Synthesis, characterization, and controlled nitric oxide release from S-nitrosothiol-derivatized fumed silica polymer filler particles

A new type of nitric oxide (NO)-releasing material is described that utilizes S-nitrosothiols anchored to tiny fumed silica (FS) particles as the NO donor system. The synthetic procedures suitable for tethering three different thiol species (cysteine, N-acetylcysteine, and N-acetylpenicillamine) to the surface of FS polymer filler particles are detailed. The thiol-derivatized particles are converted to their corresponding S-nitrosothiols by reaction with t-butylnitrite. The total NO loading on the resulting particles range from 21-138 nmol/mg for the three different thiol-derivatized materials [S-nitrosocysteine-(NO-Cys)-FS, S-nitroso-N-acetylcysteine (SNAC)-FS, and S-nitroso-N-acetylpenicillamine (SNAP)-FS], with SNAP-FS yielding the highest NO loading. NO can be generated from these particles when suspended in solution via the addition of copper(II) ions, ascorbate, or irradiation with visible light. The SNAC-FS and SNAP-FS particles can be blended in polyurethane and silicone rubber matrixes to create films that release NO at controlled rates. Polyurethane films containing SNAC-FS submerged in phosphate-buffered saline (pH 7.4) generate NO surface fluxes approximately 0.1-0.7x10(-10) mol cm-2 min-1 and SNAP-FS films generate NO fluxes of approximately 0-7.5x10(-10) mol cm-2 min-1 upon addition of increasing amounts of copper ions. Silicone rubber films containing SNAC-FS or SNAP-FS do not liberate NO upon exposure to copper ions or ascorbate in phosphate-buffered saline solution. However, such films are shown to release NO at rates proportional to increasing intensities of visible light impinging on the films. Such photoinitiated NO release from these composite materials offers the first NO-releasing hydrophobic polymers with an external on/off trigger to control NO generation.     

Nitric oxide-releasing S-nitrosothiol-modified xerogels

The synthesis, material characterization, and in vitro biocompatibility of S-nitrosothiol (RSNO)-modified xerogels are described. Thiol-functionalized xerogel films were formed by hydrolysis and co-condensation of 3-mercaptopropyltrimethoxysilane (MPTMS) and methyltrimethoxysilane (MTMOS) sol–gel precursors at varying concentrations. Subsequent thiol nitrosation via acidified nitrite produced RSNO-modified xerogels capable of generating nitric oxide (NO) for up to 2 weeks under physiological conditions. Xerogels also exhibited NO generation upon irradiation with broad-spectrum light or exposure to copper, with NO fluxes proportional to wattage and concentration, respectively. Xerogels were capable of storing up to ～1.31 μmol NO mg^?1, and displayed negligible fragmentation over a 2-week period. Platelet and bacterial adhesion to nitrosated films was reduced compared to non-nitrosated controls, confirming the antithrombotic and antibacterial properties of the NO-releasing materials. Fibroblast cell viability was maintained on the xerogel surfaces illustrating the promise of RSNO-modified xerogels as biomedical device coatings.     

Inhibition of implant-associated infections via nitric oxide release

The in vivo antibacterial activity of nitric oxide (NO)-releasing xerogel coatings was evaluated against an aggressive subcutaneous Staphylococcus aureus infection in a rat model. The NO-releasing implants were created by coating a medical-grade silicone elastomer with a sol-gel-derived (xerogel) film capable of storing NO. Four of the bare or xerogel-coated silicone materials were subcutaneously implanted into male rats. Ten rats were administered 10 microl of a 10(8) cfuml(-1)S. aureus colony directly into the subcutaneous pocket with the implant prior to wound closure. Infection was quantitatively and qualitatively evaluated after 8d of implantation with microbiological and histological methods, respectively. A 82% reduction in the number of infected implants was achieved with the NO-releasing coating. Histology revealed that the capsule formation around infected bare silicone rubber controls was immunoactive and that a biofilm may have formed. Capsule formation in response to NO-releasing implants had greater vascularity in comparison with uninoculated or untreated controls. These results suggest that NO-releasing coatings may dramatically reduce the incidence of biomaterial-associated infection.     

