N-Acetyl Cysteine-Decorated Nitric Oxide-Releasing Interface for Biomedical Applications.

Biomedical devices are vulnerable to infections and biofilm formation, leading to extended hospital stays, high expenditure, and increased mortality. Infections are clinically treated via the administration of systemic antibiotics, leading to the development of antibiotic resistance. A multimechanistic strategy is needed to design an effective biomaterial with broad-spectrum antibacterial potential. Recent approaches have investigated the fabrication of innately antimicrobial biomedical device surfaces in the hope of making the antibiotic treatment obsolete. Herein, we report a novel fabrication strategy combining antibacterial nitric oxide (NO) with an antibiofilm agent N-acetyl cysteine (NAC) on a polyvinyl chloride surface using polycationic polyethylenimine (PEI) as a linker. The designed biomaterial could release NO for at least 7 days with minimal NO donor leaching under physiological conditions. The proposed surface technology significantly reduced the viability of Gram-negative Escherichia coli (>97%) and Gram-positive Staphylococcus aureus (>99%) bacteria in both adhered and planktonic forms in a 24 h antibacterial assay. The composites also exhibited a significant reduction in biomass and extra polymeric substance accumulation in a dynamic environment over 72 h. Overall, these results indicate that the proposed combination of the NO donor with mucolytic NAC on a polymer surface efficiently resists microbial adhesion and can be used to prevent device-associated biofilm formation.

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.

Preventing Staphylococci Surgical Site Infections with a Nitric Oxide-Releasing Poly(lactic acid-co-glycolic acid) Suture Material.

Of the 27 million surgeries performed in the United States each year, a reported 2.6% result in a surgical site infection (SSI), and Staphylococci species are commonly the culprit. Alternative therapies, such as nitric oxide (NO)-releasing biomaterials, are being developed to address this issue. NO is a potent antimicrobial agent with several modes of action, including oxidative and nitrosative damage, disruption of bacterial membranes, and dispersion of biofilms. For targeted antibacterial effects, NO is delivered by exogenous donor molecules, like S-nitroso-N-acetylpenicillamine (SNAP). Herein, the impregnation of SNAP into poly(lactic-co-glycolic acid) (PLGA) for SSI prevention is reported for the first time. The NO-releasing PLGA copolymer is fabricated and characterized by donor molecule loading, leaching, and the amount remaining after ethylene oxide sterilization. The swelling ratio, water uptake, static water contact angle, and tensile strength are also investigated. Furthermore, its cytocompatibility is tested against 3T3 mouse fibroblast cells, and its antimicrobial efficacy is assessed against multiple Staphylococci strains. Overall, the NO-releasing PLGA copolymer holds promise as a suture material for eradicating surgical site infections caused by Staphylococci strains. SNAP impregnation affords robust antibacterial properties while maintaining the cytocompatibility and mechanical integrity.

In vivo assessment of dual-function submicron textured nitric oxide releasing catheters in a 7-day rabbit model.

Catheter-induced thrombosis is a major contributor to infectious and mechanical complications of biomaterials that lead to device failure. Herein, a dualfunction submicron textured nitric oxide (NO)-releasing catheter was developed. The hemocompatibility and antithrombotic activity of vascular catheters were evaluated in both 20 h in vitro blood loop and 7 d in vivo rabbit model. Surface characterization assessments via atomic force microscopy show the durability of the submicron pattern after incorporation of NO donor S-nitroso-N-acetylpenicillamine (SNAP). The SNAP-doped catheters exhibited prolonged and controlled NO release mimicking the levels released by endothelium. Fabricated catheters showed cytocompatibility when evaluated against BJ human fibroblast cell lines. After 20h in vitro evaluation of catheters in a blood loop, textured-NO catheters exhibited a 13-times reduction in surface thrombus formation compared to the control catheters, which had 83% of the total area covered by clots. After the 7 d in vivo rabbit model, analysis on the catheter surface was examined via scanning electron microscopy, where significant reduction of platelet adhesion, fibrin mesh, and thrombi can be observed on the NO-releasing textured surfaces. Moreover, compared to relative controls, a 63% reduction in the degree of thrombus formation within the jugular vein was observed. Decreased levels of fibrotic tissue decomposition on the jugular vein and reduced platelet adhesion and thrombus formation on the texture of the NO-releasing catheter surface are indications of mitigated foreign body response. This study demonstrated a biocompatible and robust dual-functioning textured NO PU catheter in limiting fouling-induced complications for longer-term blood-contacting device applications. STATEMENT OF SIGNIFICANCE: Catheter-induced thrombosis is a major contributor to infectious and mechanical complications of biomaterials that lead to device failure. This study demonstrated a robust, biocompatible, dual-functioning textured nitric oxide (NO) polyurethane catheter in limiting fouling-induced complications for longer-term blood-contacting device applications. The fabricated catheters exhibited prolonged and controlled NO release that mimics endothelium levels. After the 7 d in vivo model, a significant reduction in platelet adhesion, fibrin mesh, and thrombi was observed on the NO-releasing textured catheters, along with decreased levels of fibrotic tissue decomposition on the jugular vein. Results illustrate that NO-textured catheter surface mitigates foreign body response.

