N-diazeniumdiolate Nitric Oxide Donors Research Articles

Nitric Oxide-Releasing Hemodialysis Catheter Lock Solutions.

In an attempt to address the significant morbidity, mortality, and economic cost associated with tunneled dialysis catheter (TDC) dysfunction, we report the development of nitric oxide-releasing dialysis catheter lock solutions. Catheter lock solutions with a range of NO payloads and release kinetics were prepared using low-molecular-weight N-diazeniumdiolate nitric oxide donors. Nitric oxide released through the catheter surface as a dissolved gas was maintained at therapeutically relevant levels for at least 72 h, supporting clinical translatability (interdialytic period). Slow, sustained NO release from the catheter surface prevented bacterial adhesion in vitro by 88.9 and 99.7% for Pseudomonas aeruginosa and Staphylococcus epidermidis, respectively, outperforming a burst NO-release profile. Furthermore, bacteria adhered to the catheter surface in vitro prior to lock solution use was reduced by 98.7 and 99.2% for P. aeruginosa and S. epidermidis, respectively, when using a slow releasing NO donor, demonstrating both preventative and treatment potential. The adhesion of proteins to the catheter surface, a process often preceding biofilm formation and thrombosis, was also lessened by 60-65% by sustained NO release. In vitro cytotoxicity of catheter extract solutions to mammalian cells was minimal, supporting the non-toxic nature of the NO-releasing lock solutions. The use of the NO-releasing lock solution in an in vivo TDC porcine model demonstrated decreased infection and thrombosis, enhanced catheter functionality, and improved outcome (i.e., likelihood of survival) as a result of catheter use.

Nitric Oxide-Releasing Alginates.

Low and high molecular weight alginate biopolymers were chemically modified to store and release potentially therapeutic levels of nitric oxide (NO). Carbodiimide chemistry was first used to modify carboxylic acid functional groups with a series of small molecule alkyl amines. The resulting secondary amines were subsequently converted to N-diazeniumdiolate NO donors via reaction with NO gas under basic conditions. NO donor-modified alginates stored between 0.4-0.6 μmol NO·mg-1. In aqueous solution, the NO-release kinetics were diverse (0.3-13 h half-lives), dependent on the precursor amine structure. The liberated NO showed bactericidal activity against Pseudomonas aeruginosa and Staphylococcus aureus with pathogen eradication efficiency dependent on both molecular weight and NO-release kinetics. The combination of lower molecular weight (∼5 kDa) alginates with moderate NO-release durations (half-life of ∼4 h) resulted in enhanced killing of both planktonic and biofilm-based bacteria. Toxicity against human respiratory epithelial (A549) cells proved negligible at NO-releasing alginate concentrations required to achieve a 5-log reduction in viability in the biofilm eradication assay.

Nitric Oxide Releasing Polymeric Coatings for the Prevention of Biofilm Formation.

The ability of nitric oxide (NO)-releasing polymer coatings to prevent biofilm formation is described. NO-releasing coatings on (poly(ethylene terephthalate) (PET) and silicone elastomer (SE)) were fabricated using aminosilane precursors. Pristine PET and SE were oxygen plasma treated, followed by immobilisation of two aminosilane molecules: N-(3-(trimethoxysilyl)propyl)diethylenetriamine (DET3) and N-(3-trimethoxysilyl)propyl)aniline (PTMSPA). N-diazeniumdiolate nitric oxide donors were formed at the secondary amine sites on the aminosilane molecules producing NO-releasing polymeric coatings. The NO payload and release were controlled by the aminosilane precursor, as DET3 has two secondary amine sites and PTMSPA only one. The antibacterial efficacy of these coatings was tested using a clinical isolate of Pseudomonas aeruginosa (PA14). All NO-releasing coatings in this study were shown to significantly reduce P. aeruginosa adhesion over 24 h with the efficacy being a function of the aminosilane modification and the underlying substrate. These NO-releasing polymers demonstrate the potential and utility of this facile coating technique for preventing biofilms for indwelling medical devices.

Design Considerations for Silica-Particle-Doped Nitric-Oxide-Releasing Polyurethane Glucose Biosensor Membranes.