Nitric oxide-releasing sol-gels as antibacterial coatings for orthopedic implants

To assess the benefits of nitric oxide (NO)-releasing sol-gels as potential antibacterial coatings for orthopedic devices, medical-grade stainless steel is coated with a sol-gel film of 40% N-aminohexyl-N-aminopropyltrimethoxysilane and 60% isobutyltrimethoxysilane. Upon converting the diamine groups in these films to diazeniumdiolate NO donors, the NO release from the sol-gel-coated stainless steel is evaluated at both ambient and physiological temperature. Sol-gel films incubated at 25 degrees C have a lower NO flux over the first 24 h compared to those at 37 degrees C, but release more than five times longer. The bacterial adhesion resistance of NO-releasing coatings is evaluated in vitro by exposing bare steel, sol-gel, and NO-releasing sol-gel-coated steel to cell suspensions of Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis at 25 degrees C and 37 degrees C. Cell adhesion to bare and sol-gel-coated steel is similar, while NO-releasing surfaces have significantly less bacterial adhesion for all species and temperatures investigated.     

Nitric oxide-releasing polyurethane-PEG copolymer containing the YIGSR peptide promotes endothelialization with decreased platelet adhesion

Thrombosis and intimal hyperplasia are the principal causes of small-diameter vascular graft failure. To improve the long-term patency of polyurethane vascular grafts, we have incorporated both poly(ethylene glycol) and a diazeniumdiolate nitric oxide (NO) donor into the backbone of polyurethane to improve thromboresistance. Additionally, we have incorporated the laminin-derived cell adhesive peptide sequence YIGSR to encourage endothelial cell adhesion and migration, while NO release encourages endothelial cell proliferation. NO production by polyurethane films under physiological conditions demonstrated biphasic release, in which an initial burst of 70% of the incorporated NO was released within 2 days, followed by sustained release over 2 months. Endothelial cell proliferation in the presence of the NO-releasing material was increased as compared to control polyurethane, and platelet adhesion to polyethylene glycol-containing polyurethane was decreased significantly with the addition of the NO donor.     

Antibacterial properties of nitric oxide-releasing sol-gels

The antibacterial characteristics of nitric oxide (NO)-releasing sol-gel coatings are described. The NO release from these surfaces is steady over short periods (approximately 1 h) and measurable over several days. The ability of NO to prevent bacterial adhesion is evaluated by exposing controls and NO-releasing sol-gels to approximately 10(8) colony-forming units (cfu)/mL saline suspensions of Pseudomonas aeruginosa. Pseudomonas aeruginosa adhesion to sol-gel controls varies depending on the sol-gel formulation. Sol-gel surfaces capable of NO release decrease bacterial adhesion by 30% to 95% relative to controls. The contact angle measurements of control and NO-releasing surfaces are similar, supporting NO's action as an antibacterial agent against bacterial adhesion.     

Antibacterial nitric oxide-releasing xerogels: cell viability and parallel plate flow cell adhesion studies

The ability of nitric oxide (NO)-releasing xerogels to reduce adhesion of Pseudomonas aeruginosa under flowing conditions was evaluated using a parallel plate flow chamber. At a controlled bacterial suspension flow rate of 0.2mL/min, the NO-releasing xerogels reduced bacterial adhesion in a flux-dependent fashion, with an NO flux of approximately 21pmolcm(-2)s(-1) reducing P. aeruginosa adhesion by approximately 65% compared to controls. Fluorescent viability staining indicated that bacteria adhered to NO-releasing xerogels were killed within 7h. Quantitative cell-plating viability studies showed that the extent of bactericidal activity was dependent on the total amount of NO released, with 750nmolcm(-2) killing >90% more adhered bacteria than xerogels releasing 25nmolcm(-2). Thus, NO-releasing xerogels were shown to both inhibit P. aeruginosa adhesion and kill adhered bacteria cells, two important steps toward designing anti-infective biomaterial coatings.     

Superhydrophobic nitric oxide-releasing xerogels

Superhydrophobic nitric oxide (NO)-releasing xerogels were prepared by spray-coating a fluorinated silane/silica composite onto N-diazeniumdiolate NO donor-modified xerogels. The thickness of the superhydrophobic layer was used to extend NO release durations from 59 to 105 h. The resulting xerogels were stable, maintaining superhydrophobicity for up to 1 month (the longest duration tested) when immersed in solution, with no leaching of silica or undesirable fragmentation detected. The combination of superhydrophobicity and NO release reduced viable Pseudomonas aeruginosa adhesion by >2-logs. The killing effect of NO was demonstrated at longer bacterial contact times, with superhydrophobic NO-releasing xerogels resulting in 3.8-log reductions in adhered viable bacteria vs. controls. With no observed toxicity to L929 murine fibroblasts, NO-releasing superhydrophobic membranes may be valuable antibacterial coatings for implants as they both reduce adhesion and kill bacteria that do adhere.     

