Analysis of the broad-spectrum potential of nitric oxide for antibacterial activity against clinically isolated drug-resistant bacteria.

The development of drug-resistant microorganisms is taking a heavy toll on the biomedical world. Clinical infections are costly and becoming increasingly dangerous as bacteria that once responded to standard antibiotic treatment are developing resistance mechanisms that require innovative treatment strategies. Nitric oxide (NO) is a gaseous molecule produced endogenously that has shown potent antibacterial capabilities in numerous research studies. Its multimechanistic antibacterial methods prevent the development of resistance and have shown potential as an alternative to antibiotics. However, there has yet to be a direct comparison study evaluating the antibacterial properties of NO against antibiotic susceptible and antibiotic-resistant clinically isolated bacterial strains. Herein, standardized lab and clinically isolated drug-resistant bacterial strains are compared side-by-side for growth and viability following treatment with NO released from S-nitrosoglutathione (GSNO), an NO donor molecule. Evaluation of growth kinetics revealed complete killing of E. coli lab and clinical strains at 17.5 mM GSNO, though 15 mM displayed >50% killing and significantly reduced metabolic activity, with greater dose dependence for membrane permeability. Clinical P. aeruginosa showed greater susceptibility to GSNO during growth curve studies, but metabolic activity and membrane permeability demonstrated similar effects for 12.5 mM GSNO treatment of lab and clinical strains. MRSA lab and clinical strains exhibited total killing at 17.5 mM treatment, though metabolic activity was decreased, and membrane permeation began at 12.5 mM for both strains. Lastly, both S. epidermidis strains were killed by 15 mM GSNO, with sensitivities in metabolic activity and membrane permeability at 12.5 mM GSNO. The mirrored antibacterial effects seen by the lab and clinical strains of two Gram-negative and two Gram-positive bacteria reveal the translational success of NO as an antibacterial therapy and potential alternative to standard antibiotic treatment.

Engineering Nitric Oxide-Releasing Antimicrobial Dental Coating for Targeted Gingival Therapy.

Bacterial biofilms play a central role in the development and progression of periodontitis, a chronic inflammatory condition that affects the oral cavity. One solution to current treatment constraints is using nitric oxide (NO)─with inherent antimicrobial properties. In this study, an antimicrobial coating is developed from the NO donor S-nitroso-N-acetylpenicillamine (SNAP) embedded within polyethylene glycol (PEG) to prevent periodontitis. The SNAP-PEG coating design enabled a controlled NO release, achieving tunable NO levels for more than 24 h. Testing the SNAP-PEG composite on dental floss showed its effectiveness as a uniform and bioactive coating. The coating exhibited antibacterial properties against Streptococcus mutans and Escherichia coli, with inhibition zones measuring up to 7.50 ± 0.28 and 14.80 ± 0.46 mm2, respectively. Furthermore, SNAP-PEG coating materials were found to be stable when stored at room temperature, with 93.65% of SNAP remaining after 28 d. The coatings were biocompatible against HGF and hFOB 1.19 cells through a 24 h controlled release study. This study presents a facile method to utilize controlled NO release with dental antimicrobial coatings comprising SNAP-PEG. This coating can be easily applied to various substrates, providing a user-friendly approach for targeted self-care in managing gingival infections associated with periodontitis.

Antimicrobial efficacy of a nitric oxide-releasing ampicillin conjugate catheter lock solution on clinically-isolated antibiotic-resistant bacteria.