Bioinspired superhydrophobic surfaces with silver and nitric oxide-releasing capabilities to prevent device-associated infections and thrombosis.

Bacteria-associated infections and thrombus formation are the two major complications plaguing the application of blood-contacting medical devices. Therefore, functionalized surfaces and drug delivery for passive and active antifouling strategies have been employed. Herein, we report the novel integration of bio-inspired superhydrophobicity with nitric oxide release to obtain a functional polymeric material with anti-thrombogenic and antimicrobial characteristics. The nitric oxide release acts as an antimicrobial agent and platelet inhibitor, while the superhydrophobic components prevent non-specific biofouling. Widely used medical-grade silicone rubber (SR) substrates that are known to be susceptible to biofilm and thrombus formation were dip-coated with fluorinated silicon dioxide (SiO2) and silver (Ag) nanoparticles (NPs) using an adhesive polymer as a binder. Thereafter, the resulting superhydrophobic (SH) SR substrates were impregnated with S-nitroso-N-acetylpenicillamine (SNAP, an NO donor) to obtain a superhydrophobic, Ag-bound, NO-releasing (SH-SiAgNO) surface. The SH-SiAgNO surfaces had the lowest amount of viable adhered E. coli (> 99.9 % reduction), S. aureus (> 99.8 % reduction), and platelets (> 96.1 % reduction) as compared to controls while demonstrating no cytotoxic effects on fibroblast cells. Thus, this innovative approach is the first to combine SNAP with an antifouling SH polymer surface that possesses the immense potential to minimize medical device-associated complications without using conventional systemic anticoagulation and antibiotic treatments.

S-Nitroso-N-acetylpenicillamine impregnated latex: A new class of barrier contraception for the prevention of intercourse-associated UTIs.

Urinary tract infections (UTIs) are some of the most common infections seen in humans, affecting over half of the female population. Though easily and quickly treatable, if gone untreated for too long, UTIs can lead to narrowing of the urethra as well as bladder and kidney infections. Due to the disease potential, it is crucial to mitigate the development of UTIs throughout healthcare. Unfortunately, sexual activity and the use of condoms have been identified as common risk factors for the development of sexually acquired UTIs. Therefore, this study outlines a potential alteration to existing condom technology to decrease the risk of developing sexually acquired UTIs using S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide (NO) donor. Herein, varying concentrations of SNAP are integrated into commercialized condoms through a facile solvent swelling method. Physical characterization studies showed that 72%-100% of the ultimate tensile strength was maintained with lower SNAP concentrations, validating the modified condom's mechanical integrity. Additionally, the evaluation of room-temperature storage stability via NO release analysis outlined a lack of special storage conditions needed compared to commercial products. Moreover, these samples exhibited >90% relative cell viability and >96% bacterial killing, proving biocompatibility and antimicrobial properties. SNAP-Latex maintains the desired condom durability while demonstrating excellent potential as an effective new contraceptive technology to mitigate the occurrence of sexually acquired UTIs.

Varying material thickness of silicone rubber for tunable nitric oxide release.

Silicone rubber (SR), a common medical-grade polymer used in medical devices, has previously been modified for nitric oxide (NO) releasing capabilities. However, the effects of material properties such as film thickness on NO release kinetics are not well explored. In this study, SR is used in the first analysis of how a polymer's thickness affects the storage and uptake of an NO donor and subsequent release properties. Observed NO release trends show that a polymer's thickness results in tunable NO release. These results indicate how crucial a polymer's thickness is to optimize the NO release in an efficient and effective method.

Catalytic effect of transition metal-doped medical grade polymer on S-nitrosothiol decomposition and its biological response.

