, Staphylococcus aureus, and Listeria monocytogenes and demonstrated its potential applications for food packaging.

Characterization of an S-nitroso-N-acetylpenicillamine–based nitric oxide releasing polymer from a translational perspective

ABSTRACT Due to the role of nitric oxide (NO) in regulating a variety of biological functions in humans, numerous studies on different NO releasing/generating materials have been published over the past two decades. Although NO has been demonstrated to be a strong antimicrobial and potent antithrombotic agent, NO-releasing (NOrel) polymers have not reached the clinical setting. While increasing the concentration of the NO donor in the polymer is a common method to prolong the NO release, this should not be at the cost of mechanical strength or biocompatibility of the original material. In this work, it was shown that the incorporation of S-nitroso-penicillamine (SNAP), an NO donor molecule, into Elast-eon E2As (a copolymer of mixed soft segments of polydimethylsiloxane and poly(hexamethylene oxide)), does not adversely impact the physical and biological attributes of the base polymer. Incorporating 10 wt% of SNAP into E2As reduces the ultimate tensile strength by only 20%. The inclusion of SNAP did not significantly affect the surface chemistry or roughness of E2As polymer. Ultraviolet radiation, ethylene oxide, and hydrogen peroxide vapor sterilization techniques retained approximately 90% of the active SNAP content and did not affect the NO-release profile over an 18-day period. Furthermore, these NOrel materials were shown to be biocompatible with the host tissues as observed through hemocompatibility and cytotoxicity analysis. In addition, the stability of SNAP in E2As was studied under a variety of storage conditions, as they pertain to translational potential of these materials. SNAP-incorporated E2As stored at room temperature for over six months retained 87% of its initial SNAP content. Stored and fresh films exhibited similar NO release kinetics over an 18-day period. Combined, the results from this study suggest that SNAP-doped E2As polymer is suitable for commercial biomedical applications due to the reported physical and biological characteristics that are important for commercial and clinical success. GRAPHICAL ABSTRACT

Attenuation of thrombosis and bacterial infection using dual function nitric oxide releasing central venous catheters in a 9 day rabbit model

UNLABELLED Two major problems with implanted catheters are clotting and infection. Nitric oxide (NO) is an endogenous vasodilator as well as natural inhibitor of platelet adhesion/activation and an antimicrobial agent, and NO-releasing polymers are expected to have similar properties. Here, NO-releasing central venous catheters (CVCs) are fabricated using Elast-eon™ E2As polymer with both diazeniumdiolated dibutylhexanediamine (DBHD/NONO) and poly(lactic-co-glycolic acid) (PLGA) additives, where the NO release can be modulated and optimized via the hydrolysis rate of the PLGA. It is observed that using a 10% w/w additive of a PLGA with ester end group provides the most controlled NO release from the CVCs over a 14d period. The optimized DBHD/NONO-based catheters are non-hemolytic (hemolytic index of 0%) and noncytotoxic (grade 0). After 9d of catheter implantation in the jugular veins of rabbits, the NO-releasing CVCs have a significantly reduced thrombus area (7 times smaller) and a 95% reduction in bacterial adhesion. These results show the promise of DBHD/NONO-based NO releasing materials as a solution to achieve extended NO release for longer term prevention of clotting and infection associated with intravascular catheters. STATEMENT OF SIGNIFICANCE Clotting and infection are significant complications associated with central venous catheters (CVCs). While nitric oxide (NO) releasing materials have been shown to reduce platelet activation and bacterial infection in vitro and in short-term animal models, longer-term success of NO-releasing materials to further study their clinical potential has not been extensively evaluated to date. In this study, we evaluate diazeniumdiolate based NO-releasing CVCs over a 9d period in a rabbit model. The explanted NO-releasing CVCs were found to have significantly reduced thrombus area and bacterial adhesion. These NO-releasing coatings can improve the hemocompatibility and bactericidal activity of intravascular catheters, as well as other medical devices (e.g., urinary catheters, vascular grafts).