Antibiotic lock therapy (ALT) is standard clinical practice for treating bacteremia linked with catheter-related bloodstream infections (CRBSIs). However, this strategy frequently fails against multi-drug-resistant bacteria in clinical settings. In this study, a novel approach to utilize a nitric oxide (NO) donor S-nitroso-N-acetyl-penicillamine (SNAP)-conjugated to ampicillin antibiotic (namely SNAPicillin) as a catheter lock solution is presented. The conjugate of two antimicrobial agents is anticipated to overcome the challenges of bacterial infection caused by antibiotic-resistant bacteria in ALT applications. Nitric oxide release from the SNAPicillin lock solution at varying concentrations was measured at 0 and 24 h time points in a catheter model system, which revealed tunable NO release at physiological levels. The clinical strains of E. coli (CDC AR-0089) and S. marcescens (CDC AR-0099) were screened using a zone of inhibition assay against standard antibiotics which confirmed the antibiotic resistance in bacteria. The minimum inhibitory concentration (MIC) testing of SNAPicillin unveiled the lowest MIC value for SNAPicillin against both E. coli and S. marcescens (1 and 2 mM of SNAPicillin, respectively) with an 8.24- and 4.28-log reduction in bacterial load compared to controls, respectively. In addition, while the ampicillin-treated biofilm demonstrated resistance toward the antibiotic, SNAPicillin led to >99% reduction in exterminating biofilm buildup on polymeric catheter surfaces. Lastly, the SNAPicillin lock solution was determined to be biocompatible via hemolysis and cell compatibility studies. Together, these results emphasize the promising potential of SNAPicillin lock solution with the dual-action of NO and ampicillin in overcoming bacterial challenges on medical devices like central venous catheters and other medical device interfaces.

Biomimetic catheter surface with dual action NO-releasing and generating properties for enhanced antimicrobial efficacy.

Infection of indwelling catheters is a common healthcare problem, resulting in higher morbidity and mortality. The vulnerable population reliant on catheters post-surgery for food and fluid intake, blood transfusion, or urinary incontinence or retention is susceptible to hospital-acquired infection originating from the very catheter. Bacterial adhesion on catheters can take place during the insertion or over time when catheters are used for an extended period. Nitric oxide-releasing materials have shown promise in exhibiting antibacterial properties without the risk of antibacterial resistance which can be an issue with conventional antibiotics. In this study, 1, 5, and 10 wt % selenium (Se) and 10 wt % S-nitrosoglutathione (GSNO)-incorporated catheters were prepared through a layer-by-layer dip-coating method to demonstrate NO-releasing and NO-generating capability of the catheters. The presence of Se on the catheter interface resulted in a 5 times higher NO flux in 10% Se-GSNO catheter through catalytic NO generation. A physiological level of NO release was observed from 10% Se-GSNO catheters for 5 d, along with an enhanced NO generation via the catalytic activity as Se was able to increase NO availability. The catheters were also found to be compatible and stable when subjected to sterilization and storage, even at room temperature. Additionally, the catheters showed a 97.02% and 93.24% reduction in the adhesion of clinically relevant strains of Escherichia coli and Staphylococcus aureus, respectively. Cytocompatibility testing of the catheter with 3T3 mouse fibroblast cells supports the material's biocompatibility. These findings from the study establish the proposed catheter as a prospective antibacterial material that can be translated into a clinical setting to combat catheter-related infections.

Recent Developments in Multifunctional Antimicrobial Surfaces and Applications toward Advanced Nitric Oxide-Based Biomaterials.

Implant-associated infections arising from biofilm development are known to have detrimental effects with compromised quality of life for the patients, implying a progressing issue in healthcare. It has been a struggle for more than 50 years for the biomaterials field to achieve long-term success of medical implants by discouraging bacterial and protein adhesion without adversely affecting the surrounding tissue and cell functions. However, the rate of infections associated with medical devices is continuously escalating because of the intricate nature of bacterial biofilms, antibiotic resistance, and the lack of ability of monofunctional antibacterial materials to prevent the colonization of bacteria on the device surface. For this reason, many current strategies are focused on the development of novel antibacterial surfaces with dual antimicrobial functionality. These surfaces are based on the combination of two components into one system that can eradicate attached bacteria (antibiotics, peptides, nitric oxide, ammonium salts, light, etc.) and also resist or release adhesion of bacteria (hydrophilic polymers, zwitterionic, antiadhesive, topography, bioinspired surfaces, etc.). This review aims to outline the progress made in the field of biomedical engineering and biomaterials for the development of multifunctional antibacterial biomedical devices. Additionally, principles for material design and fabrication are highlighted using characteristic examples, with a special focus on combinational nitric oxide-releasing biomedical interfaces. A brief perspective on future research directions for engineering of dual-function antibacterial surfaces is also presented.

