Roussin's Black Salt Research Articles

Motor-Cargo Structured Nanotractors for Augmented NIR Phototherapy via Gas-Boosted Tumor Penetration and Respiration-Impaired Mitochondrial Dysfunction.

Tumor microenvironment, characterized by dense extracellular matrix and severe hypoxia, has caused pronounced resistance to photodynamic therapy (PDT). Herein, it has designed an artificial nitric oxide (NO) nanotractor with a unique "motor-cargo" structure, where a photoswitching upconversion nanoparticle (UCNP) core serves as the optical engine to harvest NIR light and asymmetrically coated mesoporous silica (SiO2) shell acts as a cargo unit to load nitric oxide (NO) fuel molecule (RBS, Roussin's black salt) and PDT photosensitizer (ZnPc, zinc phthalocyanine). Upon illumination by 980nm light, the UCNP emits blue light to excite RBS salt and release NO gas. On one hand, NO is used as the driving force to propel the particle with a high speed of ≈194µms-1 that generates significant rupture stress (over 0.95kPa) on cell membrane to promote cellular endocytosis and intratumoral penetration. On the other hand, NO enables to alleviate tumor hypoxia by inhibiting cellular respiration as an oxygen conserver. When the excitation is subsequently switched to 808nm light, the UCNP emits red light, triggering ZnPc to produce large amount of reactive oxygen species for PDT treatment. This study explores Janus-typed nanostructures for cell-particle interaction and gas-assisted phototherapy, opening avenues for versatile bioapplications.

Preliminary investigation of nitric oxide release from upconverted nanoparticles excited at 808 nm near-infrared for brain tumors

Upconverted UCNPs@mSiO2-NH2 nanoparticles were synthesized via thermal decomposition while employing the energy resonance transfer principle and the excellent near-infrared (NIR) light conversion property of up-conversion. The 808 nm NIR-excited photocontrolled nitric oxide (NO) release platform was successfully developed by electrostatically loading photosensitive NO donor Roussin's black salt (RBS) onto UCNPs@mSiO2-NH2, enabling the temporal, spatial, and dosimetric regulation of NO release for biological applications of NO. The release of NO ranged from 0.015⁓0.099 mM under the conditions of 2.0 W NIR excitation power, 20 min of irradiation time, and UCNPs@mSiO2-NH2&RBS concentration of 0.25⁓1.25 mg/mL. Therefore, this NO release platform has an anti-tumor effect. In vitro experiments showed that under the NIR light, at concentrations of 0.3 mg/mL and 0.8 mg/mL of UCNPs@mSiO2-NH2&RBS, the activity of glioma (U87) and chordoma (U–CH1) cells, as measured by CCK8 assay, was reduced to 50 %. Cell flow cytometry and Western Blot experiments showed that NO released from UCNPs@mSiO2-NH2&RBS under NIR light induced apoptosis in brain tumor cells. In vivo experiments employing glioma and chordoma xenograft mouse models revealed significant inhibition of tumor growth in the NIR and UCNPs@mSiO2-NH2&RBS group, with no observed significant side effects in the mice. Therefore, NO released by UCNPs@mSiO2-NH2&RBS under NIR irradiation can be used as a highly effective and safe strategy for brain tumor therapy.

NIR-Triggered Release of Nitric Oxide by Upconversion-Based Nanoplatforms to Enhance Osteogenic Differentiation of Mesenchymal Stem Cells for Osteoporosis Therapy.

