Nitric oxide-triggering activity of gold-, platinum- and cerium oxide-nanozymes from S-nitrosothiols and diazeniumdiolates.

Cardiovascular diseases pose a significant global health challenge, contributing to high mortality rates and impacting overall well-being and quality of life. Nitric oxide (NO) plays a pivotal role as a vasodilator, regulating blood pressure and enhancing blood flow-crucial elements in preventing cardiovascular diseases, making it a prime therapeutic target. Herein, metal-based nanozymes (NZs) designed to induce NO release from both endogenous and exogenous NO-donors are investigated. Successful synthesis of gold, platinum (Pt) and cerium oxide NZs is achieved, with all three NZs demonstrating the ability to catalyze the NO release from various NO sources, namely S-nitrosothiols and diazeniumdiolates. Pt-NZs exhibit the strongest performance among the three NZ types. Further exploration involved investigating encapsulation and coating techniques using poly(lactic-co-glycolic acid) nanoparticles as experimental carriers for Pt-NZs. Both strategies showed efficiency in serving as platforms for Pt-NZs, successfully showing the ability to trigger NO release.

Recent Developments in Pharmacological Effect, Mechanism and Application Prospect of Diazeniumdiolates.

Nitric oxide (NO) is a simple structured and unstable free radical molecule, which participates in the regulation of many pathophysiological processes. It functions both as a second messenger and as an endogenous neurotransmitter. Diazeniumdiolates (NONOates) are a series of compounds containing the functional parent nuclear structure of [N(O)NO]-, which are the most widely studied NO donors. NONOates are unstable and easy to release NO in physiological conditions. The biomedical applications and drug development of NO donor have attracted the scientists' attention in recent years. In this review, recent advances in NONOates research are highlighted in terms of chemical structures, molecular characteristics, pharmacological effects, and biomedical application prospects.

Nitric Oxide Releasing Titanium Surfaces for Antimicrobial Bone-Integrating Orthopedic Implants.

Titanium implants in orthopedic applications can fail due to infection and impaired integration into the host. Most research efforts that facilitate osseointegration of the implant have not considered infection, and vice versa. Moreover, most infection control measures involve the use of conventional antibiotics which contributes to the global epidemic of antimicrobial resistance. Nitric oxide (NO) is a promising alternative to antibiotics, and while researchers have investigated NO releasing coatings, there are few reports on the function/robustness or the mechanism of NO release. Our comprehensive mechanistic study has allowed us to design, characterize, and optimize NO releasing coatings to achieve maximum antimicrobial efficacy toward bacteria with minimum cytotoxicity to human primary osteoblasts in vitro. As the antibiotic era is coming to an end and the future of infection control continues to demand new alternatives, the coatings described herein represent a promising therapeutic strategy for use in orthopedic surgeries.

Biosynthesis and Structure-Activity Relationship Investigations of the Diazeniumdiolate Antifungal Agent Fragin.

Only a few natural products incorporating a diazeniumdiolate moiety have been isolated, and these compounds usually display a broad range of biological activities. Only recently has the first diazeniumdiolate natural product biosynthetic gene cluster been identified in Burkholderia cenocepacia H111, which produces the fungicide (-)-fragin and the signal molecule rac-valdiazen. In this study, l-valine was identified as the initial substrate of (-)-fragin biosynthesis with the aid of feeding experiments using isotopically labelled amino acid. The formation of the diazeniumdiolate was chemically studied with several proposed intermediates. Our results indicate that the functional group is formed during an early stage of the biosynthesis. Furthermore, an oxime compound was identified as a degradation product of (-)-fragin and was also observed in the crude extract of the wild-type strain. Moreover, a structure-activity relationship analysis revealed that each moiety of (-)-fragin is essential for its biological activity.

Comparison of Diazeniumdiolated Dialkylhexanediamines as Nitric Oxide Release Agents on Nonthrombogenicity in an Extracorporeal Circulation Model.

