Development of an Intracellular Nitric Oxide-Donating Cell-Penetrating Polypeptide as an Immunogenic Cell Death Inducer.

Recently, nitric oxide (NO) has been shown to induce immunogenic cell death (ICD) in tumor cells through endoplasmic reticulum (ER) stress and mitochondrial outer membrane permeabilization (MOMP). However, NO is unstable, making direct delivery difficult. In this study, we developed a cell-penetrating polypeptide-based NO donor, poly(l-guanidine) (PLG). Given that the guanidine structure can be catalyzed by reactive oxygen species (ROS) to produce NO, helical PLG plays three roles: spontaneous cell penetration, intracellular ROS generation to produce NO, and induction of ICD. The results revealed that helical PLG generates NO inside the cell by self-inducible guanidine oxidation and that NO effectively elicits ICD by ER stress- and MOMP-dependent intertwined mechanisms.

Wearable nitric oxide-releasing antibacterial insert for preventing device-associated infections.

Medical device-associated infections are a pervasive global healthcare concern, often leading to severe complications. Bacterial biofilms that form on indwelling medical devices, such as catheters, are significant contributors to infections like bloodstream and urinary tract infections. This study addresses the challenge of biofilms on medical devices by introducing a portable antimicrobial catheter insert (PACI) designed to be efficient, biocompatible, and anti-infective. The PACI utilizes nitric oxide (NO), known for its potent antimicrobial properties, to deter bacterial adhesion and biofilm formation. To achieve this, a photoinitiated NO donor, S-nitroso-N-acetylpenicillamine (SNAP), is covalently linked to a polydimethylsiloxane (PDMS) polymer. This design allows for higher NO loading for long-term impact and prevents premature donor leaching, a common challenge with SNAP-blended polymers. The SNAP-PDMS material was applied to a side-glowing fiber optic and connected to a wearable light module emitting 450 nm light, creating a functional antimicrobial insert. Activation of the fiber optic, accomplished with a one-click mechanism, enables real-time NO release, maintaining controlled NO levels for a minimum of 24  hours. The therapeutic levels of NO released via photocatalysis from the PACI demonstrated remarkable efficacy, with >90 % reduction in bacterial viability against S. aureus, S. epidermidis, and P. mirabilis without any cytotoxic impact on mammalian cells. This study underscores the potential of the NO-releasing insert in clinical settings, providing a portable and adaptable solution for preventing catheter-associated infections.

Nitric oxide in the cardio-cerebrovascular system: Source, regulation and application.

Nitric oxide (NO) plays a crucial role as a messenger or effector in the body, yet it presents a dual impact on cardio-cerebrovascular health. Under normal physiological conditions, NO exhibits vasodilatory effects, regulates blood pressure, inhibits platelet aggregation, and offers neuroprotective actions. However, in pathological situations, excessive NO production contributes to or worsens inflammation within the body. Moreover, NO may combine with reactive oxygen species (ROS), generating harmful substances that intensify physical harm. This paper succinctly reviews pertinent literature to clarify the in vivo and in vitro origins of NO, its regulatory function in the cardio-cerebrovascular system, and the advantages and disadvantages associated with NO donor drugs, NO delivery systems, and vascular stent materials for treating cardio-cerebrovascular disease. The findings provide a theoretical foundation for the application of NO in cardio-cerebrovascular diseases.

Lignin-Based Visible Light-Triggered Nitric Oxide Nanogenerator for Antibacterial Applications.

Nitric oxide (NO) has received growing attention as an effective antibacterial agent with broad-spectrum activity and a low risk of resistance. However, it remains challenging to develop effective, controllable, and biocompatible NO-releasing materials. Here, we report a novel NO nanogenerator (AL-BNN6-PEG) self-assembled by lignin, a UV-absorbing and hydrophobic NO donor (N,N'-disec-butyl-N,N'-dinitroso-1,4-phenylenediamine, BNN6), and PEG-DSPE2000. It was discovered that upon visible light irradiation (450-460 nm), BNN6 can be decomposed by lignin within micellar nanoparticles via a photoinduced electron transfer mechanism in the aqueous medium. Lignin not only served as a sustainable carrier, enhancing the water dispersity of BNN6, but also acted as a biocompatible photosensitizer, triggering BNN6 decomposition with the concomitant release of NO. As a result, the micellar nanoparticles displayed superior antibacterial effects against Gram-negative and Gram-positive bacteria upon visible light illumination. Moreover, MTT assay revealed the negligible cytotoxic effect of the micellar nanoparticles to the mouse fibroblast cells (L929). This research provides more insight into the BNN6 decomposition mechanism and demonstrates a straightforward, effective, and biocompatible strategy for controlled NO-mediated antibacterial applications.