Reduced foreign body response at nitric oxide-releasing subcutaneous implants

The tissue response to nitric oxide (NO)-releasing subcutaneous implants is presented. Model implants were created by coating silicone elastomer with diazeniumdiolate-modified xerogel polymers capable of releasing NO. The host tissue response to such implants was evaluated at 1, 3, and 6 weeks and compared to that of uncoated silicone elastomer blanks and xerogel-coated controls incapable of releasing NO. Delivery of NO (approximately 1.35 micromol/cm2 of implant surface area) reduced foreign body collagen capsule ("scar tissue") thickness by >50% compared to uncoated silicone elastomer after 3 weeks. The chronic inflammatory response at the tissue/implant interface was also reduced by >30% at NO-releasing implants after 3 and 6 weeks. Additionally, CD-31 immunohistochemical staining revealed approximately 77% more blood vessels in proximity to NO-releasing implants after 1 week compared to controls. These findings suggest that conferring NO release to subcutaneous implants may promote effective device integration into healthy vascularized tissue, diminish foreign body capsule formation, and improve the performance of indwelling medical devices that require constant mass transport of analytes (e.g., implantable sensors).     

Preparation and characterization of hydrophobic polymeric films that are thromboresistant via nitric oxide release

The preparation of hydrophobic polymer films (polyurethane and poly(vinyl chloride)) containing nitric oxide (NO)-releasing diazeniumdiolate functions is reported as a basis for improving the thromboresistivity of such polymeric materials for biomedical applications. Several different approaches for preparing NO-releasing polymer films are presented, including: (1) dispersion of diazeniumdiolate molecules within the polymer matrix; (2) covalent attachment of the diazeniumdiolate to the polymer backbone; and (3) ion-pairing of a diazeniumdiolated heparin species to form an organic soluble complex that can be blended into the polymer. Each approach is characterized in terms of NO release rates and in vitro biocompatibility. Results presented indicate that the polymer films prepared by each approach release NO for variable periods of time (10-72 h), although they differ in the mechanism, location and amount of NO released. In vitro platelet adhesion studies demonstrate that the localized NO release may prove to be an effective strategy for improving blood compatibility of polymer materials for a wide range of medical devices.     

Carboxyl-ebselen-based layer-by-layer films as potential antithrombotic and antimicrobial coatings

A carboxyl-ebselen-based layer-by-layer (LbL) film was fabricated by alternatively assembling carboxyl-ebselen immobilized polyethylenimine (e-PEI) and alginate (Alg) onto substrates followed by salt annealing and cross-linking. The annealed films exhibiting significantly improved stability are capable of generating nitric oxide (NO) from endogeneous S-nitrosothiols (RSNOs) in the presence of a reducing agent. The NO generation behaviors of different organoselenium species in solution phase are compared and the annealing mechanism to create stable LbL films is studied in detail. An LbL film coated polyurethane catheter is capable of generating physiological levels of NO from RSNOs even after blood soaking for 24 h, indicating potential antithrombotic applications of the coating. Further, the LbL film is also demonstrated to be capable of reducing living bacterial surface attachment and killing a broad spectrum of bacteria, likely through generation of superoxide (O(2)(·-)) from oxygen. This type of film is expected to have potential application as an antithrombotic and antimicrobial coating for different biomedical device surfaces.     

Morphological analysis of the antimicrobial action of nitric oxide on Gram-negative pathogens using atomic force microscopy

Atomic force microscopy (AFM) was used to study the morphological changes of two Gram-negative pathogens, Pseudomonas aeruginosa and Escherichia coli, after exposure to nitric oxide (NO). The time-dependent effects of NO released from a xerogel coating and the concentration-dependent effects rendered by a small molecule that releases NO in a bolus were examined and compared. Bacteria exhibited irregular and degraded exteriors. With NO-releasing surfaces, an increase in surface debris and disorganized adhesion patterns were observed compared to controls. Analysis of cell surface topography revealed that increasing membrane roughness correlated with higher doses of NO. At a lower total dose, NO delivered via a bolus resulted in greater membrane roughness than NO released from a surface via a sustained flux. At sub-inhibitory levels, treatment with amoxicillin, an antibiotic known to compromise the integrity of the cell wall, led to morphologies resembling those resulting from NO treatment. Our observations indicate that cell envelope deterioration is a visible consequence of NO-exposure for both Gram-negative species studied.     