Stem cell therapy is an attractive approach to bone tissue regeneration in osteoporosis (OP); however, poor cell engraftment and survival within injured tissues limits its success in clinical settings. Nitric oxide (NO) is an important signaling molecule involved in various physiological processes, with emerging evidence supporting its diverse roles in modulating stem cell behavior, including survival, migration, and osteogenic differentiation. To control and enhance osteogenic differentiation of mesenchymal stem cells (MSCs) for OP therapy, we designed a near-infrared (NIR) light-triggered NO-releasing nanoplatform based on upconversion nanoparticles (UCNPs) that converts 808-nm NIR light into visible light, stimulating NO release by light control. We demonstrate that the UCNP nanoplatforms can encapsulate a light-sensitive NO precursor, Roussin's black salt (RBS), through the implementation of a surface mesoporous silica coating. Upon exposure to 808-nm irradiation, NO is triggered by the controlled upconversion of UCNP visible light at the desired time and location. This controlled release mechanism facilitates photoregulated differentiation of MSCs toward osteogenic lineage and avoids thermal effects and phototoxicity on cells, thus offering potential therapeutic applications for treating OP invivo. Following the induction of osteogenic differentiation, the UCNP nanoplatforms exhibit the capability to serve as nanoprobes for the real-time detection of differentiation through enzymatic digestion and fluorescence recovery of UCNPs, enabling assessment of the therapeutic efficacy of OP treatment. Consequently, these UCNP-based nanoplatforms present a novel approach to control and enhance osteogenic differentiation of MSCs for OP therapy, simultaneously detecting osteogenic differentiation for evaluating treatment effectiveness.

Roussin's black salt decorated Cu-doped ZnO nanoparticles for bacterial inhibition

RBS@Cu:ZnO NPs are promising antibacterial agents and exhibit high and controllable antibacterial performance due to the synergistic effect of Cu-doped ZnO and released nitric oxide under light irradiation.

Mass Spectrometric Identification of [4Fe-4S](NO)x Intermediates of Nitric Oxide Sensing by Regulatory Iron-Sulfur Cluster Proteins.

Nitric oxide (NO) can function as both a cytotoxin and a signalling molecule. In both cases, reaction with iron-sulfur (Fe-S) cluster proteins plays an important role because Fe-S clusters are reactive towards NO and so are a primary site of general NO-induced damage (toxicity). This sensitivity to nitrosylation is harnessed in the growing group of regulatory proteins that function in sensing of NO via an Fe-S cluster. Although information about the products of cluster nitrosylation is now emerging, detection and identification of intermediates remains a major challenge, due to their transient nature and the difficulty in distinguishing spectroscopically similar iron-NO species. Here we report studies of the NO-sensing Fe-S cluster regulators NsrR and WhiD using non-denaturing mass spectrometry, in which non-covalent interactions between the protein and Fe/S/NO species are preserved. The data provide remarkable insight into the nitrosylation reactions, permitting identification, for the first time, of protein-bound mono-, di- and tetranitrosyl [4Fe-4S] cluster complexes ([4Fe-4S](NO), [4Fe-4S])(NO)2 and [4Fe-4S](NO)4 ) as intermediates along pathways to formation of product Roussin's red ester (RRE) and Roussin's black salt (RBS)-like species. The data allow the nitrosylation mechanisms of NsrR and WhiD to be elucidated and clearly distinguished.

Dioxygen controls the nitrosylation reactions of a protein-bound [4Fe4S] cluster.

Iron-sulfur clusters are exceptionally tuneable protein cofactors, and as one of their many roles they are involved in biological responses to nitrosative stress. Both iron-sulfur proteins and synthetic model clusters are extremely sensitive to nitrosylation, tending towards rapid multi-step reaction and cluster degradation. Reaction of protein-bound iron-sulfur clusters with nitric oxide can be stopped at partial nitrosylation in vivo, and repair of protein-bound nitrosylated clusters is possible in the cellular environment. We have used a combination of infrared, EPR, and UV-visible spectroscopies to show that a model [4Fe4S] cluster-containing protein, A. ferroxidans high potential iron-sulfur protein (HiPIP), reacts with NO to give a product mixture dominated by Roussin's Black Salt (RBS) and Roussin's Red Ester (RRE) species. We have shown that O2 plays a critical role in controlling the major product of nitrosylation, with RBS-like products favoured under strictly anaerobic conditions and RRE favoured in the presence of trace O2. Moreover, addition of trace O2 to anaerobically nitrosylated samples induces conversion of RBS-like products to RRE. These findings may have implications for mechanisms of iron-sulfur cluster repair following nitrosative stress, suggest a crucial role for trace O2, and provide an important link between nitrosylation chemistry of iron-sulfur proteins and the well-established reactivity of synthetic iron-sulfur clusters.