Development of Nitric Oxide-Donating Netarsudil Derivatives as a Synergistic Therapy for Glaucoma with Reduced Ocular Irritation.

Based on the synergistic therapeutic effect of nitric oxide (NO) and Rho-associated protein kinase (ROCK) inhibitors on glaucoma, a series of NO-donating Netarsudil derivatives were designed, synthesized, and their activities in vitro and in vivo were evaluated. Among them, (S)-10e released an appropriate amount of NO in aqueous humor in vitro and displayed potent ROCK inhibition. Topical administration of (S)-10e significantly lowered intraocular pressure in an acute ocular hypertension rabbit model and protected retinal ganglion cells in a magnetic microbead occlusion mouse model. A metabolism investigation revealed that (S)-10e released 7a, a metabolite after NO releasing, and 13, an active metabolite of (S)-Netarsudil, in rabbit eyes. Notably, introducing an NO donor moiety attenuated ROCK inhibition-induced ocular irritation in an sGC-independent manner, suggesting that the attenuated conjunctival hyperemia effect of (S)-10e is related to the NO-induced protein S-nitrosation of phosphodiesterase 3A (PDE3A). Overall, (S)-10e is a promising candidate for glaucoma treatment.

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.

Harnessing Nitric Oxide-Donating Benzofuroxans for Targeted Inhibition of Carbonic Anhydrase IX in Cancer.

We describe here the design and antitumor evaluation of benzofuroxan-based nitric oxide (NO)-donor hybrid derivatives targeting human carbonic anhydrases (hCAs) IX and XII. The most effective compounds, 27 and 28, demonstrated potent dual action, exhibiting low nanomolar inhibition constants against hCA IX and significant NO release. Notably, compound 27 showed significant antiproliferative effects against various cancer cell lines, particularly renal carcinoma A-498 cells. In these cells, it significantly reduced the expression of CA IX and iron-regulatory proteins, inducing apoptosis via mitochondrial caspase activity and ferroptosis pathways, as evidenced by increases in ROS, nitrite, and down-regulated expression of ferritin-encoding genes. In three-dimensional tumor models, compound 27 effectively reduced spheroid size and viability. In vivo toxicity studies in mice indicated that the compounds were well-tolerated, with no significant alterations in kidney function. These findings underscore the potential of benzofuroxan-based CA inhibitors for further preclinical evaluations as therapeutic agents targeting renal cell carcinoma.

Recent Advancements in Polymeric N-Nitrosamine-Based Nitric Oxide (NO) Donors and their Therapeutic Applications.

Nitric oxide (NO), a gasotransmitter, is known for its wide range of effects in vasodilation, cardiac relaxation, and angiogenesis. This diatomic free radical also plays a pivotal role in reducing the risk of platelet aggregation and thrombosis. Furthermore, NO demonstrates promising potential in cancer therapy as well as in antibacterial and antibiofilm activities at higher concentrations. To leverage their biomedical activities, numerous NO donors have been developed. Among these, N-nitrosamines are emerging as a notable class, capable of releasing NO under suitable photoirradiation and finding a broad range of therapeutic applications. This review discusses the design, synthesis, and biological applications of polymeric N-nitrosamines, highlighting their advantages over small molecular NO donors in terms of stability, NO payload, and target-specific delivery. Additionally, various small-molecule N-nitrosamines are explored to provide a comprehensive overview of this burgeoning field. We anticipate that this review will aid in developing next-generation polymeric N-nitrosamines with improved physicochemical properties.

Cytotoxic and pro-oxidant profile of a photosensitive ruthenium nitrosyl candidate for NO delivery in healthy human fibroblasts.