Characterization and ornidazole release in vitro of a novel composite film prepared with chitosan/poly(vinyl alcohol)/alginate

To create a moist environment for rapid wound healing, a new C-P-A film with sustained antibacterial capacity had been developed by the casting/solvent evaporation method. This new type of C-P-A film consists of a chitosan top layer and sodium alginate sublayer separated by an ornidazole-incorporated poly(vinyl alcohol) layer, exhibited perfect binding characteristics among the three layers. Physical characterization of the C-P-A film showed that the triple-layerd film had excellent light transmittance, control of water vapor transmission rate, and fluid drainage ability promotion, compared with the single-layer film. From the in vitro release studies, about 90% of OD was released from the composite films within 60 min, and no significant difference was observed in cumulative release percentage with increases in the drug content. The composite film at low concentration of OD (1.0 mg/cm2) showed effective antimicrobial activity in the cultures of Staphylococcus aureus and Escherichia coli in agar plates. The results obtained in this work indicated that the new type of C-P-A composite film incorporated with ornidazole has the potential for wound dressing application.     

In vivo evaluation of vaginal films for mucosal delivery of nitric oxide

Nitric oxide (NO)-releasing vaginal films were developed and evaluated as a potential advanced treatment option for female sexual arousal disorder (FSAD). The polymeric films containing s-nitrosoglutathione (GSNO), an endogenous NO donor, were prepared using the reduced-pressure drying method. The surface morphology, thermal/mechanical properties, stability, loading efficiency and physicodynamic properties were characterized and the pharmacological activities were evaluated through in vitro and in vivo studies. The GSNO films were homogeneous and transparent, and showed suitable mucoadhesiveness and mechanical properties. The release profiles of NO from the GSNO films followed the first-order kinetic pattern and NO activated the NO–cGMP signaling pathway in vaginal cells. The GSNO films significantly enhanced the duration of action of GSNO and vagina blood perfusion in the rat model without causing any cytotoxic effects. The NO-releasing vaginal films might be used as a promising treatment device against FSAD.     

Nitric Oxide-Releasing Macromolecular Systems in Anticoagulant Therapeutics

Nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H₂S) are considered endogenous gaseous signaling molecules that play crucial roles in regulating physiological and pathological pathways. Among these molecules, NO has demonstrated unique advantages and clear efficacy in the treatment of blood coagulation in cardiovascular diseases. As a result, the development of NO-releasing materials for anticoagulant therapeutics has garnered significant attention. However, unlike traditional anticoagulant treatments such as heparin and oral anticoagulants, NO, a gaseous molecule under ambient conditions, cannot be administered orally, through injection, or via the skin. Consequently, researchers have focused on developing NO-releasing macromolecular systems by combining various donors with polymeric materials for use in anticoagulant therapeutics. This minireview aims to highlight the methods and advancements in constructing such NO-releasing macromolecular systems and exploring their applications in anticoagulant therapeutics.     

Silica sol-gel for the controlled release of antibiotics. I. Synthesis, characterization, and in vitro release

Room temperature processed silica sol-gel (xerogel) was investigated as a novel controlled release carrier of antibiotics (vancomycin). Xerogel characteristics, in vitro release properties, and bactericidal efficacy of the released antibiotic were determined. The xerogel/vancomycin composite showed a long-term sustained release (up to 6 weeks). In addition, bactericidal efficacy of released vancomycin was retained. The kinetics of release and the amount released were dose dependent. The initial, first-order release was followed by a near-zero-order release. The time to transition from the first- to zero-order release increased with vancomycin load (from 2 to 3 weeks with load increase from 2.2 to 11.1 mg/g). Regardless of the load, about 70% of the original vancomycin content was released by the transitional point, and the cumulative release after 6 weeks of immersion was about 90%. This study, combined with other reports documenting biocompatibility and controlled resorbability of the xerogel/drug composite in vivo, suggests that silica xerogel is a promising controlled release material for the treatment of bone infections.     