Nitric Oxide-Releasing Silicone Oil with Tunable Payload for Antibacterial Applications.

Bacterial infections are a hurdle to the application of medical devices, and in the United States alone, more than one million infection cases are reported annually from indwelling medical devices. Infections not only affect the function of medical devices but also risk the lives and health of patients. Nitric oxide (NO) has been used as an antibacterial therapy that kills bacteria without causing resistance and provides many therapeutic effects such as anti-inflammation, antithrombosis, and angiogenesis. Silicone oils have been widely utilized in manufacturing consumer goods, healthcare products, and medical products. Specifically, liquid silicone oils are used as a medical lubricant that creates lubricated interfaces between medical devices and the exterior physiological environment to improve the performance of medical devices. Herein, we report the first primary S-nitrosothiol-based NO-releasing silicone oil (RSNO-Si) that exhibits proactive antibacterial effects. S-nitrosothiol silicone oils (RSNO-Si) were synthesized and the NO payloads ranged from 34.0 to 603.9 µM. The increased NO payload induced higher-viscosity RSNO-Si oils, as RSNO0.1-Si, RSNO0.5-Si, and RSNO1-Si had viscosities of 12.8 ± 0.1 cP, 32.0 ± 0.2 cP, and 35.1 ± 0.3 cP, respectively. RSNO-Si-SR interfaces were fabricated by infusing silicone rubber (SR) in RSNO-Si oil, and the resulting RSNO-Si-SR disks demonstrated NO release without NO donor leaching. RSNO0.1-Si-SR, RSNO0.5-Si-SR, and RSNO1-Si-SR exhibited maximum NO flux at 0.8, 6.5, and 21.5 × 10 -10 mol cm-2 min-1 in 24 h, respectively. RSNO-Si-SR disks also demonstrated 97.45, 95.40, and 96.08% of inhibition against S. aureus in a 4 h bacterial adhesion assay. Considering the easy synthesis, simple fabrication of non-leaching NO-releasing interfaces, tunable payloads, NO flux levels, and antimicrobial effects, RSNO-Si oils exhibited their potential use as platform chemicals for creating antimicrobial medical device surfaces and other antibacterial materials.

Smartphone compatible nitric oxide releasing insert to prevent catheter-associated infections.

A large fraction of nosocomial infections is associated with medical devices that are deemed life-threatening in immunocompromised patients. Medical device-related infections are a result of bacterial colonization and biofilm formation on the device surface that affects >1 million people annually in the US alone. Over the past few years, light-based antimicrobial therapy has made substantial advances in tackling microbial colonization. Taking the advantage of light and antibacterial properties of nitric oxide (NO), for the first time, a robust, biocompatible, anti-infective approach to design a universal disposable catheter disinfection insert (DCDI) that can both prevent bacterial adhesion and disinfect indwelling catheters in situ is reported. The DCDI is engineered using a photo-initiated NO donor molecule, incorporated in polymer tubing that is mounted on a side glow fiber optic connected to an LED light source. Using a smartphone application, the NO release from DCDI is photoactivated via white light resulting in tunable physiological levels of NO for up to 24 h. When challenged with microorganisms S. aureus and E. coli, the NO-releasing DCDI statistically reduced microbial attachment by >99% versus the controls with just 4 h of exposure. The DCDI also eradicated ∼97% of pre-colonized bacteria on the CVC catheter model demonstrating the ability to exterminate an established catheter infection. The smart, mobile-operated novel universal antibacterial device can be used to both prevent catheter infections or can be inserted within an infected catheter to eradicate the bacteria without complex surgical interventions. The therapeutic levels of NO generated via illuminating fiber optics can be the next-generation biocompatible solution for catheter-related bloodstream infections.

Long-Term Storage Stability and Nitric Oxide Release Behavior of (N-Acetyl-S-nitrosopenicillaminyl)-S-nitrosopenicillamine-Incorporated Silicone Rubber Coatings.