Ruthenium nitrosyl (Ru-NO) complexes are of interest as photoactive nitric oxide (NO) donor candidates for local therapeutic applications. NO plays a crucial regulatory role in skin homeostasis, concentration-dependently affecting processes like the proliferation, apoptosis, autophagy and redox balance. In this context, we investigated HE-10, a ruthenium-based photoinducible NO donor, for its pro-oxidant and cytotoxic effects under light and dark conditions in VH10 human foreskin fibroblast cells. We also tested its intracellular and extracellular NO-releasing function. Our study reveals a significant dose-dependent cytotoxic effect of HE-10, an increase in intracellular reactive oxygen and nitrogen species, and the occurrence of apoptosis in skin fibroblast cells. Furthermore, exposure to both increasing doses of HE-10 and white LED light led to substantial cellular events, including a significant induction of autophagy and G2/M phase cell cycle arrest. Paradoxically, these effects were not solely attributable to NO release based on DAF2-DA NO probe results, suggesting that intracellular photochemical reactions additional to NO photolysis contribute to HE-10's biological activity. This study shows that HE-10 exhibits both cytotoxic and potential therapeutic effects, depending on concentration and light exposure. These findings are crucial for developing targeted Ru-NO complex treatments for skin diseases and potentially certain types of skin cancer, where controlled NO release could be beneficial.

Effect of Nitrosyl Iron Complex with 3,4-Dichlorothiophenolyls on the Level of Cyclic Nucleotide In Vitro.

The effect of a promising NO donor, a binuclear nitrosyl iron complex (NIC) with 3,4-dichlorothiophenolyls [Fe2(SC6H3Cl2)2(NO)4], on the adenylate cyclase and soluble guanylate cyclase enzymatic systems was studied. In in vitro experiments, this complex increased the concentration of important secondary messengers, such as cAMP and cGMP. An increase of their level by 2.4 and 4.5 times, respectively, was detected at NIC concentration of 0.1 mM. The ligand of the complex, 3,4-dichlorothiophenol, produced a less pronounced effect on adenylate cyclase. It was shown that the effect of this complex on the activity of soluble guanylate cyclase was comparable to the effect of anionic nitrosyl complex with thiosulfate ligands that exhibits vasodilating and cardioprotective properties.

Evaluation of Nitric Oxide-Donating Properties of 11H-indeno[1,2-b]quinoxalin-11-one Oxime (IQ-1) by Electron Paramagnetic Resonance Spectroscopy.

IQ-1 (11H-indeno[1,2-b]quinoxalin-11-one oxime) is a specific c-Jun N-terminal kinase (JNK) inhibitor with anticancer and neuro- and cardioprotective properties. Because aryloxime derivatives undergo cytochrome P450-catalyzed oxidation to nitric oxide (NO) and ketones in liver microsomes, NO formation may be an additional mechanism of IQ-1 pharmacological action. In the present study, electron paramagnetic resonance (EPR) of the Fe2+ complex with diethyldithiocarbamate (DETC) as a spin trap and hemoglobin (Hb) was used to detect NO formation from IQ-1 in the liver and blood of rats, respectively, after IQ-1 intraperitoneal administration (50 mg/kg). Introducing the spin trap and IQ-1 led to signal characteristics of the complex (DETC)2-Fe2+-NO in rat liver. Similarly, the introduction of the spin trap components and IQ-1 resulted in an increase in the Hb-NO signal for both the R- and the T-conformers in blood samples. The density functional theory (DFT) calculations were in accordance with the experimental data and indicated that the NO formation of IQ-1 through the action of superoxide anion radical is thermodynamically favorable. We conclude that the administration of IQ-1 releases NO during its oxidoreductive bioconversion in vivo.

Multifunctional Molecular Hybrids Photoreleasing Nitric Oxide: Advantages, Pitfalls, and Opportunities.

The multifaceted role nitric oxide (NO) plays in human physiology and pathophysiology has opened new scenarios in biomedicine by exploiting this free radical as an unconventional therapeutic against important diseases. The difficulties in handling gaseous NO and the strict dependence of the biological effects on its doses and location have made the light-activated NO precursors, namely NO photodonors (NOPDs), very appealing by virtue of their precise spatiotemporal control of NO delivery. The covalent integration of NOPDs and additional functional components within the same molecular skeleton through suitable linkers can lead to an intriguing class of multifunctional photoactivatable molecular hybrids. In this Perspective, we provide an overview of the recent advances in these molecular constructs, emphasizing those merging NO photorelease with targeting, fluorescent reporting, and phototherapeutic functionalities. We will highlight the rational design behind synthesizing these molecular hybrids and critically describe the advantages, drawbacks, and opportunities they offer in biomedical research.

Nitric oxide releasing coatings for the prevention of viral and bacterial infections.