X‐Ray‐Controlled Generation of Peroxynitrite Based on Nanosized LiLuF4:Ce3+ Scintillators and their Applications for Radiosensitization

Peroxynitrite (ONOO- ), the reaction product derived from nitric oxide (NO) and superoxide (O2 -• ), is a potent oxidizing and nitrating agent that modulates complex biological processes and promotes cell death. Therefore, it can be expected that the overproduction of ONOO- in tumors can be an efficient approach in cancer therapy. Herein, a multifunctional X-ray-controlled ONOO- generation platform based on scintillating nanoparticles (SCNPs) and UV-responsive NO donors Roussin's black salt is reported, and consequently the mechanism of their application in enhanced therapeutic efficacy of radiotherapy is illustrated. Attributed to the radioluminescence and high X-ray-absorbing property of SCNPs, the nanocomposite can produce NO and O2 -• simultaneously when excited by X-ray irradiation. Such simultaneous release of NO and O2 -• ensures the efficient X-ray-controlled generation of ONOO- in tumors. Meanwhile, the application of X-rays as the excitation source can achieve better penetration depth and induce radiotherapy in this nanotherapeutic platform. It is found that the X-ray-controlled ONOO- -generation platform can efficiently improve the radiotherapy efficiency via directly damaging DNA, downregulating the expression of the DNA-repair enzyme, and overcoming the hypoxia-associated resistance in radiotherapy. Therefore, this SCNP-based platform may provide a new combinatorial strategy of ONOO- and radiotherapy to improve cancer treatment.

Mass spectrometric detection of iron nitrosyls, sulfide oxidation and mycothiolation during nitrosylation of the NO sensor [4Fe-4S] NsrR.

The bacterial nitric oxide (NO)-sensing transcriptional regulator NsrR binds a [4Fe-4S] cluster that enables DNA-binding and thus repression of the cell's NO stress response. Upon exposure to NO, the cluster undergoes a complex nitrosylation reaction resulting in a mixture of iron-nitrosyl species, which spectroscopic studies have indicated are similar to well characterized low molecular weight dinitrosyl iron complex (DNIC), Roussin's Red Ester (RRE) and Roussin's Black Salt (RBS). Here we report mass spectrometric studies that enable the unambiguous identification of NsrR-bound RRE-type species, including a persulfide bound form that results from the oxidation of cluster sulfide. In the presence of the low molecular weight thiols glutathione and mycothiol, glutathionylated and mycothiolated forms of NsrR were readily formed.

Controllable release of nitric oxide and doxorubicin from engineered nanospheres for synergistic tumor therapy.

In this paper, core-shell structured UCNPs(DOX)@CS-RBS nanospheres have been designed and synthesized via a step-by-step procedure. The stimuli-responsive UCNPs(DOX)@CS-RBS nanospheres act as nanocarriers for controllable drug delivery towards cancer therapy. The encapsulated UCNPs can efficiently absorb NIR photons and convert them into visible light to trigger NO release. Meanwhile, the entrapped DOX can be released from the swollen nanospheres caused by stretched oleoyl-CS chains at lowered pH typical of intracellular environment. Synergistic cancer therapy will be achieved through the combination of NO and DOX releases based on NO dose-dependent mechanisms. This study provides new drug nanocarriers with high antitumor efficacy for synergistic cancer therapy.

Nitrosylation of Nitric-Oxide-Sensing Regulatory Proteins Containing [4Fe-4S] Clusters Gives Rise to Multiple Iron-Nitrosyl Complexes.