Physical incorporation of nitric oxide (NO) releasing materials in biomedical grade polymer matrices to fabricate antimicrobial coatings and devices is an economically viable process. However, achieving long-term NO release with a minimum or no leaching of the NO donor from the polymer matrix is still a challenging task. Herein, (N-acetyl-S-nitrosopenicillaminyl)-S-nitrosopenicillamine (SNAP-SNAP), a penicillamine dipeptide NO-releasing molecule, is incorporated into a commercially available biomedical grade silicone rubber (SR) to fabricate a NO-releasing coating (SNAP-SNAP/SR). The storage stabilities of the SNAP-SNAP powder and SNAP-SNAP/SR coating were analyzed at different temperatures. The SNAP-SNAP/SR coatings with varying wt % of SNAP-SNAP showed a tunable and sustained NO release for up to 6 weeks. Further, S-nitroso-N-acetylpenicillamine (SNAP), a well-explored NO-releasing molecule, was incorporated into a biomedical grade silicone polymer to fabricate a NO-releasing coating (SNAP/SR) and a comparative analysis of the NO release and S-nitrosothiol (RSNO) leaching behavior of 10 wt % SNAP-SNAP/SR and 10 wt % SNAP/SR was studied. Interestingly, the 10 wt % SNAP-SNAP/SR coatings exhibited ∼36% higher NO release and 4 times less leaching of NO donors than the 10 wt % SNAP/SR coatings. Further, the 10 wt % SNAP-SNAP/SR coatings exhibited promising antibacterial properties against Staphylococcus aureus and Escherichia coli due to the persistent release of NO. The 10 wt % SNAP-SNAP/SR coatings were also found to be biocompatible against NIH 3T3 mouse fibroblast cells. These results corroborate the sustained stability and NO-releasing properties of the SNAP-SNAP in a silicone polymer matrix and demonstrate the potential for the SNAP-SNAP/SR polymer in the fabrication of long-term indwelling biomedical devices and implants to enhance biocompatibility and resist device-related infections.

Nitric Oxide Release and Antibacterial Efficacy Analyses of S-Nitroso-N-Acetyl-Penicillamine Conjugated to Titanium Dioxide Nanoparticles.

Therapeutic agents can be linked to nanoparticles to fortify their selectivity and targeted delivery while impeding systemic toxicity and efficacy loss. Titanium dioxide nanoparticles (TiNPs) owe their rise in biomedical sciences to their versatile applicability, although the lack of inherent antibacterial properties limits its application and necessitates the addition of bactericidal agents along with TiNPs. Structural modifications can improve TiNP's antibacterial impact. The antibacterial efficacy of nitric oxide (NO) against a broad spectrum of bacterial strains is well established. For the first time, S-nitroso-N-acetylpenicillamine (SNAP), an NO donor molecule, was covalently immobilized on TiNPs to form the NO-releasing TiNP-SNAP nanoparticles. The TiNPs were silanized with 3-aminopropyl triethoxysilane, and N-acetyl-d-penicillamine was grafted to them via an amide bond. The nitrosation was carried out by t-butyl nitrite to conjugate the NO-rich SNAP moiety to the surface. The total NO immobilization was measured to be 127.55 ± 4.68 nmol mg-1 using the gold standard chemiluminescence NO analyzer. The NO payload can be released from the TiNP-SNAP under physiological conditions for up to 20 h. The TiNP-SNAP exhibited a concentration-dependent antimicrobial efficiency. At 5 mg mL-1, more than 99.99 and 99.70% reduction in viable Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacteria, respectively, were observed. No significant cytotoxicity was observed against 3T3 mouse fibroblast cells at all the test concentrations determined by the CCK-8 assay. TiNP-SNAP is a promising and versatile nanoparticle that can significantly impact the usage of TiNPs in a wide variety of applications, such as biomaterial coatings, tissue engineering scaffolds, or wound dressings.

Prevention of medical device infections via multi-action nitric oxide and chlorhexidine diacetate releasing medical grade silicone biointerfaces.

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

A Multidisciplinary Experiment to Characterize Antifouling Biocompatible Interfaces via Quantification of Surface Protein Adsorption.