When blood from a patient is circulated through extracorporeal circuits (ECCs), such as in cardiopulmonary bypass or extracorporeal life support, platelets in the blood are activated and form a thrombus. This is prevented clinically with a range of different systemic anticoagulation agents (e.g., heparin); however, this increases a patient's risk of hemorrhage. Previous work with nitric oxide (NO) releasing materials using the combined diazeniumdiolated diamine, N-N-di-N'-butyl-1,6-hexanediamine (DBHD), and a polymer-linked thrombin inhibitor, argatroban (AG), showed significant nonthrombogenicity in ECCs using a 4 h rabbit model. Herein, we evaluated if diazeniumdiolated N-N-di-N'-propyl-1,6-hexanediamine (DPHDN2O2), which has a slightly lower degree of lipophilicity compared to DBHDN2O2, would provide similar nonthrombogenicity as the AG/DBHDN2O2-polymer-coated circuits. While DPHDN2O2 releases NO at a higher flux rate than DBHDN2O2 when coated (within CarboSil polymer) on the inner wall of polyvinyl chloride tubing, neither coated circuit significantly affected animal hemodynamics. Both diazeniumdiolated diamines, in combination with immobilized AG or alone, significantly reduced thrombus formation similarly in the 4 h rabbit model (vs uncoated control): AG/DBHDN2O2: 0.12 ± 0.03 cm2; DBHDN2O2: 2.57 ± 0.82 cm2; AG/DPHDN2O2: 0.68 ± 0.22 cm2; DPHDN2O2: 1.87 + 1.26 cm2; uncoated control: 6.95 ± 0.82 cm2. AG/DPHDN2O2 was no different than AG/DBHDN2O in preserving platelet count and function. In addition, AG did not leach into the systemic circulation as the total clotting times were insignificantly different from the baseline values (AG/DPHDN2O2: 12.7 + 0.5 s (n = 3); AG/DBHDN2O2: 12.3 + 0.7 s (n = 3); baseline: 13.9 + 0.3 s (n = 13)). Based on these results, both DPHDN2O2 and DPHDN2O2 are good candidates as NO donor molecules for creating nonthrombogenic polymer coatings for ECCs.

Biosynthesis of the N-N-Bond-Containing Compound l-Alanosine.

The formation of a N-N bond is a unique biochemical transformation, and nature employs diverse biosynthetic strategies to activate nitrogen for bond formation. Among molecules that contain a N-N bond, biosynthetic routes to diazeniumdiolates remain enigmatic. We here report the biosynthetic pathway for the diazeniumdiolate-containing amino acid l-alanosine. Our work reveals that the two nitrogen atoms in the diazeniumdiolate of l-alanosine arise from glutamic acid and aspartic acid, and we clarify the early steps of the biosynthetic pathway by using both in vitro and in vivo approaches. Our work demonstrates a peptidyl-carrier-protein-based mechanism for activation of the precursor l-diaminopropionate, and we also show that nitric oxide can participate in non-enzymatic diazeniumdiolate formation. Furthermore, we demonstrate that the gene alnA, which encodes a fusion protein with an N-terminal cupin domain and a C-terminal AraC-like DNA-binding domain, is required for alanosine biosynthesis.

Nitric Oxide (NO)-Releasing Macromolecules: Rational Design and Biomedical Applications.

Nitric oxide (NO) has been recognized as a ubiquitous gaseous transmitter and the therapeutic potential has nowadays received increasing interest. However, NO cannot be easily directly administered due to its high reactivity in air and high concentration-dependent physiological roles. As such, a plethora of NO donors have been developed that can reversibly store and release NO under specific conditions. To enhance the stability and modulate the NO release profiles, small molecule-based NO donors were covalently linked to polymeric scaffolds, rendering them with multifunctional integration, prolonged release durations, and optimized therapeutic outcomes. In this minireview, we highlight the recent achievements of NO-releasing macromolecules in terms of chemical design and biomedical applications. We hope that more efforts could be devoted to this emerging yet promising field.

Novel primary amine diazeniumdiolates-Chemical and biological characterization.

Hit, Lead & Candidate Discovery Diazeniumdiolates, also known as NONOates, are extensively used in biochemical, physiological, and pharmacological studies due to their ability to release nitric oxide (NO. ) and/or their congeneric nitroxyl (HNO). The purpose of this work was to synthesize a series of primary amine-based diazeniumdiolates as HNO/NO donors and to determine their efficacy as anticancer and antifungal agents in vivo. The seven compounds (3a-3g) were successfully synthesized and characterized, one of which had been previously reported in the literature (3g). Two compounds showed anti-proliferative effects against ovarian (ES2 and SKOV3) and AML monocyte-derived cancer cells (THP-1) when tested with standard MTT assays. Compounds 3a and 3g demonstrated reduced ovarian cancer cell proliferation when treated at doses from 0.033 to 1.0 mg/mL at the 24 hr time point. These compounds also exhibited moderate and selective antifungal activity against Fusarium oxysporum f.sp. lycopersici, one cause of opportunistic infections of immunocompromised patients, inhibiting the growth of the fungi at LD50 at 10 mg/mL. A third compound (3e) did not exhibit similar activities, possibly due to the alkyl chain. Our results suggest that the primary amine diazeniumdiolates may offer a versatile platform for the development of HNO/NO donors for biomedical applications.

