Real-time Chemiluminescence Research Articles

S-nitrosothiol-terminated Pluronic F127: Influence of microstructure on nitric oxide release

Hypothesis: Nitric oxide (NO)-releasing Pluronic F127 hydrogels (F127) containing dissolved S-nitrosothiols or pendant N-diazeniumdiolate (NONOate) groups have been described. The NO charging of these hydrogels is usually limited by their low stability or disruption of the micellar packing. S-nitrosothiol-terminated F127 may emerge as a new strategy for allowing NO delivery at different rates in biomedical applications.Experiments: Terminal hydroxyl groups of F127 were esterified and reduced to produce F127-mercaptopropionate (HS-F127-SH), which was subsequently S-nitrosated to generate S-nitrosothiol-terminated F127 (ONS-F127-SNO). Micro-differential scanning calorimetry, 1H NMR spin-spin relaxation (T2), temperature-dependent small-angle X-ray scattering, and cryo-transmission electron microscopy, were used to determine the micellar packing structure, while real-time chemiluminescence NO detection and UV–Vis spectrophotometry were used to evaluate the kinetics of NO release.Findings: HS-F127-SH micellization and gelation processes were analogous to native F127, however, with a decreased short-range ordering of the micelles. ONS-F127-SNO hydrogels released NO thorough a preferentially intramicellar SNO dimerization reaction. Increasing ONS-F127-SNO concentration reduces the rate of SNO dimerization and increases the overall rate of NO release to the gas phase, opening up new possibilities for tailoring NO delivery from F127-based hydrogels.

S-nitrosothiol-terminated poly(vinyl alcohol): Nitric oxide release and skin blood flow response

Polymeric biomaterials capable of delivering nitric oxide (NO) topically can be used to enhance skin blood flow (SkBF) and accelerate wound healing. Herein, we used reversible addition-fragmentation chain transfer radical (RAFT) polymerization to synthesize the first poly(vinyl alcohol) (PVA) functionalized with terminal NO-releasing S-nitrosothiol (RSNO) groups for topical NO delivery. This strategy was based on the synthesis of a precursor amino-terminated PVA (PVA-NH2), which was next functionalized with iminothiolane yielding 4-imino-4-amino-PVA-butane-1-thiol (PVA-SH), and finally S-nitrosated yielding S-nitroso 4-imino-4-amino-PVA-butane-1-thiol (PVA-SNO). Real-time chemiluminescence NO detection showed that blended films of pure PVA with PVA-SNO with mass ratios 30:70, 50:50 and 70:30 release NO with initial rates ranging from 1 to 12 nmol g−1 min−1, and lead to a 2 to 10-fold dose-response increase in the SkBF, after topical application on the ventral forearm of volunteers. These results show that PVA-SNO is a potential platform for topical NO delivery in biomedical applications.

An Activatable Chemiluminescent Probe for Sensitive Detection of γ-Glutamyl Transpeptidase Activity in Vivo.

Activatable chemiluminescent probes that show enhanced chemiluminescence upon interaction with a molecular target of interest have offered promising tools for sensing and bioimaging in terms of low background, high sensitivity, and improved penetration depth in biological tissues. Here, we reported a γ-glutamyl transpeptidase (GGT) activatable chemiluminescent probe for real-time detection of GGT activity in vitro and in living mice. The probe was designed by caging an electron-withdrawing acrylic group-substituted Schaap's phenoxy-dioxetane with a GGT-recognitive substrate (γ-Glu) and a self-immolative linker (p-aminobenzyl alcohol), which was initially chemiluminescence off. Upon interaction with GGT, strong chemiluminescence with a more than 800-fold turn-on ratio could be achieved in aqueous solution, allowing to specifically detect GGT activity with ultrahigh signal-to-background ratio and sensitivity in vitro and in live cells. We demonstrated that the probe was reliable to quantify the GGT in serum, permitting to accurately report the elevated levels of GGT in lipopolysaccharide-treated mouse serum. Moreover, through real-time chemiluminescence imaging of GGT activity, the designed probe was feasible to detect GGT-positive tumors in living mice after intravenous systemic administration. This study demonstrates the high potential of GGT-activatable chemiluminescent probe for serum assays and molecular imaging, which might find wide applications in diagnosis of GGT-related diseases.