Novel biomaterial development is a rapidly growing field that is crucial because biomaterial fouling, due to rapid and irreversible protein adsorption, leads to cellular responses and potentially detrimental consequences such as surface thrombosis, biofilm formation, or inflammation. Therefore, biomaterial technology's fundamentals, like material biocompatibility, are critical in undergraduate education. Exposing undergraduate students to biomaterials and biomedical engineering through interdisciplinary experiments allows them to integrate knowledge from different fields to analyze multidisciplinary results. In this practical laboratory experiment, undergraduate students will characterize surface properties (contact and sliding angle measurements) for the antifouling polydimethylsiloxane (PDMS) polymer using a goniometer and a smartphone, as well as quantify protein adsorption on antifouling surfaces via a colorimetric assay kit to develop their understanding of antifouling surface characteristics, UV-vis spectroscopy, and colorimetric assays. The antifouling PDMS polymer is prepared by silicone oil infusion and compared to untreated control PDMS. The polymer hydrophobicity was demonstrated by static water contact angles of ~99° and 102° for control and antifouling PDMS surfaces, respectively. The control PDMS sliding angle (>90°) was significantly reduced to 9° after antifouling preparation. After 24 h incubation of polymer samples in a 200 mg/mL bovine serum albumin (BSA) solution, the surface adsorbed BSA was quantified using a colorimetric assay. The adsorbed protein on the fouling PDMS controls (29.1 ± 7.0 µg/cm2) was reduced by ~79% on the antifouling PDMS surface (6.2 ± 0.9 µg/cm2). Students will gain experience in materials science, biomedical engineering, chemistry, and biology concepts and better understand the influence of material properties on biological responses for biomaterial interfaces.

Reduction in Foreign Body Response and Improved Antimicrobial Efficacy via Silicone-Oil-Infused Nitric-Oxide-Releasing Medical-Grade Cannulas.

Foreign body response and infection are two universal complications that occur with indwelling medical devices. In response, researchers have developed different antimicrobial and antifouling surface strategies to minimize bacterial colonization and fibrous encapsulation. In this study, the nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP) and silicone oil were impregnated into silicone rubber cannulas (SR-SNAP-Si) using a solvent swelling method to improve the antimicrobial properties and decrease the foreign body response. The fabricated SR-SNAP-Si cannulas demonstrated a stable, prolonged NO release, exhibited minimal SNAP leaching, and maintained sliding angles < 15° for 21 days. SR-SNAP-Si cannulas displayed enhanced antimicrobial efficacy against Staphylococcus aureus in a 7-day biofilm bioreactor study, reducing the viability of adhered bacteria by 99.2 ± 0.2% compared to unmodified cannulas while remaining noncytotoxic toward human fibroblast cells. Finally, SR-SNAP-Si cannulas were evaluated for the first time in a 14- and 21-day subcutaneous mouse model, showing significantly enhanced biocompatibility compared to control cannulas by reducing the thickness of fibrous encapsulation by 60.9 ± 6.1 and a 60.8 ± 10.5% reduction in cell density around the implant site after 3 weeks. Thus, this work demonstrates that antifouling, NO-releasing surfaces can improve the lifetime and safety of indwelling medical devices.

Therapeutic Delivery of Nitric Oxide Utilizing Saccharide-Based Materials.

As a gasotransmitter, nitric oxide (NO) regulates physiological pathways and demonstrates therapeutic effects such as vascular relaxation, anti-inflammation, antiplatelet, antithrombosis, antibacterial, and antiviral properties. However, gaseous NO has high reactivity and a short half-life, so NO delivery and storage are critical questions to be solved. One way is to develop stable NO donors and the other way is to enhance the delivery and storage of NO donors from biomaterials. Most of the researchers studying NO delivery and applications are using synthetic polymeric materials, and they have demonstrated significant therapeutic effects of these NO-releasing polymeric materials on cardiovascular diseases, respiratory disease, bacterial infections, etc. However, some researchers are exploring saccharide-based materials to fulfill the same tasks as their synthetic counterparts while avoiding the concerns of biocompatibility, biodegradability, and sustainability. Saccharide-based materials are abundant in nature and are biocompatible and biodegradable, with wide applications in bioengineering, drug delivery, and therapeutic disease treatments. Saccharide-based materials have been implemented with various NO donors (like S-nitrosothiols and N-diazeniumdiolates) via both chemical and physical methods to deliver NO. These NO-releasing saccharide-based materials have exhibited controlled and sustained NO release and demonstrated biomedical applications in various diseases (respiratory, Crohn's, cardiovascular, etc.), skin or wound applications, antimicrobial treatment, bone regeneration, anticoagulation, as well as agricultural and food packaging. This review aims to highlight the studies in methods and progress in developing saccharide-based NO-releasing materials and investigating their potential applications in biomedical, bioengineering, and disease treatment.