Improved hemocompatibility of silicone rubber extracorporeal tubing via solvent swelling-impregnation of S-nitroso-N-acetylpenicillamine (SNAP) and evaluation in rabbit thrombogenicity model

UNLABELLED Blood-contacting devices, including extracorporeal circulation (ECC) circuits, can suffer from complications due to platelet activation and thrombus formation. Development of nitric oxide (NO) releasing polymers is one method to improve hemocompatibility, taking advantage of the ability of low levels of NO to prevent platelet activation/adhesion. In this study a novel solvent swelling method is used to load the walls of silicone rubber tubing with the NO donor S-nitroso-N-acetylpenicillamine (SNAP). This SNAP-silicone rubber tubing exhibits an NO flux of ca. 1×10(-10)molcm(-2)min(-1), which mimics the range of NO release from the normal endothelium, which is stable for at least 4h. Images of the tubing before and after swelling, obtained via scanning electron microscopy, demonstrate that this swelling method has little effect on the surface properties of the tubing. The SNAP-loaded silicone rubber and silicone rubber control tubing are used to fabricate ECC circuits that are evaluated in a rabbit model of thrombogenicity. After 4h of blood flow, the SNAP-loaded silicone rubber circuits were able to preserve the blood platelet count at 64% of baseline (vs. 12% for silicone rubber control). A 67% reduction in the degree of thrombus formation within the thrombogenicity chamber was also observed. This study demonstrates the ability to improve the hemocompatibility of existing/commercial silicone rubber tubing via a simple solvent swelling-impregnation technique, which may also be applicable to other silicone-based blood-contacting devices. STATEMENT OF SIGNIFICANCE Localized nitric oxide (NO) release can be achieved from biomedical grade polymers doped with S-nitroso-N-acetylpenicillamine (SNAP). Despite the promising in vitro and in vivo biocompatibility results reported for these NO releasing polymers, many of these materials may face challenges in being translated to clinical applications, especially in the areas of polymer processing and manufacturing. In this study, we report a solvent swelling-impregnation technique to incorporate SNAP into extracorporeal circuit (ECC) tubing. These NO-releasing ECCs were able to attenuate the activation of platelets and maintain their functionality, while significantly reducing the extent of thrombus formation during 4h blood flow in the rabbit model of thrombogenicity.

A multi-defense strategy: Enhancing bactericidal activity of a medical grade polymer with a nitric oxide donor and surface-immobilized quaternary ammonium compound

Although the use of biomedical devices in hospital-based care is inevitable, unfortunately, it is also one of the leading causes of the nosocomial infections, and thus demands development of novel antimicrobial materials for medical device fabrication. In the current study, a multi-defense mechanism against Gram-positive and Gram-negative bacteria is demonstrated by combining a nitric oxide (NO) releasing agent with a quaternary ammonium antimicrobial that can be covalently grafted to medical devices. Antibacterial polymeric composites were fabricated by incorporating an NO donor, S-nitroso-N-acetyl-penicillamine (SNAP) in CarboSil® polymer and top coated with surface immobilized benzophenone based quaternary ammonium antimicrobial (BPAM) small molecule. The results suggest that SNAP and BPAM individually have a different degree of toxicity towards Gram-positive and Gram-negative bacteria, while the SNAP-BPAM combination is effective in reducing both types of adhered viable bacteria equally well. SNAP-BPAM combinations reduced the adhered viable Pseudomonas aeruginosa by 99.0% and Staphylococcus aureus by 99.98% as compared to the control CarboSil films. Agar diffusion tests demonstrate that the diffusive nature of NO kills bacteria beyond the direct point of contact which the non-leaching BPAM cannot achieve alone. This is important for potential application in biofilm eradication. The live-dead bacteria staining shows that the SNAP-BPAM combination has more attached dead bacteria (than live) as compared to the controls. The SNAP-BPAM films have increased hydrophilicity and higher NO flux as compared to the SNAP films useful for preventing blood protein and bacterial adhesion. Overall the combination of SNAP and BPAM imparts different attributes to the polymeric composite that can be used in the fabrication of antimicrobial surfaces for various medical device applications. STATEMENT OF SIGNIFICANCE A significant increase in the biomedical device related infections (BDRIs), inability of the currently existing antimicrobial strategies to combat them and a proportional rise in the associated morbidity demands development of novel antimicrobial surfaces. Some of the major challenges associated with the currently used therapeutics are: antibiotic resistance and cytotoxicity. In the current study, engineered polymeric composites with multi-defense mechanism were fabricated to kill bacteria via both active and passive mode. This was done by incorporating a nitric oxide (NO) donor S-nitroso-N-acetypenicillamine (SNAP) in a medical grade polymer (CarboSil®) and a benzophenone based quaternary ammonium antimicrobial small molecule (BPAM) was surface immobilized as the top layer. The developed biomaterial was tested with Gram-positive and Gram-negative strains and was found to be effective against both the strains resulting in up to 99.98% reduction in viable bacterial count. This preventative strategy can be used to fabricate implantable biomedical devices (such as catheters, stents, extracorporeal circuits) to not only significantly limit biofilm formation but also to reduce the antibiotic dose which are usually given post infections.