Nitric oxide (NO)-release from polymer metal composites is achieved through the incorporation of NO donors such as S-nitrosothiols (RSNO). Several studies have shown that metal nanoparticles catalytically decompose RSNO to release NO. In polymer composites, the NO surface flux from the surface can be modulated by the application of metal nanoparticles with a varying degree of catalytic activity. In this study, we compare the NO-releasing polymer composite design strategy - demonstrating how different ways of incorporating RSNO and metal nanoparticles can affect NO flux, donor leaching, or biological activity of the films. The first approach included blending both the RSNO and metal nanoparticle in the matrix (non-layered), while the second approach involved dip-coating metal nanoparticle/polymer layer on the RSNO-containing polymer composite (layered). Secondly, we compare both designs with respect to metal nanoparticles, including iron (Fe), copper (Cu), nickel (Ni), zinc (Zn), and silver (Ag). Differential NO surface flux is observed for each metal nanoparticle, with the Cu-containing polymer composites showing the highest flux for layered composites, whereas Fe demonstrated the highest NO flux for non-layered composites in 24 h. Additionally, a comparative study on NO flux modulation via the choice of metal nanoparticles is shown. Furthermore, mouse fibroblast cell viability when exposed to leachates from the polymer metal composites was dependent on (1) the design of the polymer composite where the layered approach performed better than non-layered composites (2) diffusion of metal nanoparticles from the composites plays a key role. Antibacterial activity on methicillin-resistant Staphylococcus aureus was also dependent on individual metal nanoparticles and flux levels in a 24 h in vitro CDC bioreactor study. Therefore, the study establishes the need for a layered polymer metal composite strategy that synergizes NO flux without negatively affecting biocompatibility.

In Situ Photo-crosslinkable Hyaluronic Acid/Gelatin Hydrogel for Local Nitric Oxide Delivery.

An increasing number of studies have shown that the local release of nitric oxide (NO) from hydrogels stimulates tissue regeneration by modulating cell proliferation, angiogenesis, and inflammation. The potential biomedical uses of NO-releasing hydrogels can be expanded by enabling their application in a fluid state, followed by controlled gelation triggered by an external factor. In this study, we engineered a hydrogel composed of methacrylated hyaluronic acid (HAGMA) and thiolated gelatin (GELSH) with the capacity for in situ photo-cross-linking, coupled with localized NO release. To ensure a gradual and sustained NO release, we charged the hydrogels with poly(l-lactic-co-glycolic acid) (PLGA) nanoparticles functionalized with S-nitrosoglutathione (GSNO), safeguarding SNO group integrity during photo-cross-linking. The formation of thiol-ene bonds via the reaction between GELSH's thiol groups and HAGMA's vinyl groups substantially accelerated gelation (by a factor of 6) and increased the elastic modulus of hydrated hydrogels (by 1.9-2.4 times). HAGMA/GELSH hydrogels consistently released NO over a 14 day duration, with the release of NO depending on the hydrogels' equilibrium swelling degree, determined by the GELSH-to-HAGMA ratio. Biocompatibility assessments confirmed the suitability of these hydrogels for biological applications as they display low cytotoxicity and stimulated fibroblast adhesion and proliferation. In conclusion, in situ photo-cross-linkable HAGMA/GELSH hydrogels, loaded with PLGA-GSNO nanoparticles, present a promising avenue for achieving localized and sustained NO delivery in tissue regeneration applications.

Advances in Nitric Oxide-Releasing Hydrogels for Biomedical Applications.

Hydrogels provide a plethora of advantages to biomedical treatments due to their highly hydrophilic nature and tissue-like mechanical properties. Additionally, the numerous and widespread endogenous roles of nitric oxide have led to an eruption in research developing biomimetic solutions to the many challenges the biomedical world faces. Though many design factors and fabrication details must be considered, utilizing hydrogels as nitric oxide delivery vehicles provides promising materials in several applications. Such applications include cardiovascular therapy, vasodilation and angiogenesis, antimicrobial treatments, wound dressings, and stem cell research. Herein, a recent update on the progress of NO-releasing hydrogels is presented in depth. In addition, considerations for the design and fabrication of hydrogels and specific biomedical applications of nitric oxide-releasing hydrogels are discussed.

Chemical Modification of Tiopronin for Dual Management of Cystinuria and Associated Bacterial Infections.