S-Nitroso-N-acetyl-l-cysteine Ethyl Ester (SNACET) Catheter Lock Solution to Reduce Catheter-Associated Infections.

Antimicrobial-lock therapy is an economically viable strategy to prevent/reduce the catheter-related bloodstream infections (CRBSI) that are associated with central venous catheters (CVCs). Herein, we report the synthesis and characterization of the S-nitroso-N-acetyl-l-cysteine ethyl ester (SNACET), a nitric oxide (NO)-releasing molecule, and for the first time its application as a catheter lock solution to combat issues of bacterial infection associated with indwelling catheters. Nitric oxide is an endogenous gasotransmitter that exhibits a wide range of biological properties, including broad-spectrum antimicrobial activity. The storage stability of the SNACET and the NO release behavior of the prepared lock solution were analyzed. SNACET lock solutions with varying concentrations exhibited tuneable NO release at physiological levels for >18 h, as measured using chemiluminescence. The SNACET lock solutions were examined for their efficacy in reducing microbial adhesion after 18 h of exposure toStaphylococcus aureus (Gram-positive bacteria) andEscherichia coli (Gram-negative bacteria). SNACET lock solutions with 50 and 75 mM concentrations were found to reduce >99% (ca. 3-log) of the adhered S. aureus and E. coli adhesion to the catheter surface after 18 h. The SNACET lock solutions were evaluated in a more challenging in vitro model to evaluate the efficacy against an established microbial infection on catheter surfaces using the same bacteria strains. A >90% reduction in viable bacteria on the catheter surfaces was observed after instilling the 75 mM SNACET lock solution within the lumen of the infected catheter for only 2 h. These findings propound that SNACET lock solution is a promising biocidal agent and demonstrate the initiation of a new platform technology for NO-releasing lock solution therapy for the inhibition and treatment of catheter-related infections.

Tailoring nitric oxide release with additive manufacturing to create antimicrobial surfaces.

The current use of implantable and indwelling medical is limited due to potential microbial colonization leading to severe ailments. The aim of this work is to develop bioactive polymers that can be customized based on patient needs and help prevent bacterial infection. Potential benefits of additive manufacturing technology are integrated with the antimicrobial properties of nitric oxide (NO) to develop NO-releasing biocompatible polymer interfaces for addressing bacterial infections. Using filament-based additive manufacturing and polycarbonateurethane-silicone (PCU-Sil) a range of films possessing unique porosities (Disk-60, Disk-40, solid, capped) were fabricated. The films were impregnated with S-nitroso-N-acetyl-penicillamine (SNAP) using a solvent-swelling process. The Disk-60 porous films had the greatest amount of SNAP (19.59 wt%) as measured by UV-vis spectroscopy. Scanning electron microscopy and energy-dispersive X-ray spectroscopy confirmed an even distribution of SNAP throughout the polymer. The films exhibited structure-based tunable NO-release at physiological levels ranging from 7-14 days for solid and porous films, as measured by chemiluminescence. The antibacterial efficacy of the films was studied against Staphylococcus aureus using 24 h in vitro bacterial adhesion assay. The results demonstrated a >99% reduction of viable bacteria on the surface of all the NO-releasing films compared to unmodified PCU-Sil controls. The combination of 3D-printing technology with NO-releasing properties represents a promising technique to develop customized medical devices (such as 3D-scaffolds, catheters, etc.) with distinct NO-release levels that can provide antimicrobial properties and enhanced biocompatibility.