The reaction of protein‐bound iron–sulfur (Fe‐S) clusters with nitric oxide (NO) plays key roles in NO‐mediated toxicity and signaling. Elucidation of the mechanism of the reaction of NO with DNA regulatory proteins that contain Fe‐S clusters has been hampered by a lack of information about the nature of the iron‐nitrosyl products formed. Herein, we report nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT) calculations that identify NO reaction products in WhiD and NsrR, regulatory proteins that use a [4Fe‐4S] cluster to sense NO. This work reveals that nitrosylation yields multiple products structurally related to Roussin's Red Ester (RRE, [Fe2(NO)4(Cys)2]) and Roussin's Black Salt (RBS, [Fe4(NO)7S3]. In the latter case, the absence of 32S/34S shifts in the Fe−S region of the NRVS spectra suggest that a new species, Roussin's Black Ester (RBE), may be formed, in which one or more of the sulfide ligands is replaced by Cys thiolates.

Nitrosylation of Nitric‐Oxide‐Sensing Regulatory Proteins Containing [4Fe‐4S] Clusters Gives Rise to Multiple Iron–Nitrosyl Complexes

AbstractThe reaction of protein‐bound iron–sulfur (Fe‐S) clusters with nitric oxide (NO) plays key roles in NO‐mediated toxicity and signaling. Elucidation of the mechanism of the reaction of NO with DNA regulatory proteins that contain Fe‐S clusters has been hampered by a lack of information about the nature of the iron‐nitrosyl products formed. Herein, we report nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT) calculations that identify NO reaction products in WhiD and NsrR, regulatory proteins that use a [4Fe‐4S] cluster to sense NO. This work reveals that nitrosylation yields multiple products structurally related to Roussin's Red Ester (RRE, [Fe2(NO)4(Cys)2]) and Roussin's Black Salt (RBS, [Fe4(NO)7S3]. In the latter case, the absence of 32S/34S shifts in the Fe−S region of the NRVS spectra suggest that a new species, Roussin's Black Ester (RBE), may be formed, in which one or more of the sulfide ligands is replaced by Cys thiolates.

Controllable Generation of Nitric Oxide by Near‐Infrared‐Sensitized Upconversion Nanoparticles for Tumor Therapy

NaYbF4:Tm@NaYF4:Yb/Er upconversion nanoparticles are synthesized and then integrated with light‐sensitive nitric oxide (NO) donors (Roussin's black salt) to construct a novel near‐infrared (NIR)‐triggered on‐demand NO delivery platform. This nanocompound can absorb 980 nm NIR photons, convert them into higher energy photons and then transfer the energy to the NO donors, resulting in an efficient release of NO. By manipulating the output power of the 980‐nm NIR light, NO‐concentration‐dependent biological effects for cancer therapy can be fine‐tuned, which is investigated and confirmed in vitro. High concentrations of NO can directly kill cancer cells and low concentrations of NO can act as a potent P‐glycoprotein (P‐gp) modulator to overcome multi‐drug resistance (MDR) if combined with chemotherapy.

Visible light-triggered nitric oxide release from near-infrared fluorescent nanospheric vehicles.

Bifunctional Ag2S QDs@CS-RBS nanospheres with ultralow toxicity for biomedical use were fabricated. These are capable of releasing NO under irradiation of visible light and emitting in near-infrared (NIR) region fluorescence excited by NIR laser under physiological environment. Glutathiose (GSH)-capped Ag2S QDs were synthesized and encapsulated by chitosan (CS) to form NIR fluorescent Ag2S QDs@CS nanospheres. A type of iron/sulfur/-nitrosyl cluster, Roussin's black salt anion Fe4S3(NO)7(-) (RBS) was conjugated with the Ag2S QDs@CS to obtain Ag2S QDs@CS-RBS nanospheres. A variety of characterization methods were employed to analyze the nanospheres. In vitro cell imaging and in vivo mice imaging experiments demonstrated that the Ag2S QDs@CS-RBS nanospheres could emit readily observable NIR fluorescence and deliver NO in living cells and small animals. The NIR imaging of the Ag2S QDs@CS nanospheres would not interfere with the light-triggered NO release from them, as the excitation lasers needed for these two functions are in different wavelength regions. This work provides new perspectives for the application of multifunctional nano-structured materials in diagnostics and bioimaging.