Poly-ε-caprolactone/polysulfhydrylated polyester blend: A platform for topical and degradable nitric oxide-releasing materials

Polymeric biomaterials for implantable medical devices are subject to demands that go beyond passive biocompatibility. Inhibition of thrombus formation, reduced inflammatory response and the eventual complete bioabsorption of the implant are central challenges for emerging polymeric biomaterials. Nitric oxide (NO) released at the biomaterial/tissue interface may inhibit platelet adhesion and improve tissue integration in blood-contacting/implanted devices. In addition, local NO release may increase vasodilation, thereby improving ischemic conditions in topical applications. In this study, we present a novel degradable NO-releasing polymeric platform based on blended poly-ε-caprolactone (PCL)/polysulfhydrylated polyester (PSPE). PCL formed miscible blends with PSPE up to 45 wt% of PSPE. S-nitrosation of the PCL/PSPE blends converted the surface PSPE component into a NO-releasing polynitrosated polyester (PNPE). Real-time chemiluminescence NO detection showed that blends with different PCL:PSPE ratios allow surface S-nitrosothiol concentrations in the range of 0.5–1.5 μmol/cm2. Topical application of blended PCL/PNPE films on the healthy skin led to a 6–10-fold increase in the skin blood flow due to local NO delivery, as shown by in vivo laser-Doppler flowmetry. Blended PCL/PSPE films displayed higher hydrophilicity and a 2–11-fold increase in the hydrolytic mass loss, compared to pure PCL films. Therefore, PCL/PSPE blend is a potential biomaterial platform for local NO delivery and more quickly degradable biomedical devices.

Chemiluminescence imaging of UVA induced reactive oxygen species in mouse skin using L-012 as a probe

Reactive oxygen species (ROS) caused by ultraviolet A (UVA) can be reduced by treating with antioxidants and photoprotective reagents. Here we reported a real-time chemiluminescence (CL) imaging method which was simple, non-invasive and sensitive to evaluate UVA-induced ROS generation and the efficacy of sunscreens and antioxidants in vivo. The in vitro experiments indicated that l-ascorbic acid, live SPSC01 yeast, and its intracellular metabolites can suppress the intensity of CL signals in the presence of hydrogen peroxide, which proved the good antioxidant ability of them. Meanwhile, we used 8-amino-5-chloro-7-phenylpyrido[3,4-d] pyridazine-1,4(2H,3H) dione (L-012) as a high sensitive CL probe for in vivo imaging of ROS generated by UVA irradiation. The CL intensity was reduced after treated with l-ascorbic acid and SPSC01 yeast intracellular metabolites, consistent with the in vitro results. Additionally, the in vivo protective capability of two azobenzene compounds as sunscreens was confirmed further through the suppression of CL signals of UVA-induced ROS in mouse skin by this method.

Real-time chemiluminescence imaging using nano-lantern probes.

Chemiluminescence imaging can be performed without excitation light sources at various spatial levels ranging from a single cell to the whole body. Thus far, chemiluminescence imaging has been primarily performed with long exposure times because of weak signals, resulting in low temporal resolution. Recently, the brightest-known chemiluminescent proteins--Nano-lantern and Nano-lantern-based functional indicators--have been developed. Nano-lantern probes break the limitation of temporal resolution and enable chemiluminescence imaging of living samples such as cells, plants, and small animals at video rates. This unit describes one protocol for observation of a freely moving unshaved mouse transplanted with Nano-lantern-expressing tumor cells, and another for compatible use of optogenetic tools and a Nano-lantern calcium indicator. Both protocols utilize the synchronization of illumination and camera acquisition sessions, thereby enabling real-time chemiluminescence imaging.