Nitric oxide (NO)-releasing polymers have proven useful for improving the biocompatibility of in vivo glucose biosensors. Unfortunately, leaching of the NO donor from the polymer matrix remains a critical design flaw of NO-releasing membranes. Herein, a toolbox of NO-releasing silica nanoparticles (SNPs) was utilized to systematically evaluate SNP leaching from a diverse selection of biomedical-grade polyurethane sensor membranes. Glucose sensor analytical performance and NO-release kinetics from the sensor membranes were also evaluated as a function of particle and polyurethane (PU) chemistries. Particles modified with N-diazeniumdiolate NO donors were prone to leaching from PU membranes due to the zwitterionic nature of the NO donor modification. Leaching was minimized (<5% of the entrapped silica over 1 month) in low water uptake PUs. However, SNP modification with neutral S-nitrosothiol (RSNO) NO donors lead to biphasic leaching behavior. Particles with low alkanethiol content (<3.0 wt % sulfur) leached excessively from a hydrogel PU formulation (HP-93A-100 PU), while particles with greater degrees of thiol modification did not leach from any of the PUs tested. A functional glucose sensor was developed using an optimized HP-93A-100 PU membrane doped with RSNO-modified SNPs as the outer, glucose diffusion-limiting layer. The realized sensor design responded linearly to physiological concentrations of glucose (minimum 1-21 mM) over 2 weeks incubation in PBS and released NO at >0.8 pmol cm-2 s-1 for up to 6 days with no detectable (<0.6%) particle leaching.

Functionalized Mesoporous Silica via an Aminosilane Surfactant Ion Exchange Reaction: Controlled Scaffold Design and Nitric Oxide Release.

Nitric oxide-releasing mesoporous silica nanoparticles (MSNs) were prepared using an aminosilane-template surfactant ion exchange reaction. Initially, bare silica particles were synthesized under basic conditions in the presence of cetyltrimethylammonium bromide (CTAB). These particles were functionalized with nitric oxide (NO) donor precursors (i.e., secondary amines) via the addition of aminosilane directly to the particle sol and a commensurate ion exchange reaction between the cationic aminosilanes and CTAB. N-Diazeniumdiolate NO donors were formed at the secondary amines to yield NO-releasing MSNs. Tuning of the ion exchange-based MSN modification approach allowed for the preparation of monodisperse particles ranging from 30 to 1100 nm. Regardless of size, the MSNs stored appreciable levels of NO (0.4–1.5 μmol mg–1) with tunable NO release durations (1–33 h) dependent on the aminosilane modification. Independent control of NO release properties and particle size was achieved, demonstrating the flexibility of this novel MSN synthesis over conventional co-condensation and surface grafting strategies.

Anti-biofilm action of nitric oxide-releasing alkyl-modified poly(amidoamine) dendrimers against Streptococcus mutans

The effect of nitric oxide (NO)-releasing dendrimer hydrophobicity on Streptococcus mutans killing and biofilm disruption was examined at pH 7.4 and 6.4, the latter relevant to dental caries. Generation 1 (G1) poly(amidoamine) (PAMAM) dendrimers were modified with alkyl epoxides to generate propyl-, butyl-, hexyl-, octyl-, and dodecyl-functionalized dendrimers. The resulting secondary amines were reacted with NO to form N-diazeniumdiolate NO donor-modified dendrimer scaffolds (total NO ∼1μmol/mg). The bactericidal action of the NO-releasing dendrimers against both planktonic and biofilm-based S. mutans proved greatest with increasing alkyl chain length and at lower pH. Improved bactericidal efficacy at pH 6.4 was attributed to increased scaffold surface charge that enhanced dendrimer–bacteria association and ensuing membrane damage. For shorter alkyl chain (i.e., propyl and butyl) dendrimer modifications, increased antibacterial action at pH 6.4 was due to faster NO-release kinetics from proton-labile N-diazeniumdiolate NO donors. Octyl- and dodecyl-modified PAMAM dendrimers proved most effective for eradicating S. mutans biofilms with NO release mitigating dendrimer scaffold cytotoxicity. Statement of significanceWe report the antibacterial and anti-biofilm efficacy of dual-action nitric oxide (NO)-releasing dendrimers against S. mutans, an etiological agent in dental caries. This work was undertaken to enhance the anti-biofilm action of these scaffolds by employing various alkyl chain modifications. Furthermore, we evaluated the ability of NO to eradicate cariogenic biofilms. We found that at the lower pH associated with dental caries (pH ∼6.4), NO has a more pronounced antibacterial effect for alkyl modifications less capable of biofilm penetration and membrane disruption. Of greatest significance, we introduce dendrimers as a new macromolecular antibacterial agent against the cariogenic bacteria S. mutans.