Nitric Oxide Releasing Materials Triggered by Near-Infrared Excitation Through Tissue Filters

Novel materials for the phototherapeutic release of the bioregulator nitric oxide (nitrogen monoxide) are described. Also reported is a method for scanning these materials with a focused NIR beam to induce photouncaging while minimizing damage from local heating. The new materials consist of poly(dimethylsiloxane) composites with near-infrared-to-visible upconverting nanoparticles (UCNPs) that are cast into a biocompatible polymer disk (PD). These PDs are then impregnated with the photochemical nitric oxide precursor Roussin's black salt (RBS) to give UCNP_RBS_PD devices that generate NO when irradiated with 980 nm light. When the UCNP_RBS_PD composites were irradiated with NIR light through filters composed of porcine tissue, physiologically relevant NO concentrations were released, thus demonstrating the potential of such devices for minimally invasive phototherapeutic applications.

NIR‐Triggered Release of Caged Nitric Oxide using Upconverting Nanostructured Materials

Lanthanide-doped upconversion nanoparticles (UCNPs) mediate nitric oxide uncaging from Roussin's black salt anion Fe4S3(NO)7− using NIR light from a simple diode laser operating continuously at 980 nm. This result offers a new paradigm for applications of multi-photon absorption in the therapeutic delivery of NO.

Understanding the Unusually Straight: A Search For MO Insights into Linear {FeNO}7 Units

Ferrous-nitrosyl {FeNO}(7) complexes, whether S = 1/2 or 3/2, generally exhibit bent FeNO angles of around 140-145 degrees . There are, however, a handful of exceptions, which are characterized by linear or quasi-linear FeNO units. Presented herein is a relatively comprehensive DFT-based MO analysis of these unusual {FeNO}(7) complexes. DFT-derived FeNO bending potentials indicate that the unusual, experimentally observed quasi-linear geometries indeed correspond to minimum-energy structures on the potential energy surfaces of the isolated molecules/ions. Walsh diagram analyses support our earlier suggestion that the linearity of the {FeNO}(7) units in question is most commonly attributable metal d(sigma)-p(sigma) mixing resulting from the lack of a ligand trans to the NO. Importantly, this effect explains the linearity of both S = 1/2 {FeNO}(7) complexes such as [Fe(CN)(4)(NO)](2-)and Fe(dtc-Me(2))(2)(NO) (dtc-Me(2) = N,N'-dimethyldithiocarbamate) and S = 3/2 complexes such as [Fe(S(t)Bu)(3)(NO)](-). However, Roussin's black salt anion, [Fe(4)(mu-S)(3)(NO)(7)](-), which also contains a linear {FeNO}(7) unit, entails additional, special metal-ligand orbital interactions. The well-known brown-ring complex [Fe(H(2)O)(5)(NO)](2+) also contains a linear {FeNO}(7) unit; the linearity in this case is attributable to the weakness of the trans water ligand.

Antimicrobial Activity of the Iron-Sulfur Nitroso Compound Roussin's Black Salt [Fe4S3(NO)7] on the Hyperthermophilic ArchaeonPyrococcus furiosus

The iron-sulfur nitroso compound [Fe(4)S(3)(NO)(7)](-) is a broad-spectrum antimicrobial agent that has been used for more than 100 years to combat pathogenic anaerobes. Known as Roussin's black salt (RBS), it contains seven moles of nitric oxide, the release of which was always assumed to mediate its cytotoxicity. Using the hyperthermophilic archaeon Pyrococcus furiosus, it is demonstrated through growth studies, membrane analyses, and scanning electron microscopy that nitric oxide does not play a role in RBS toxicity; rather, the mechanism involves membrane disruption leading to cell lysis. Moreover, insoluble elemental sulfur (S(0)), which is reduced by P. furiosus to hydrogen sulfide, prevents cell lysis by RBS. It is proposed that S(0) also directly interacts with the membranes of P. furiosus during its transfer into the cell, ultimately for reduction by a cytosolic NADPH sulfur reductase. RBS is proposed to be a new class of inorganic antimicrobial agent that also has potential use as an inert cell-lysing agent.