Cystinuria is an inherited autosomal recessive disease of the kidneys of recurring nature that contributes to frequent urinary tract infections due to bacterial growth and biofilm formation surrounding the stone microenvironment. In the past, commonly used strategies for managing cystinuria involved the use of (a) cystine crystal growth inhibitors such as l-cystine dimethyl ester and lipoic acid, and (b) thiol-based small molecules such as N-(2-mercaptopropionyl) glycine, commonly known as tiopronin, that reduce the formation of cystine crystals by reacting with excess cystine and generating more soluble disulfide compounds. However, there is a dearth of simplistic chemical approaches that have focused on the dual treatment of cystinuria and the associated microbial infections. This work strategically exploited a single chemical approach to develop a nitric oxide (NO)-releasing therapeutic compound, S-nitroso-2-mercaptopropionyl glycine (tiopronin-NO), for the dual management of cystine stone formation and the related bacterial infections. The results successfully demonstrated that (a) the antibacterial activity of NO rendered tiopronin-NO effective against the stone microenvironment inhabitants, Escherichia coli and Pseudomonas aeruginosa, and (b) tiopronin-NO retained the ability to undergo disulfide exchange with cystine while being reported to be safe against canine kidney and mouse fibroblast cells. Thus, the synthesis of such a facile molecule aimed at the dual management of cystinuria and related infections is unprecedented in the literature.

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.

Synthesis and Characterization of Nitric Oxide-Releasing Ampicillin as a Potential Strategy for Combatting Bacterial Biofilm Formation.

Biofilm formation on biomaterial interfaces and the development of antibiotic-resistant bacteria have decreased the effectiveness of traditional antibiotic treatment of infections. In this project, ampicillin, a commonly used antibiotic, was conjugated with S-nitroso-N-acetylpenicillamine (SNAP), an S-nitrosothiol compound (RSNO) used for controlled nitric oxide (NO) release. This novel multifunctional molecule is the first of its kind to provide combined antibiotic and NO treatment of infectious pathogens. Characterization of the molecule included NMR, FTIR, and mass spectrometry. NO release behavior was also measured and compared to pure, unmodified SNAP. When evaluating the antimicrobial efficacy, the synthesized SNAPicillin molecule showed the lowest MIC value against Gram-negative Pseudomonas aeruginosa and Gram-positive methicillin-resistant Staphylococcus aureus compared to ampicillin and SNAP alone. SNAPicillin also displayed enhanced biofilm dispersal and killing of both bacterial strains when treating a 48 h biofilm preformed on a polymer surface. The antibacterial results combined with the biocompatibility of the molecule show great promise for infection prevention and treatment of polymeric interfaces to reduce medical device-related infections.

Nanoarchitectonics of nitric oxide releasing supramolecular structures for enhanced antibacterial efficacy under visible light irradiation.

Light-controlled therapies offer a promising strategy to prevent and suppress infections caused by numerous bacterial pathogens. Excitation of exogenously supplied photosensitizers (PS) at specific wavelengths elicits levels of reactive oxygen intermediates toxic to bacteria. Porphyrin-based supramolecular nanostructure frameworks (SNF) are effective PS with unique physicochemical properties that have led to their widespread use in photomedicine. Herein, we developed a nitric oxide (NO) releasing, biocompatible, and stable porphyrin-based SNF (SNF-NO), which was achieved through a confined noncovalent self-assembly process based on π-π stacking. Characterization of the SNFs via scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analysis showed the formation of three-dimensional, well-defined octahedral structures. These SNF-NO were shown to exhibit a red shift due to the noncovalent self-assembly of porphyrins, which also show extended light absorption to broadly cover the entire visible light spectrum to enhance photodynamic therapy (PDT). Under visible light irradiation (46 J cm-2), the SNF generates high yields of singlet oxygen (1O2) radicals, hydroxyl radicals (HO), superoxide radicals (O2), and peroxynitrite (ONOO-) radicals that have shown potential to enhance antimicrobial photodynamic therapy (APDT) against Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) and Gram-negative Escherichia coli (E. coli). The resulting SNFs also exhibit significant biofilm dispersion and a decrease in biomass production. The combination of robust photosensitizer SNFs with nitric oxide-releasing capabilities is dynamic in its ability to target pathogenic infections while remaining nontoxic to mammalian cells. The engineered SNFs have enormous potential for treating and managing microbial infections.

Derivatization of graphene oxide nanosheets with tunable nitric oxide release for antibacterial biomaterials.