Kinetic-dependent Killing of Oral Pathogens with Nitric Oxide

Nitric oxide (NO)–releasing silica nanoparticles were synthesized via the co-condensation of tetramethyl orthosilicate with aminosilanes and subsequent conversion of secondary amines to N-diazeniumdiolate NO donors. A series of ~150 nm NO-releasing particles with different NO totals and release kinetics (i.e., half-lives) were achieved by altering both the identity and mol% composition of the aminosilane precursors. Independent of identical 2 h NO-release totals, enhanced antibacterial action was observed against the periodontopathogens Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis with extended NO-release kinetics at pH 7.4. Negligible bactericidal effect was observed against cariogenic Streptococcus mutans at pH 7.4, even when using NO-releasing silica particles with greater NO-release totals. However, antibacterial activity was observed against S. mutans at lower pH (6.4). This result was attributed to more rapid proton-initiated decomposition of the N-diazeniumdiolate NO donors and greater NO-release payloads. The data suggest a differential sensitivity to NO between cariogenic and periodontopathogenic bacteria with implications for the future development of NO-releasing oral care therapeutics.

Anti-Biofilm Efficacy of Dual-Action Nitric Oxide-Releasing Alkyl Chain Modified Poly(amidoamine) Dendrimers.

Herein, we describe the synthesis of nitric oxide (NO)-releasing alkyl chain modified poly(amidoamine) (PAMAM) dendrimers of various sizes (i.e., generations). Generation 1 (G1) through generation 4 (G4) dendrimers were modified with either short (i.e., butyl) or medium (i.e., hexyl) alkyl chains via a ring-opening reaction. The resulting secondary amines were subsequently modified with N-diazeniumdiolate NO donors to establish NO payloads of ∼1.0 μmol/mg. The bactericidal efficacy of these dendrimers was evaluated against Gram-negative and Gram-positive biofilms, including antibiotic-resistant strains. The anti-biofilm action of the dendrimer biocides was found to be dependent on dendrimer generation, bacterial Gram class, and alkyl chain length, with the most effective biofilm eradication occurring when antibacterial agents were capable of efficient biofilm infiltration. The addition of NO release markedly enhanced anti-biofilm activity of dendrimers incapable of effective biofilm penetration.

Nitric oxide-releasing quaternary ammonium-modified poly(amidoamine) dendrimers as dual action antibacterial agents.

Herein we describe the synthesis of nitric oxide (NO)-releasing quaternary ammonium (QA)-functionalized generation 1 (G1) and generation 4 (G4) poly(amidoamine) (PAMAM) dendrimers. Dendrimers were modified with QA moieties of different alkyl chain lengths (i.e., methyl, butyl, octyl, dodecyl) via a ring-opening reaction. The resultant secondary amines were then modified with N-diazeniumdiolate NO donors to yield NO-releasing QA-modified PAMAM dendrimers capable of spontaneous NO release (payloads of ~0.75 μmol/mg over 4 h). The bactericidal efficacy of individual (i.e., non-NO-releasing) and dual action (i.e., NO-releasing) QA-modified PAMAM dendrimers was evaluated against Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa bacteria. Bactericidal activity was found to be dependent on dendrimer generation, QA alkyl chain length, and bacterial Gram class for both systems. Shorter alkyl chains (i.e., methylQA, butylQA) demonstrated increased bactericidal activity against P. aeruginosa versus S. aureus for both generations, with NO release markedly enhancing overall killing.

Nitric oxide-releasing chitosan oligosaccharides as antibacterial agents

Secondary amine-functionalized chitosan oligosaccharides of different molecular weights (i.e., ∼2500, 5000, 10,000) were synthesized by grafting 2-methyl aziridine from the primary amines on chitosan oligosaccharides, followed by reaction with nitric oxide (NO) gas under basic conditions to yield N-diazeniumdiolate NO donors. The total NO storage, maximum NO flux, and half-life of the resulting NO-releasing chitosan oligosaccharides were controlled by the molar ratio of 2-methyl aziridine to primary amines (e.g., 1:1, 2:1) and the functional group surrounding the N-diazeniumdiolates (e.g., polyethylene glycol (PEG) chains), respectively. The secondary amine-modified chitosan oligosaccharides greatly increased the NO payload over existing biodegradable macromolecular NO donors. In addition, the water-solubility of the chitosan oligosaccharides enabled their penetration across the extracellular polysaccharides matrix of Pseudomonas aeruginosa biofilms and association with embedded bacteria. The effectiveness of these chitosan oligosaccharides at biofilm eradication was shown to depend on both the molecular weight and ionic characteristics. Low molecular weight and cationic chitosan oligosaccharides exhibited rapid association with bacteria throughout the entire biofilm, leading to enhanced biofilm killing. At concentrations resulting in 5-log killing of bacteria in Pseudomonas aeruginosa (P. aeruginosa) biofilms, the NO-releasing and control chitosan oligosaccharides elicited no significant cytotoxicity to mouse fibroblast L929 cells in vitro.