Association of mitochondria with microtubules inhibits mitochondrial fission by precluding assembly of the fission protein Dnm1.

Mitochondria are organized as tubular networks in the cell and undergo fission and fusion. Although several of the molecular players involved in mediating mitochondrial dynamics have been identified, the precise cellular cues that initiate mitochondrial fission or fusion remain largely unknown. In fission yeast (Schizosaccharomyces pombe), mitochondria are organized along microtubule bundles. Here, we employed deletions of kinesin-like proteins to perturb microtubule dynamics and used high-resolution and time-lapse fluorescence microscopy, revealing that mitochondrial lengths mimic microtubule lengths. Furthermore, we determined that compared with WT cells, mutant cells with long microtubules exhibit fewer mitochondria, and mutant cells with short microtubules have an increased number of mitochondria because of reduced mitochondrial fission in the former and elevated fission in the latter. Correspondingly, upon onset of closed mitosis in fission yeast, wherein interphase microtubules assemble to form the spindle within the nucleus, we observed increased mitochondrial fission. We found that the consequent rise in the mitochondrial copy number is necessary to reduce partitioning errors during independent segregation of mitochondria between daughter cells. We also discovered that the association of mitochondria with microtubules physically impedes the assembly of the fission protein Dnm1 around mitochondria, resulting in inhibition of mitochondrial fission. Taken together, we demonstrate a mechanism for the regulation of mitochondrial fission that is dictated by the interaction between mitochondria and the microtubule cytoskeleton.

Development of S-Nitroso-N-Acetylpenicillamine Impregnated Medical Grade Polyvinyl Chloride for Antimicrobial Medical Device Interfaces.

In the clinical setting, polyvinyl chloride (PVC) accounts for 25% of all polymers used in medical device applications. However, medical devices fabricated with plasticized PVC, such as endotracheal tubes, extracorporeal circuits (ECCs), or intravenous catheters, can lead to thrombosis and infection complications. Mortality associated with hospital associated infections (HAIs) exceed 100,000 deaths each year. One method to overcome these challenges is to develop bioactive polymers with nitric oxide (NO) release. Nitric oxide exhibits many physiological roles including antibacterial, antithrombic, and anti-inflammatory activity. In this study, plasticized Tygon PVC tubing was impregnated with a NO donor molecule, S-nitroso-N-acetylpenicillamine (SNAP), via a simple solvent-swelling-impregnation method, where polymer samples were submerged in a SNAP impregnation solvent (methanol, acetone, plasticizer), rinsed, and dried. An additional topcoat of a biocompatible CarboSil 2080A (CB) was applied to reduce SNAP leaching. The SNAP-PVC-CB was characterized for NO release using chemiluminescence, leaching with UV-vis spectroscopy, surface characterization with scanning electron microscopy, tensile strength analysis, stability during storage and sterilization, and antimicrobial properties in vitro. The SNAP-PVC-CB exhibited an NO flux of 4.29 ± 0.80 × 10-10 mol cm-2 min-1 over the initial 24 h under physiological conditions and continued to release physiological levels of NO for up to 14 d (incubated in PBS buffer at 37 °C). The addition of the CB topcoat reduced the total SNAP leaching by 60% during incubation. Mechanical properties and surface topography remained similar to the original PVC after SNAP impregnation and application of the CB topcoat. After ethylene oxide sterilization and 1 month of storage, the SNAP-PVC-CB demonstrated excellent SNAP stability (ca. 90% SNAP remaining). In a 24 h antibacterial assay, SNAP-PVC reduced viable bacteria colonization (ca. 1 log reduction) of S. aureus and E. coli compared to PVC controls. This simple method for SNAP impregnation of medical grade plasticized PVC holds great potential for improving the biocompatibility of postfabricated, plasticized PVC medical devices.

Increasing the Lifetime of Insulin Cannula with Antifouling and Nitric Oxide Releasing Properties.