Graphene oxide (GO) nanosheets are a promising class of carbon-based materials suitable for application in the construction of medical devices. These materials have inherent antimicrobial properties based on sheet size, but these effects must be carefully traded off to maintain biocompatibility. Chemical modification of functional groups to the lattice structure of GO nanosheets enables unique opportunities to introduce new surface properties to bolster biological effects. Herein, we have developed nitric oxide (NO)-releasing GO nanosheets via immobilization of S-nitrosothiol (RSNO) moieties to GO nanosheets (GO-[NH]x -SNO). These novel RSNO-based GO nanosheets were characterized for chemical functionality via Fourier transform infrared spectroscopy, x-ray photoelectron spectroscopy, and colorimetric assays for functional group quantification. Stoichiometric control of the available RSNO groups functionalized onto the nanosheets was studied using chemiluminescence-based NO detection methods, showing highly tunable NO release kinetics. Studies of electrical stimulation and subsequent electrochemical reduction of the nanosheets demonstrated further tunability of the NO release based on stimuli. Finally, nanosheets were evaluated for cytotoxicity and antibacterial effects, showing strong cytocompatibility with human fibroblasts in parallel to broad antibacterial and anti-biofilm effects against both Gram-positive and Gram-negative strains. In summary, derivatized GO-(NH)x -SNO nanosheets were shown to have tunable NO release properties, enabling application-specific tailoring for diverse biomedical applications such as antimicrobial coatings and composite fillers for stents, sensors, and other medical devices.

Superhydrophobic and Conductive Foams with Antifouling and Oil-Water Separation Properties.

Hybrid organic-inorganic materials are attracting enormous interest in materials science due to the combination of multiple advantageous properties of both organic and inorganic components. Taking advantage of a simple, scalable, solvent-free hard-sacrificial method, we report the successful fabrication of three-dimensional hybrid porous foams by integrating two types of fillers into a poly(dimethylsiloxane) (PDMS) framework. These fillers consist of hydrophobic electrically conductive graphene (GR) nanoplatelets and hydrophobic bactericidal copper (Cu) microparticles. The fillers were utilized to create the hierarchical rough structure with low-surface-energy properties on the PDMS foam surfaces, leading to remarkable superhydrophobicity/superoleophilicity with contact angles of 158 and 0° for water and oil, respectively. The three-dimensional interconnected porous foam structures facilitated high oil adsorption capacity and excellent reusability as well as highly efficient oil/organic solvent-water separation in turbulent, corrosive, and saline environments. Moreover, the introduction of the fillers led to a significant improvement in the electrical conductivity and biofouling resistance (vs whole blood, fibrinogen, platelet cells, and Escherichia coli) of the foams. We envision that the developed composite strategy will pave a facile, scalable, and effective way for fabricating novel multifunctional hybrid materials with ideal properties that may find potential use in a broad range of biomedical, energy, and environmental applications.

A dual-action nitric oxide-releasing slippery surface for extracorporeal organ support: Dynamic in vitro hemocompatibility evaluation.

Numerous biomaterials have been developed for application in blood-contacting medical devices to prevent thrombosis; however, few materials have been applied to full-scale devices and evaluated for hemocompatibility under clinical blood flow conditions. We applied a dual-action slippery liquid-infused (LI) nitric oxide (NO)-releasing material modification (LINO) to full-scale blood circulation tubing for extracorporeal lung support and evaluated the tubing ex vivo using swine whole blood circulated for 6 h at a clinically relevant flow. LINO tubing was compared to unmodified tubing (CTRL) and isolated LI and NO-releasing modifications (n = 9/group). The primary objective was to evaluate safety and blood compatibility of this approach, prior to progression to in vivo testing of efficacy in animal models. The secondary objective was to evaluate coagulation outcomes relevant to hemocompatibility. No untoward effects of the coating, such as elevated methemoglobin fraction, were observed. Additionally, LINO delayed platelet loss until 6 h versus the reduction in platelet count in CTRL at 3 h. At 6 h, LINO significantly reduced the concentration of platelets in an activated P-selectin expressing state versus CTRL (32 ± 1% decrease, p = .02). Blood clot deposition was significantly reduced on LINO blood pumps (p = .007) and numerically reduced on tubing versus CTRL. Following blood exposure, LINO tubing continued to produce a measurable NO-flux (0.20 ± 0.06 × 10-10  mol cm-2  min-1 ). LINO is a potential solution to reduce circuit-related bleeding and clotting during extracorporeal organ support, pending future extended testing in vivo using full-scale extracorporeal lung support devices.

Biomimetic gasotransmitter-releasing alginate beads for biocompatible antimicrobial therapy.