Reactions of Synthetic [2Fe-2S] and [4Fe-4S] Clusters with Nitric Oxide and Nitrosothiols

The interaction of nitric oxide (NO) with iron-sulfur cluster proteins results in degradation and breakdown of the cluster to generate dinitrosyl iron complexes (DNICs). In some cases the formation of DNICs from such cluster systems can lead to activation of a regulatory pathway or the loss of enzyme activity. In order to understand the basic chemistry underlying these processes, we have investigated the reactions of NO with synthetic [2Fe-2S] and [4Fe-4S] clusters. Reaction of excess NO(g) with solutions of [Fe2S2(SR)4](2-) (R = Ph, p-tolyl (4-MeC6H4), or 1/2 (CH2)2-o-C6H4) cleanly affords the respective DNIC, [Fe(NO)2(SR)2](-), with concomitant reductive elimination of the bridging sulfide ligands as elemental sulfur. The structure of (Et4N)[Fe(NO)2(S-p-tolyl)2] was verified by X-ray crystallography. Reactions of the [4Fe-4S] clusters, [Fe4S4(SR)4](2-) (R = Ph, CH2Ph, (t)Bu, or 1/2 (CH2)-m-C6H4) proceed in the absence of added thiolate to yield Roussin's black salt, [Fe4S3(NO)7](-). In contrast, (Et4N)2[Fe4S4(SPh)4] reacts with NO(g) in the presence of 4 equiv of (Et4N)(SPh) to yield the expected DNIC. For all reactions, we could reproduce the chemistry effected by NO(g) with the use of trityl-S-nitrosothiol (Ph3CSNO) as the nitric oxide source. These results demonstrate possible pathways for the reaction of iron-sulfur clusters with nitric oxide in biological systems and highlight the importance of thiolate-to-iron ratios in stabilizing DNICs.

Structure and UV–vis spectroscopy of roussin black salt [Fe 4S 3(NO) 7] −

The calculations of the electronic structure, geometry and electronic spectra of the Roussin's black salt anion (RBS) were carried out with the RB3LYP and UB3LYP methods. The geometry was determined by the RB3LYP method. The electronic structure of RBS may be described as composed of the one {Fe(NO)} 7 unit, in which ferric ion ( S=5/2) is antiferromagnetically coupled to a NO − ligand ( S=1) giving S=3/2; such a unit is antiferromagnetically coupled to three {Fe(NO) 2} 9 units, each of them is formed by a ferric ion ( S=5/2) antiferromagnetically coupled to two NO − ligands with S=1. The resulting total spin of RBS is S=0. The S 2− bridges mediate in the antiferromagnetic coupling. The calculated spectrum of RBS shows agreement with the experimental spectra. The calculated transitions are mainly of charge transfer character. The photochemical behaviour is interpreted.

Nitrosative stress induced cytotoxicity in Giardia intestinalis.

To investigate the antigiardial properties of the nitrosating agents: sodium nitrite, sodium nitroprusside and Roussin's black salt. Use of confocal laser scanning microscopy and flow cytometry indicated permeabilization of the plasma membrane to the anionic fluorophore, DiBAC4(3) [bis(1,3-dibutylbarbituric acid) trimethine oxonol]. Loss of plasma membrane electrochemical potential was accompanied by loss of regulated cellular volume control. Changes in ultrastructure revealed by electron microscopy and capacity for oxygen consumption, were also consequences of nitrosative stress. Roussin's black salt (RBS), active at micromolar concentrations was the most potent of the three agents tested. These multitargeted cytotoxic agents affected plasma membrane functions, inhibited cellular functions in Giardia intestinalis and led to loss of viability. Nitrosative damage, as an antigiardial strategy, may have implications for development of chemotherapy along with suggesting natural host defence mechanisms.