Enhanced photochemical nitric oxide release from a flutamide derivative incorporated in Pluronic F127 micelles

The incorporation of nitric oxide (NO) photodonors into nontoxic vehicles may allow for the tunable control of topical NO release to treat pathological conditions associated with the impairment of NO production in the dermal vasculature. Pluronic F127 is a PEO–PPO–PEO triblock copolymer that is used as a nontoxic vehicle that undergoes thermally reversible micellization and gelation. In this study, we synthesized a thermally stable flutamide derivative, N-(3-aminopropyl)-3-(trifluoromethyl)-4-nitrobenzenamine (ATN), which is capable of releasing NO upon irradiation with visible light, as shown by real-time chemiluminescence NO detection. We demonstrated that the incorporation of ATN into the hydrophobic nuclei of F127 micelles leads to an enhancement of its photochemical NO release, which is consistent with the photodecomposition route associated with the nitro-to-nitrite photorearrangement of ATN. This mechanism underscores a new role of the F127 micelles as concentrating “nanoreactors” for hydrophobic NO releasing drugs. In this case, this effect may represent a new strategy for enhancing photochemical NO release from ATN, which makes the micellar F127/ATN solutions promising for topical NO application.

Comparative study of β-glucan induced respiratory burst measured by nitroblue tetrazolium assay and real-time luminol-enhanced chemiluminescence assay in common carp (Cyprinus carpio L.)

The respiratory burst is an important feature of the immune system. The increase in cellular oxygen uptake that marks the initiation of the respiratory burst is followed by the production of reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide which plays a role in the clearance of pathogens and tissue regeneration processes. Therefore, the respiratory burst and associated ROS constitute important indicators of fish health status. This paper compares two methods for quantitation of ROS produced during the respiratory burst in common carp: the widely used, single-point measurement based on the intracellular reduction of nitroblue tetrazolium (NBT) and a real-time luminol-enhanced assay based on the detection of native chemiluminescence. Both assays allowed for detection of dose-dependent changes in magnitude of the respiratory burst response induced by β-glucans in head kidney cells of carp. However, whereas the NBT assay was shown to detect the production of only superoxide anions, the real-time luminol-enhanced assay could detect the production of both superoxide anions and hydrogen peroxide. Only the chemiluminescence assay could reliably record the production of ROS on a real-time scale at frequent and continual time intervals for time course experiments, providing more detailed information on the respiratory burst response. The real-time chemiluminescence assay was used to measure respiratory burst activity in macrophage and neutrophilic granulocyte-enriched head kidney cell fractions and total head kidney cell suspensions and proved to be a fast, reliable, automated multiwell microplate assay to quantitate fish health status modulated by β-glucans.

Biodegradable S-nitrosothiol tethered multiblock polymer for nitric oxide delivery

A biodegradable multiblock polymer containing the S-nitrosothiol moiety was synthesized via a 4 step process using non-aqueous reaction conditions. The structural and morphological properties of the material were characterized via1H NMR, GPC, DSC, TGA, and SAXS. The nitric oxide (NO) storage capacity of the polymer was found to be 0.42 ± 0.01 mmol NO per g polymer using UV spectroscopy, and real-time chemiluminescence measurements demonstrated a controlled and extended NO release profile. The degradation rate of the material was the most rapid during the first week followed by a slower linear release rate. Preliminary biocompatibility testing of the material using ISO protocols indicated no cell lysis in vitro. Taken together, this material has the requisite properties to control initial biological responses and the longevity to provide longer term mechanical support.

In Situ Real-Time Chemiluminescence Imaging of Reactive Oxygen Species Formation from Cardiomyocytes

We have applied the highly sensitive chemiluminescence (CL) imaging technique to investigate the in situ ROS formation in cultured monolayers of rat H9c2 cardiomyocytes. Photon emission was detected via an innovative imaging system after incubation of H9c2 cells in culture with luminol and horseradish peroxidase (HRP), suggesting constitutive formation of ROS by the cardiomyocytes. Addition of benzo(a)pyrene-1,6-quinone (BPQ) to cultured H9c2 cells resulted in a 4-5-fold increase in the formation of ROS, as detected by the CL imaging. Both constitutive and BPQ-stimulated CL responses in cultured H9c2 cells were sustained for up to 1 hour. The CL responses were completely abolished in the presence of superoxide dismutase and catalase, suggesting the primary involvement of superoxide and hydrogen peroxide (H2O2). In contrast to BPQ-mediated redox cycling, blockage of mitochondrial electron transport chain by either antimycin A or rotenone exerted marginal effects on the ROS formation by cultured H9c2 cells. Upregulation of cellular antioxidants for detoxifying both superoxide and H2O2 by 3H-1,2-dithiole-3-thione resulted in marked inhibition of both constitutive and BPQ-augmented ROS formation in cultured H9c2 cells. Taken together, we demonstrate the sensitive detection of ROS by CL imaging in cultured cardiomyocytes.