Healthcare associated infections (HCAI) represent a significant burden worldwide contributing to morbidity and mortality and result in substantial economic consequences equating to billions annually. Although the impacts of HCAI have been felt for many years, the coronavirus pandemic has had a profound effect, escalating rates of HCAI, even with extensive preventative measures such as vaccination, personal protective equipment, and deep cleaning regimes. Therefore, there is an urgent need for new solutions to mitigate this serious health emergency. In this paper, the fabrication of nitric oxide (NO) releasing dual action polymer coatings for use in healthcare applications is described. The coatings are doped with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) and release high payloads of NO in a sustained manner for in excess of 50 hours. These coatings are extensively characterized in multiple biologically relevant solutions and the antibacterial/antiviral efficacy is studied. For the first time, we assess antibacterial activity in a time course study (1, 2, 4 and 24 h) in both nutrient rich and nutrient poor conditions. Coatings exhibit excellent activity against Pseudomonas aeruginosa and methicillin resistant Staphylococcus aureus (MRSA), with up to complete reduction observed over 24 hours. Additionally, when tested against SARS-CoV-2, the coatings significantly reduced active virus in as little as 10 minutes. These promising results suggest that these coatings could be a valuable addition to existing preventative measures in the fight against HCAIs.

Synthesis and Characterization of a Sustained Nitric Oxide-Releasing Orthodontic Elastomeric Chain for Antimicrobial Action.

The acidic byproducts of bacteria in plaque around orthodontic brackets contribute to white spot lesion (WSL) formation. Nitric oxide (NO) has antibacterial properties, hindering biofilm formation and inhibiting the growth of oral microbes. Materials that mimic NO release could prevent oral bacteria-related pathologies. This study aims to integrate S-nitroso-acetylpenicillamine (SNAP), a promising NO donor, into orthodontic elastomeric ligatures, apply an additional polymer coating, and evaluate the NO-release kinetics and antimicrobial activity against Streptococus mutans. SNAP was added to clear elastomeric chains (8 loops, 23 mm long) at three concentrations (50, 75, 100 mg/mL, and a control). Chains were then coated, via electrospinning, with additional polymer (Elastollan®) to aid in extending the NO release. NO flux was measured daily for 30 days. Samples with 75 mg/mL SNAP + Elastollan® were tested against S. mutans for inhibition of biofilm formation on and around the chain. SNAP was successfully integrated into ligatures at each concentration. Only the 75 mg/mL SNAP chains maintained their elasticity. After polymer coating, samples exhibited a significant burst of NO on the first day, exceeding the machine's reading capacity, which gradually decreased over 29 days. Ligatures also inhibited S. mutans growth and biofilm formation. Future research will assess their mechanical properties and cytotoxicity. This study presents a novel strategy to address white spot lesion (WSL) formation and bacterial-related pathologies by utilizing nitric oxide-releasing materials. Manufactured chains with antimicrobial properties provide a promising solution for orthodontic challenges, showing significant potential for academic-industrial collaboration and commercial viability.

Evaluation of Anticancer Activity of Nucleoside-Nitric Oxide Photo-Donor Hybrids.

Herein, we report the synthesis of a new hybrid compound based on a 2'-deoxyuridine nucleoside conjugated with a NO photo-donor moiety (dU-t-NO) via CuAAC click chemistry. Hybrid dU-t-NO, as well as two previously reported 2'-deoxyadenosine based hybrids (dAdo-S-NO and dAdo-t-NO), were evaluated for their cytotoxic and cytostatic activities in selected cancer cell lines. dAdo-S-NO and dAdo-t-NO hybrids displayed higher activity with respect to dU-t-NO. All hybrids showed effective release of NO in the micromolar range. The photochemical behavior of the newly reported hybrid, dU-t-NO, was studied in the RKO colon carcinoma cell line, whereas the dAdo-t-NO hybrid was tested in both colon carcinoma RKO and hepatocarcinoma Hep 3B2.1-7 cell lines to evaluate the potential effect of NO released upon irradiation on cell viability. A customized irradiation apparatus for in vitro experiments was also designed.

Discovery of NO Donor-Aurovertin Hybrids as Dual Ferroptosis and Apoptosis Inducers for Treating Triple Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a highly lethal malignancy, and its clinical management encounters severe challenges due to its high metastatic propensity and the absence of effective therapeutic targets. To improve druggability of aurovertin B (AVB), a natural polyketide with a significant antiproliferative effect on TNBC, a series of NO donor/AVB hybrids were synthesized and tested for bioactivities. Among them, compound 4d significantly inhibited the proliferation and metastasis of TNBC in vitro and in vivo with better safety than that of AVB. The structure-activity relationship analysis suggested that the types of NO donor and the linkers had considerable effects on the activities. Mechanistic investigations unveiled that 4d induced apoptosis and ferroptosis by the reduction of mitochondrial membrane potential and the down-regulation of GPX4, respectively. The antimetastatic effect of 4d was associated with the upregulation of DUSP1. Overall, these compelling results underscore the tremendous potential of 4d for treating TNBC.