In vitro platelet adhesion on polymeric surfaces with varying fluxes of continuous nitric oxide release

Nitric oxide (NO) is released by endothelial cells that line the inner walls of healthy blood vessels at fluxes ranging from 0.5 x 10(-10) to 4.0 x 10(-10) mol cm(-2) min(-1), and this continuous NO release contributes to the extraordinary thromboresistance of the intact endothelium. To improve the biocompatibility of blood-contacting devices, a biomimetic approach to release/generate NO at polymer/blood interfaces has been pursued recently (with NO donors or NO generating catalysts doped within polymeric coatings) and this concept has been shown to be effective in preventing platelet adhesion/activation via several in vivo animal studies. However, there are no reports to date describing any quantitative in vitro assay to evaluate the blood compatibilities of such NO release/generating polymers with controlled NO fluxes. Such a methodology is desired to provide a preliminary assessment of any new NO-releasing material, in terms of the effectiveness of given NO fluxes and NO donor amounts on platelet activity before the more complex and costly in vivo testing is carried out. In this article, we report the use of a lactate dehydrogenase assay to study in vitro platelet adhesion on such NO-releasing polymer surfaces with varying NO fluxes. Reduced platelet adhesion was found to correlate with increasing NO fluxes. The highest NO flux tested, 7.05 (+/-0.25) x 10(-10) mol cm(-2) min(-1), effectively reduced platelet adhesion to nearly 20% of its original level (from 14.0 (+/-2.1) x 10(5) cells cm(-2) to 2.96 (+/-0.18) x 10(5) cells cm(-2)) compared to the control polymer coating without NO release capability.     

H2S and HS− donor NaHS releases nitric oxide from nitrosothiols, metal nitrosyl complex, brain homogenate and murine L1210 leukaemia cells

Nitrosoglutathione [(GSNO), 500 nmol/l] relaxed the norepinephrine precontracted rat aortic rings. The relaxation effect was pronouncedly enhanced by H₂S- and HS⁻-donor NaHS (30 μmol/l) at 7.5 pH but not at 6.3 pH. To study molecular mechanism of this effect, we investigated whether NaHS can release NO from NO donors. Using an electron paramagnetic resonance spectroscopy method of spin trap and by measuring the NO oxidation product, which is nitrite, by the Griess reaction, we report that NaHS released NO from nitrosothiols, namely from GSNO, S-nitroso-N-acetyl-dl-penicillamine (SNAP), from metal nitrosyl complex nitroprusside (SNP) and from rat brain homogenate and murine L1210 leukaemia cells. From the observation that the releasing effect was more pronounced at 8.0 pH than 6.0 pH, we suppose that HS⁻, rather than H₂S, is responsible for the NO-releasing effect. Since in mammals, H₂S and HS⁻ are produced endogenously, we assume that their effect to release NO from nitrosothiols and from metal nitrosyl complexes are responsible for some of their biological activities and that this mechanism may be involved in S-nitrosothiol-signalling reactions.     

Fabrication and characterization of novel bacterial cellulose/alginate/gelatin biocomposite film

Hydrogels from bacterial, algal, and animal cells—bacterial cellulose (BC), alginate, and gelatin, respectively—were combined to fabricate a biocomposite film (BCAGG) via an eco-friendly casting technique. In addition, glycerol was added as a plasticizer to improve the elasticity and water absorption capacity of the film. In this study, BC pellicles were simply deconstructed into fibrils suspension and then reconstructed into films with a supplement of alginate, gelatin and glycerol. The physical appearance of fabricated films resembled native BC but possessed improved ductility, enhanced flexibility, higher water uptake ability and better biocompatibility. The film was found to resist tearing under suture pullout strength in a hydrated state. In vitro cytotoxicity tests showed that the film was cytocompatible. A cell study using a human keratinocyte culture demonstrated enhanced cell adhesion, spreading, and proliferation on the BCAGG film compared with BC/alginate film. The BCAGG film therefore has significant potential for use in biomedical applications, particularly in dermal treatment, skin tissue regeneration, and wound healing.     

Antimicrobial effects of an NO-releasing poly(ethylene vinylacetate) coating on soft-tissue implants in vitro and in a murine model

Infection of surgical meshes used in abdominal wall reconstructions often leads to removal of the implant and increases patient morbidity due to repetitive operations and hospital administrations. Treatment with antibiotics is ineffective due to the biofilm mode of growth of the infecting bacteria and bears the risk of inducing antibiotic resistance. Hence there is a need for alternative methods to prevent and treat mesh infection. Nitric oxide (NO)-releasing coatings have been demonstrated to possess bactericidal properties in vitro. It is the aim of this study to assess possible benefits of a low concentration NO-releasing carbon-based coating on monofilament polypropylene meshes with respect to infection control in vitro and in vivo. When applied on surgical meshes, NO-releasing coatings showed significant bactericidal effect on in vitro biofilms of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and CNS. However, using bioluminescent in vivo imaging, no beneficial effects of this NO-releasing coating on subcutaneously implanted surgical meshes in mice could be observed.