Nitric Oxide-Releasing Xerogel-Based Fiber-Optic pH Sensors

A xerogel-based optical pH sensor capable of releasing low levels of nitric oxide (NO) and measuring changes in solution pH is reported. Through simple dip-coating procedures, aminoalkoxysilane-based xerogel films modified with N-diazeniumdiolate NO donor precursors and the fluorescent pH indicator seminaphthorhodamine-1 carboxylate (SNARF-1) were sequentially deposited onto optical fibers. The resulting sensors were characterized by fast and linear response to pH throughout the physiological range (pH 7.0-7.8). Real-time chemiluminescence measurements confirmed that the presence of the overlying SNARF-1-containing TMOS layer did not have an inhibitory effect on N-diazeniumdiolate formation or NO release, and the NO-releasing coatings were capable of maintaining NO fluxes >0.4 pmol/cm(2) s up to 16 h. In vitro blood compatibility studies using porcine platelets confirmed the expected thromboresistivity of the NO-releasing xerogel coatings.

Tumor necrosis factor-induced, superoxide-mediated neutrophil accumulation in cold ischemic/reperfused rat liver

The mechanisms of hepatic ischemia/reperfusion injury are complicated and multifactorial. This study was designed to examine superoxide generation and neutrophil accumulation in cold ischemic-reperfused rat livers after elimination of Kupffer cells and to determine the role of superoxide/tumor necrosis factor (TNF) interactions. Rat Kupffer cells were eliminated by liposome-encapsulated dichloromethylene diphosphonate injected intravenously. Livers from control and treated rats were isolated and preserved in University of Wisconsin solution (4°C) for 0, 12, and 24 hours and then perfused for 60 minutes with oxygenated Krebs-Henseleit bicarbonate buffer (37°C) by adding neutrophils into the perfusate. Superoxide generation was measured by using real-time chemiluminescence (CL) during perfusion, and neutrophil accumulation was assessed by measuring myeloperoxidase activity in the liver tissue. In the control livers, CL intensity markedly increased on reoxygenation, and after neutrophil infusion it increased again with a lag period of 10 minutes. Total CL intensity and myeloperoxidase activity increased with the duration of cold preservation. TNF release into the effluent perfusate was detectable only after 24 hours of preservation, and lactate dehydrogenase release was high. Elimination of Kupffer cells attenuated CL intensity and TNF and lactate dehydrogenase release and resulted in reduced myeloperoxidase activity. Electron microscopy revealed amelioration of hepatocyte swelling and endothelial cell disruption when Kupffer cells were eliminated. After 24 hours of preservation, superoxide generation was inhibited in the control livers by anti-TNF antiserum, whereas TNF release was not inhibited by superoxide dismutase. These results suggest that TNF induces superoxide generation by Kupffer cells, which mediates neutrophil accumulation and causes cellular injury in the initial phase of reperfusion.

Tumor necrosis factor-induced, superoxide-mediated neutrophil accumulation in cold ischemic/reperfused rat liver