O2-(2,4-dinitrophenyl)diazeniumdiolates derivatives: Design, synthesis, cytotoxic evaluation and reversing MDR in MCF-7/ADR cells.

A series of O2-(2,4-dinitrophenyl)diazeniumdiolates derivatives were designed, synthesized and antiproliferative activities evaluated as novel nitric oxide (NO)-releasing prodrugs that could be activated by glutathione S-transferases π (GSTπ). Most of these derivatives exhibited significant antiproliferative activities compared to the reported NO-donor prodrug JS-K, among which compounds 27 and 36 had superior potency with IC50 below 1 µM. NO released amounts detection of all derivatives indicated that the antiproliferative activities were positively correlated with the levels of intracellular NO release in HCT116 cells. The most potent compound 36 exhibited improved uncatalyzed stability of GSTπ. Additionally, 36 showed remarkably multidrug resistance reversal activity which reversed multidrug resistance of adriamycin (ADR) in MCF-7/ADR cells with IC50 from 84.94 µM to 1.13 µM.

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.

Nitric oxide for cancer therapy.

Resistance to current therapeutic interventions is a major challenge in the treatment of patients affected by cancer. While nitric oxide (NO) might have proneoplastic properties, it is now clear that at high doses, NO has a role in cancer therapeutics. Either as a single agent or in combination with other antineoplastic compounds, NO might be used to overcome tumor cell resistance to conventional treatments. The following discussion addresses the role of NO in cancer therapeutics and includes a report on the role of NO donors in the area of cancer therapeutics.

Decomposition of amino diazeniumdiolates (NONOates): molecular mechanisms.

Although diazeniumdiolates (X[N(O)NO](-)) are extensively used in biochemical, physiological, and pharmacological studies due to their ability to release NO and/or its congeneric nitroxyl, the mechanisms of these processes remain obscure. In this work, we used a combination of spectroscopic, kinetic, and computational techniques to arrive at a quantitatively consistent molecular mechanism for decomposition of amino diazeniumdiolates (amino NONOates: R2N[N(O)NO](-), where R=N(C2H5)2 (1), N(C3H4NH2)2 (2), or N(C2H4NH2)2 (3)). Decomposition of these NONOates is triggered by protonation of their [NN(O)NO](-) group with the apparent pKa and decomposition rate constants of 4.6 and 1 s(-1) for 1; 3.5 and 0.083 s(-1) for 2; and 3.8 and 0.0033 s(-1) for 3. Although protonation occurs mainly on the O atoms of the functional group, only the minor R2N(H)N(O)NO tautomer (population ~10(-7), for 1) undergoes the NN heterolytic bond cleavage (kd~10(7) s(-1) for 1) leading to amine and NO. Decompositions of protonated amino NONOates are strongly temperature-dependent; activation enthalpies are 20.4 and 19.4 kcal/mol for 1 and 2, respectively, which includes contributions from both the tautomerization and bond cleavage. The bond cleavage rates exhibit exceptional sensitivity to the nature of R substituents which strongly modulate activation entropy. At pH<2, decompositions of all three NONOates that have been investigated are subject to additional acid catalysis that occurs through di-protonation of the [NN(O)NO](-) group.

Optimized polymeric film-based nitric oxide delivery inhibits bacterial growth in a mouse burn wound model.

Nitric oxide (NO) has many biological roles (e.g. antimicrobial agent, promoter of angiogenesis, prevention of platelet activation) that make NO releasing materials desirable for a variety of biomedical applications. Localized NO release can be achieved from biomedical grade polymers doped with diazeniumdiolated dibutylhexanediamine (DBHD/N2O2) and poly(lactic-co-glycolic acid) (PLGA). In this study, the optimization of this chemistry to create film/patches that can be used to decrease microbial infection at wound sites is examined. Two polyurethanes with different water uptakes (Tecoflex SG-80A (6.2±0.7wt.%) and Tecophilic SP-60D-20 (22.5±1.1wt.%)) were doped with 25wt.% DBHD/N2O2 and 10wt.% of PLGA with various hydrolysis rates. Films prepared with the polymer that has the higher water uptake (SP-60D-20) were found to have higher NO release and for a longer duration than the polyurethane with the lower water uptake (SG-80A). The more hydrophilic polymer enhances the hydrolysis rate of the PLGA additive, thereby providing a more acidic environment that increases the rate of NO release from the NO donor. The optimal NO releasing and control SG-80A patches were then applied to scald burn wounds that were infected with Acinetobacter baumannii. The NO released from these patches applied to the wounds is shown to significantly reduce the A. baumannii infection after 24h (∼4 log reduction). The NO release patches are also able to reduce the level of transforming growth factor-ß in comparison to controls, which can enhance re-epithelialization, decrease scarring and reduce migration of bacteria. The combined DBHD/N2O2 and PLGA-doped polymer patches, which could be replaced periodically throughout the wound healing process, demonstrate the potential to reduce risk of bacterial infection and promote the overall wound healing process.