Many Type 1 diabetes patients utilize insulin pumps, which rely on a small subcutaneous insulin infusion cannula. However, insulin cannulas still suffer from infection and inflammation, which impacts the wear time of the insulin cannula, reduces the efficiency of insulin infusion, and requires frequent rotation of the insulin infusion site. Infection and inflammation of continuous insulin infusion pump therapy are growing issues and are estimated to cost billions of dollars globally each year. This study aims to develop a potent antibacterial and antifouling insulin cannula with a synergistic effect of bioinspired polymers, integrating antifouling slippery, liquid-infused porous surface technology with an active nitric oxide (NO) releasing polymer. The cannulas were developed by impregnating the NO donor molecule S-nitroso-N-acetylpenicillamine (SNAP) and silicone oil (Si) in commercial medical-grade silicone rubber (SR) tubing (SR-SNAP-Si) via a solvent-impregnation process. The efficiency of the SR-SNAP-Si to reduce protein adsorption and provide antibacterial properties against Staphylococcus aureus and Staphylococcus epidermidis were studied using in vitro bioassays. The SR-SNAP-Si cannula released NO for more than 14 d at physiological levels and were stable during storage for 30 d at room temperature. Scanning electron microscopy images revealed no observable changes to the material surface after the solvent impregnation process. The infusion of silicone oil significantly reduced the protein adsorption on the cannula by 66.40%, and the NO release reduced the viable bacterial cell adhesion of S. epidermidis and S. aureus after 24 h by 94.89% and 99.77%, respectively, as compared to SR controls. This insulin cannula provided continuous NO release and an antifouling interface for more than 14 d and exhibited significant reduction in protein and bacterial adhesion. This method of developing dual-function nitric oxide releasing and antifouling surface for subcutaneous insulin infusion cannulas holds great potential to reduce infection and inflammation associated with insulin pump delivery systems.

Rapid, label-free CD4 testing using a smartphone compatible device.

The most recent guidelines have called for a significant shift towards viral load testing for HIV/AIDS management in developing countries; however point-of-care (POC) CD4 testing still remains an important component of disease staging in multiple developing countries. Advancements in micro/nanotechnologies and consumer electronics have paved the way for mobile healthcare technologies and the development of POC smartphone-based diagnostic assays for disease detection and treatment monitoring. Here, we report a simple, rapid (30 minutes) smartphone-based microfluidic chip for automated CD4 testing using a small volume (30 µL) of whole blood. The smartphone-based device includes an inexpensive (<$5) cell phone accessory and a functionalized disposable microfluidic device. We evaluated the performance of the device using spiked PBS samples and HIV-infected and uninfected whole blood, and compared the microfluidic chip results with the manual analysis and flow cytometry results. Through t-tests, Bland-Altman analyses, and regression tests, we have shown a good agreement between the smartphone-based test and the manual and FACS analysis for CD4 count. The presented technology could have a significant impact on HIV management in developing countries through providing a reliable and inexpensive POC CD4 testing.

Rapid Real-Time Antimicrobial Susceptibility Testing with Electrical Sensing on Plastic Microchips with Printed Electrodes.

Rapid antimicrobial susceptibility testing is important for efficient and timely therapeutic decision making. Due to globally spread bacterial resistance, the efficacy of antibiotics is increasingly being impeded. Conventional antibiotic tests rely on bacterial culture, which is time-consuming and can lead to potentially inappropriate antibiotic prescription and up-front broad range of antibiotic use. There is an urgent need to develop point-of-care platform technologies to rapidly detect pathogens, identify the right antibiotics, and monitor mutations to help adjust therapy. Here, we report a biosensor for rapid (<90 min), real time, and label-free bacteria isolation from whole blood and antibiotic susceptibility testing. Target bacteria are captured on flexible plastic-based microchips with printed electrodes using antibodies (30 min), and its electrical response is monitored in the presence and absence of antibiotics over an hour of incubation time. We evaluated the microchip with Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) as clinical models with ampicillin, ciprofloxacin, erythromycin, daptomycin, gentamicin, and methicillin antibiotics. The results are compared with the current standard methods, i.e. bacteria viability and conventional antibiogram assays. The technology presented here has the potential to provide precise and rapid bacteria screening and guidance in clinical therapies by identifying the correct antibiotics for pathogens.