Dual Action Antimicrobials: Nitric Oxide Release from Quaternary Ammonium-Functionalized Silica Nanoparticles

The synthesis of quaternary ammonium (QA)-functionalized silica nanoparticles with and without nitric oxide (NO) release capabilities is described. Glycidyltrialkylammonium chlorides of varied alkyl chain lengths (i.e., methyl, butyl, octyl, and dodecyl) were tethered to the surface of amine-containing silica nanoparticles via a ring-opening reaction. Secondary amines throughout the particle were then functionalized with N-diazeniumdiolate NO donors to yield dual functional nanomaterials with surface QAs and total NO payloads of 0.3 μmol/mg. The bactericidal activities of singly (i.e., only NO-releasing or only QA-functionalized) and dual (i.e., NO-releasing and QA-functionalized) functional nanoparticles were tested against Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa . Particles with only NO release capabilities alone were found to be more effective against P. aeruginosa , while particles with only QA-functionalities exhibited greater toxicity toward S. aureus . The minimum bactericidal concentrations (MBC) of QA-functionalized particles decreased with increasing alkyl chain length against both microbes tested. Combining NO release and QA-functionalities on the same particle resulted in an increase in bactericidal efficacy against S. aureus ; however, no change in activity against P. aeruginosa was observed compared to NO-releasing particles alone.

Structurally Diverse Nitric Oxide-Releasing Poly(propylene imine) Dendrimers

Structurally diverse secondary amine-functionalized poly(propylene imine) (PPI) dendrimers capable of tunable nitric oxide (NO) release were synthesized in a straightforward, one-step manner using ring-opening or conjugate-addition reactions with propylene oxide (PO), styrene oxide (SO), acrylonitrile (ACN), poly(ethylene glycol) methyl ether acrylate (average Mn = 480) (PEG) or 1,2-epoxy-9-decene (ED). N-Diazeniumdiolate nitric oxide donors were formed on the resulting secondary amine-functionalized G2-G5 PPI dendrimers by reaction with NO gas in basic solution. The NO storage and release kinetics for the resulting dendritic scaffolds were diverse (0.9-3.8 μmol NO/mg totals and 0.3 to 4.9 h half lives), illustrating the importance of the exterior chemical modification (e.g., steric environments, hydrophobicity, etc.) on diazeniumdiolate stability/decomposition. Tunable NO release was demonstrated by combining two donor systems on the exterior of one macromolecular scaffold. Additionally, a mathematical model was developed that allows for the simulation of dual NO release kinetics using the NO release data from the two single NO donor systems. The approaches described herein extend the range and scope of NO-releasing macromolecular scaffolds by unlocking a series of materials for use as dopants in biomedical polymers or stand-alone therapeutics depending on the exterior modification.

Dendrimers as a Scaffold for Nitric Oxide Release

The preparation and characterization of nitric oxide (NO)-releasing dendrimer conjugates are reported. Generation 3 and 5 polypropylenimine dendrimers (DAB-Am-16 and DAB-Am-64) were modified at the exterior to impart different amine functionalities. The ability to store NO on a dendritic scaffold using N-diazeniumdiolate NO donors was examined via the reaction of primary amine, secondary amine, and amide functionalities with high pressures of NO (5 atm). The secondary amine dendrimer conjugates exhibited a high storage capacity for NO (up to 5.6 micromol NO/mg), greatly increasing the "payload" of released NO over existing macromolecular NO donors. The mechanism of diazeniumdiolate decomposition was proton initiated, generating NO spontaneously under physiological conditions (pH 7.4, 37 degrees C). The NO release durations (>16 h) observed for the secondary amine dendrimers were significantly longer compared to small molecule alkyl secondary amine diazeniumdiolates, thus illustrating a dendritic effect on NO release kinetics. The multivalent exterior of dendrimers allows for the future combination of NO donors and other functionalities on a single molecular scaffold, enabling diverse utility as NO storage/delivery systems.