Nitric oxide integrated polyethylenimine-based tri-block copolymer for efficient antibacterial activity.

The work demonstrated a successful synthesis of nitric oxide (NO)-releasing material and its antibacterial effect on Gram-negative Escherichia coli (E. coli), Gram-positive Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA). The polymeric support composed of thermosensitive Pluronic F68 having good biocompatibility and branched polyethylenimine (BPEI) housed N-diazeniumdiolates (NONOates) which could store and release NO under appropriate physiological condition. The developed F68-BPEI-NONOates releases a sufficient amount of NO under physiological condition to elicit effective killing of E. coli, S. aureus and MRSA. The antibacterial ability of the released NO was compared to untreated control or unmodified F68 polymer by using confocal microscopy; F68-BPEI-NONOates demonstrated excellent antibacterial activity with in vitro low cytotoxicity. TEM investigation also revealed the destruction of bacteria membrane caused by NO. The effectiveness of F68-BPEI-NONOates against resistant strains such as MRSA provides a very simple but highly efficient strategy to combat drug-resistant bacterial infections.

Quantitative detection of nitroxyl upon trapping with glutathione and labeling with a specific fluorogenic reagent.

Donors of nitroxyl (HNO) have shown promise for treatment of stroke, heart failure, alcoholism and cancer. However, comparing the pharmacological capacities of various donors is difficult without first quantifying the amount of HNO released from each donor. Detection and quantitation of HNO has been complicated by the rapid self-consumption of HNO through irreversible dimerization, poor selectivity of trapping agents against other nitrogen oxides, and/or low sensitivity towards HNO. Here, an assay is described for the trapping of HNO by glutathione (GSH) followed by labeling of GSH with the fluorogenic agent, naphthalene-2,3-dicarboxaldehyde (NDA), and subsequent quantitation by fluorescence difference. The newly developed assay was used to validate the pH-dependence of HNO release from isopropylamine NONOate (IPA/NO), which is a dual donor of HNO and NO at physiological pH. Furthermore, varied assay conditions were utilized to suggest the ratios of the products of the reaction of GSH with HNO. At intracellular concentrations of GSH, the disulfide (GSSG) was the major product, but significant concentrations of glutathione sulfinamide (GS(O)NH2) were also detected. This suggests that GS(O)NH2, which is a selective biomarker of HNO, may be produced in concentrations that are amenable to in vivo analysis.

The attenuation of platelet and monocyte activation in a rabbit model of extracorporeal circulation by a nitric oxide releasing polymer.

Nitric oxide (NO) has been shown to reduce thrombogenicity by decreasing platelet and monocyte activation by the surface glycoprotein, P-selectin and the integrin, CD11b, respectively. In order to prevent platelet and monocyte activation with exposure to an extracorporeal circulation (ECC), a nitric oxide releasing (NORel) polymeric coating composed of plasticized polyvinyl chloride (PVC) blended with a lipophilic N-diazeniumdiolate was evaluated in a 4 h rabbit thrombogenicity model using flow cytometry. The NORel polymer significantly reduced ECC thrombus formation compared to polymer control after 4 h blood exposure (2.8 +/- 0.7 NORel vs 6.7 +/- 0.4 pixels/cm(2) control). Platelet count (3.4 +/- 0.3 NORel vs 2.3 +/- 0.3 x 10(8)/ml control) and function as measured by aggregometry (71 +/- 3 NORel vs 17 +/- 6% control) were preserved after 4 h exposure in NORel versus control ECC. Plasma fibrinogen levels significantly decreased in both NORel and control groups. Platelet P-selectin mean fluorescence intensity (MFI) as measured by flow cytometry was attenuated after 4 h on ECC to ex vivo collagen stimulation (27 +/- 1 NORel vs 40 +/- 2 MFI control). Monocyte CD11b expression was reduced after 4 h on ECC with NORel polymer (87 +/- 14 NORel vs 162 +/- 30 MFI control). These results suggest that the NORel polymer coatings attenuate the increase in both platelet P-selectin and monocytic CD11b integrin expression in blood exposure to ECCs. These NO-mediated platelet and monocytic changes were shown to improve thromboresistance of these NORel-polymer-coated ECCs for biomedical devices.