Tunable Nitric Oxide Release from S-Nitroso-N-acetylpenicillamine via Catalytic Copper Nanoparticles for Biomedical Applications

The quest for novel therapies to prevent bacterial infections and blood clots (thrombosis) is of utmost importance in biomedical research due to the exponential growth in the cases of thrombosis and blood infections and the emergence of multi-drug-resistant strains of bacteria. Endogenous nitric oxide (NO) is a cellular signaling molecule that plays a pivotal role in host immunity against pathogens, prevention of clotting, and regulation of systemic blood pressure, among several other biological functions. The physiological effect of NO is dose dependent, which necessitates the study of its tunable release kinetics, which is the objective of this study. In the present study, polymer composites were fabricated by incorporating S-nitroso-N-acetylpenicillamine (SNAP) in a medical-grade polymer, Carbosil, and top-coated with varying concentrations of catalytic copper nanoparticles (Cu-NPs). The addition of the Cu-NPs increased the NO release, as well as the overall antimicrobial activity via the oligodynamic effect of Cu. SNAP (10 wt %) composites without Cu-NP coatings showed a NO flux of 1.32 ± 0.6 × 10-10 mol min-1 cm-2, whereas Cu-NP-incorporated SNAP films exhibited fluxes of 4.48 ± 0.5 × 10-10, 4.84 ± 0.3 × 10-10, and 11.7 ± 3.6 × 10-10 mol min-1 cm-2 with 1, 3, and 5 wt % Cu-NPs, respectively. This resulted in a significant reduction (up to 99.8%) in both gram-positive and gram-negative bacteria, with very low platelet adhesion (up to 92% lower) as compared to that of the corresponding controls. Copper leachates from the SNAP films were detected using the inductively coupled plasma-mass spectrometry technique and were found to be significantly lower in concentration than the recommended safety limit by the FDA. The cell viability test performed on mouse fibroblast 3T3 cells provided supportive evidence for the biocompatibility of the material in vitro.

Liquid-infused nitric oxide-releasing (LINORel) silicone for decreased fouling, thrombosis, and infection of medical devices

Recent reports on liquid-infused materials have shown promise in creating ultra-low fouling surfaces, but are limited in their ability to prevent bacterial proliferation and prevent platelet activation in blood-contacting applications. In this work, a liquid-infused nitric oxide-releasing (LINORel) material is created by incorporating the nitric oxide (NO) donor S-nitroso-acetylpenicillamine (SNAP) and silicone oil in commercial medical grade silicone rubber tubing through a solvent swelling process. This combination provides several key advantages over previous NO-releasing materials, including decreased leaching of NO donor, controlled release of NO, and maintenance of ultra-low fouling property of liquid-infused materials. The LINORel tubing reduces protein adhesion as observed using fluorescence imaging, and platelet adhesion (81.7 ± 2.5%) in vitro over a 2 h period. The LINORel combination greatly reduces bacterial adhesion and biofilm formation of two most common pathogens responsible for hospital acquired infections: gram-positive Staphylococcus aureus and gram-negative Pseudomonas aeruginosa (99.3 ± 1.9% and 88.5 ± 3.3% respectively) over a 7-day period in a CDC bioreactor environment. Overall, the LINORel approach provides a synergistic combination of active and passive non-fouling approaches to increase biocompatibility and reduce infection associated with medical devices.