HYPOTHESIS: Alginate is widely used in biomedical applications due to its high biocompatibility as well as structural and mechanical similarities to human tissue. Further, simple ionic crosslinking of alginate allows for the formation of alginate beads capable of drug delivery. S-nitrosoglutathione is a water-soluble molecule that releases nitric oxide in physiological conditions, where it acts as a potent antimicrobial gas, among other functions. As macrophages and endothelial cells endogenously produce nitric oxide, incorporating nitric oxide donors into polymers and hydrogels introduces a biomimetic approach to mitigate clinical infections, including those caused by antibiotic-resistant microorganisms. The incorporation of S-nitrosoglutathione into macro-scale spherical alginate beads is reported for the first time and shows exciting potential for biomedical applications. EXPERIMENTS: Herein, nitric oxide-releasing crosslinked alginate beads were fabricated and characterized for surface and cross-sectional morphology, water uptake, size distribution, and storage stability. In addition, the NO release was quantified by chemiluminescence and its biological effects against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus were investigated. The biocompatibility of the alginate beads was tested against 3T3 mouse fibroblast cells. FINDINGS: Overall, nitric oxide-releasing alginate beads demonstrate biologically relevant activities without eliciting a cytotoxic response, revealing their potential use as an antimicrobial material with multiple mechanisms of bacterial killing.

Nitric Oxide-Releasing Nanofibrous Scaffolds Based on Silk Fibroin and Zein with Enhanced Biodegradability and Antibacterial Properties.

Clinical applications of scaffolds and implants have been associated with bacterial infection resulting in impaired tissue regeneration. Nanofibers provide a versatile structure for both antimicrobial molecule delivery and tissue engineering. In this study, the nitric oxide (NO) donor molecule S-nitrosoglutathione (GSNO) and the natural biodegradable polymer zein (ZN) were combined with silk fibroin (SF) to develop antibacterial and biodegradable nanofibrous scaffolds for tissue engineering applications. The compatibility and intermolecular interactions of SF and ZN were studied using differential scanning calorimetry and Fourier transform infrared spectroscopy. The incorporation of ZN increased the hydrophobicity of the fibers and resulted in a more controlled and prolonged NO release profile lasting for 48 h. Moreover, the degradation kinetics of the fibers was significantly improved after blending with ZN. The results of tensile testing indicated that the addition of ZN and GSNO had a positive effect on the strength and stretchability of SF fibers and did not adversely affect their mechanical properties. Finally, due to the antibacterial properties of both NO and ZN, the SF-ZN-GSNO fibers showed a synergistically high antibacterial efficacy with 91.6 ± 2.5% and 77.5 ± 3.1% reduction in viability of adhered Staphylococcus aureus and Escherichia coli after 24 h exposure, respectively. The developed NO-releasing fibers were not only antibacterial but also non-cytotoxic and successfully enhanced the proliferation and growth of fibroblast cells, which was quantitatively studied by a CCK-8 assay and visually observed through fluorescent staining. Overall, SF-ZN-GSNO fibers developed in this study were biodegradable and highly antibacterial and showed great cytocompatibility with fibroblasts, indicating their promising potential for a range of tissue engineering and medical device applications.

Dual Action Nitric Oxide and Fluoride Ion-Releasing Hydrogels for Combating Dental Caries.

Demineralization and breakdown of tooth enamel are characterized by a condition called dental caries or tooth decay, which is caused by two main factors: (1) highly acidic food intake without proper oral hygiene and (2) overactive oral bacteria generating acidic metabolic byproducts. Fluoride treatments have been shown to help rebuild the hydroxyapatite structures that make up 98% of enamel but do not tackle the bacterial overload that continues to threaten future demineralization. Herein, we have created a dual-function Pluronic F127-alginate hydrogel with nitric oxide (NO)- and fluoride-releasing capabilities for the two-pronged treatment of dental caries. Analysis of the hydrogels demonstrated porous, shear-thinning behaviors with tunable mechanical properties. Varying the weight percent of the NO donor S-nitrosoglutathione (GSNO) within the hydrogel enabled physiologically actionable NO release over 4 h, with the fabricated gels demonstrating storage stability over 21 days. This NO-releasing capability resulted in a 97.59% reduction of viable Streptococcus mutans in the planktonic state over 4 h and reduced the preformed biofilm mass by 48.8% after 24 h. Delivery of fluoride ions was confirmed by a fluoride-sensitive electrode, with release levels resulting in the significant prevention of demineralization of hydroxyapatite discs after treatment with an acidic demineralization solution. Exposure to human gingival fibroblasts and human osteoblasts showed cytocompatibility of the hydrogel, demonstrating the potential for the successful treatment of dental caries in patients.