Spraying with encapsulated nitric oxide donor reduces weight loss and oxidative damage in papaya fruit.

The combination of nitric oxide (NO) donors with nanomaterials has emerged as a promising approach to reduce postharvest losses. The encapsulation of NO donors provides protection from rapid degradation and controlled release, enhancing the NO effectiveness in postharvest treatments. Moreover, the application method can also influence postharvest responses. In this study, two application methods were evaluated, spraying and immersion, using S-nitrosoglutathione (GSNO, a NO donor) in free and encapsulated forms on papaya fruit. Our hypothesis was that GSNO encapsulated in chitosan nanoparticles would outperform the free form in delaying fruit senescence. In addition, this study marks the pioneering characterization of chitosan nanoparticles containing GSNO within the framework of a postharvest investigation. Overall, our findings indicate that applying encapsulated GSNO (GSNO-NP-S) through spraying preserves the quality of papaya fruit during storage. This method not only minimizes weight loss, ethylene production, and softening, but also stimulates antioxidant responses, thereby mitigating oxidative damage. Consequently, it stands out as the promising technique for delaying papaya fruit senescence. This innovative approach holds the potential to enhance postharvest practices and advance sustainable agriculture.

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

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

Development of a nanozyme-based electrochemical catalyst for real-time biomarker sensing of superoxide and nitric oxide anions released from living cells and exogenous donors.

Developing quantitative biosensors of superoxide (O2•-) and nitric oxide (NO) anion is crucial for pathological research. As of today, the main challenge for electrochemical detection is to develop high-selectivity nano-mimetic materials to replace natural enzymes. In this study, the dendritic-like morphological structure of silver organic framework (Ag-MOF) was successfully synthesized via a solvothermal strategy. Owing to the introduction of polymeric composites results in improved electrical conductivity and catalytic activity, which promotes mass transfer and leads to faster electron efficiency. For monitoring the electrochemical signals of O2•- and NO, the Ag-MOF electrode substrate was produced by drop-coating, and composites were designed by cyclic voltammetric potential cycles. The designed electrode substrates demonstrate high sensitivity, wide linear concentrations of 1 nM-1000 µM and 1 nM-850 µM, and low detection limits of 0.27 nM and 0.34 nM (S/N = 3) against O2•- and NO. Aside from that, the sensor successfully monitored the cellular release of O2•-, and NO from HepG2 and RAW 264.7 living cells and has the potential to monitor exogenous NO release from donors of Diethylamine (DEA)-NONOate and sodium nitroprusside (SNP). Additionally, the developed system was applied to the analysis of O2•- and NO in real biological fluid samples, and the results were good satisfactory (94.10-99.57 ± 1.23%). The designed system provides a novel approach to obtaining a good electrochemical biosensor platform that is highly selective, stable, and flexible. Finally, the proposed method provides a quantitative way to follow the dynamic changes in O2•- and NO in biological systems.

Design and Application of an In Situ Traceable Nitric Oxide Donor for Promoting the Healing of Wound Infections.

Therapies for wound infections require medications with antibacterial and wound-healing functions. However, it remains a challenge to produce a single drug that can perform dual functions. Nitric oxide (NO), with its antibacterial and wound-healing activities, is an ideal solution to address this challenge. However, many controlled-release strategies for NO rely on external probes for tracing the release in situ, making it difficult to precisely assess the location and magnitude. To address this issue, this study describes a novel NO donor, DHU-NO1, capable of efficiently releasing NO under mild conditions (450 nm illumination). Simultaneously, DHU-NO1 generates the fluorophore Azure B (AZB), which enables direct, non-consumptive tracing of the NO release by monitoring the fluorescence and absorption changes in AZB. Given that NO can be conveniently traced, the amount of released NO can be controlled during biological applications, thereby allowing both functions of NO to be performed. When applied to the affected area, DHU-NO1, illuminated by both a simple light-emitting diode (LED) light source and natural light, achieves significant antibacterial effects against wound infections and promotes wound healing in mice. This study offers a novel and effective approach for treating wound infections.