Nitric oxide-releasing polyurethanes

Polyurethanes (PUs) are widely used polymers for a variety of biomedical devices, but their utility is limited by associated foreign body response, thrombosis, and infection. Due to its inherent properties, nitric oxide (NO) provides a promising solution for overcoming problems associated with long-term use of medical devices. Nitric oxide has been known to play a crucial role in regulating a wide range of biological functions such as angiogenesis, tissue remodeling, inflammation, wound healing, vasodilation, thrombosis, tumor formation, and infection. Therefore, NO-releasing PUs offer great potential in terms of their biomedical applications. In this chapter, the various applications, advantages, and challenges associated with NO-releasing PUs are discussed.

Characterization and In Vivo Performance of Nitric Oxide‐Releasing Extracorporeal Circuits in a Feline Model of Thrombogenicity

Infection and thrombosis are the two leading complications associated with blood contacting medical devices, and have led to the development of active materials that can delivery antibiotics or antithrombotic agents. Two key characteristics of these materials are the ability to produce controlled delivery, as well as minimal systemic delivery of the agent outside of the device site. Nitric oxide (NO) releasing materials are attractive as NO plays pivotal roles in the bodys natural defense against bacterial infection, as well as regulation of platelet adhesion and activation. This work characterizes an NO-releasing extracorporeal circuit (ECC) under flow conditions for the first time, examining the effect of incubation and application of the top coating on leaching of NO donor and NO-release kinetics. Top coated ECCs with incubation delivered ca. 1% of the total NO potential over the 4-h period, whereas uncoated ECCs delivered over 4.5% of the total NO. Incubated ECC loops maintained a flux of 1.83 ± 0.50 × 10-10 mol min-1 cm-2 for the full 4 h duration. The NO-releasing ECC loops significantly increased the time-to-clot as compared to the corresponding control (11 ± 3.6 min control, 132 ± 93.0 min NO-releasing) when evaluated in vivo in a feline animal model.

Biotemplated Synthesis and Characterization of Mesoporous Nitric Oxide-Releasing Diatomaceous Earth Silica Particles

Diatomaceous earth (DE), a nanoporous silica material composed of fossilized unicellular marine algae, possesses unique mechanical, molecular transport, optical, and photonic properties exploited across an array of biomedical applications. The utility of DE in these applications stands to be enhanced through the incorporation of nitric oxide (NO) technology shown to modulate essential physiological processes. In this work, the preparation and characterization of a biotemplated diatomaceous earth-based nitric oxide delivery scaffold are described for the first time. Three aminosilanes [(3-aminopropyl)triethoxysilane (APTES), N-(6-aminohexyl)aminomethyltriethoxysilane (AHAMTES), and 3-aminopropyldimethylethoxysilane (APDMES)] were evaluated for their ability to maximize NO loading via the covalent attachment of N-acetyl-d-penicillamine (NAP) to diatomaceous earth. The use of APTES cross-linker resulted in maximal NAP tethering to the DE surface, and NAP-DE was converted to NO-releasing S-nitroso-N-acetyl-penicillamine (SNAP)-DE by nitrosation. The total NO loading of SNAP-DE was determined by chemiluminescence to be 0.0372 ± 0.00791 μmol/mg. Retention of diatomaceous earths unique mesoporous morphology throughout the derivatization was confirmed by scanning electron microscopy. SNAP-DE exhibited 92.95% killing efficiency against Gram-positive bacteria Staphylococcus aureus as compared to the control. The WST-8-based cytotoxicity testing showed no negative impact on mouse fibroblast cells, demonstrating the biocompatible potential of SNAP-DE. The development of NO releasing diatomaceous earth presents a unique means of delivering tunable levels of NO to materials across the fields of polymer chemistry, tissue engineering, drug delivery, and wound healing.