The mechanisms of hepatic ischemia/reperfusion injury are complicated and multifactorial. This study was designed to examine superoxide generation and neutrophil accumulation in cold ischemic-reperfused rat livers after elimination of Kupffer cells and to determine the role of superoxide/tumor necrosis factor (TNF) interactions. Rat Kupffer cells were eliminated by liposome-encapsulated dichloromethylene diphosphonate injected intravenously. Livers from control and treated rats were isolated and preserved in University of Wisconsin solution (4 degrees C) for 0, 12, and 24 hours and then perfused for 60 minutes with oxygenated Krebs-Henseleit bicarbonate buffer (37 degrees C) by adding neutrophils into the perfusate. Superoxide generation was measured by using real-time chemiluminescence (CL) during perfusion, and neutrophil accumulation was assessed by measuring myeloperoxidase activity in the liver tissue. In the control livers, CL intensity markedly increased on reoxygenation, and after neutrophil infusion it increased again with a lag period of 10 minutes. Total CL intensity and myeloperoxidase activity increased with the duration of cold preservation. TNF release into the effluent perfusate was detectable only after 24 hours of preservation, and lactate dehydrogenase release was high. Elimination of Kupffer cells attenuated CL intensity and TNF and lactate dehydrogenase release and resulted in reduced myeloperoxidase activity. Electron microscopy revealed amelioration of hepatocyte swelling and endothelial cell disruption when Kupffer cells were eliminated. After 24 hours of preservation, superoxide generation was inhibited in the control livers by anti-TNF antiserum, whereas TNF release was not inhibited by superoxide dismutase. These results suggest that TNF induces superoxide generation by Kupffer cells, which mediates neutrophil accumulation and causes cellular injury in the initial phase of reperfusion. (Hepatology 1997 Jul;26(1):113-20)

Kupffer Cells Generate Superoxide Anions and Modulate Reperfusion Injury in Rat Livers After Cold Preservation

ABSTRACT This study was designed to determine the source of reactive oxygen species (ROS) and whether Kupffer cells modulate the injury of perfused rat liver after cold preservation. The livers of male Lewis rats pretreated with schizophyllan glucan (SPG) (10 mg/kg, SPG group) or gadolinium chloride (5 mg/kg; Gd group) and untreated rats (control group) were preserved in University of Wisconsin solution for 0, 12, and 24 hours at 4 degrees C and then perfused for 60 minutes with oxygenated Krebs–Henseleit bicarbonate buffer. Real–time chemiluminescence (CL) of the liver during perfusion was measured using a sensitive photomultiplier, and reperfusion injury was assessed by measuring lipid peroxidation and lactate dehydrogenase release. CL intensity reached a peak within 5 minutes of reperfusion, and superoxide dismutase completely inhibited this CL in all groups. In the control group, the total CL intensity was high after 24 hours of preservation. It significantly ( P < .05 vs. control group) increased in the SPG group, while it decreased in the Gd group after 12 and 24 hours of preservation. The total CL intensity decreased by 70% (when diphenyliodonium chloride (100 micromol/L; an NADPH oxidase inhibitor) was added to the perfusate before preservation of untreated rats. Lipid peroxidation and lactate dehydrogenase release significantly ( P < .05) deteriorated in the SPG group, while they ameliorated in the Gd group after 24 hours of preservation. These results demonstrate that Kupffer cells primarily generate superoxide anions and modulate the organ injury in the initial phase of reperfusion after cold preservation.

Kupffer cells generate superoxide anions and modulate reperfusion injury in rat livers after cold preservation

This study was designed to determine the source of reactive oxygen species (ROS) and whether Kupffer cells modulate the injury of perfused rat liver after cold preservation. The livers of male Lewis rats pretreated with schizophyllan glucan (SPG) (10 mg/kg, SPG group) or gadolinium chloride (5 mg/kg; Gd group) and untreated rats (control group) were preserved in University of Wisconsin solution for 0, 12, and 24 hours at 4 degrees C and then perfused for 60 minutes with oxygenated Krebs-Henseleit bicarbonate buffer. Real-time chemiluminescence (CL) of the liver during perfusion was measured using a sensitive photomultiplier, and reperfusion injury was assessed by measuring lipid peroxidation and lactate dehydrogenase release. CL intensity reached a peak within 5 minutes of reperfusion, and superoxide dismutase completely inhibited this CL in all groups. In the control group, the total CL intensity was high after 24 hours of preservation. It significantly (P < .05 vs. control group) increased in the SPG group, while it decreased in the Gd group after 12 and 24 hours of preservation. The total CL intensity decreased by 70% (when diphenyliodonium chloride (100 micromol/L; an NADPH oxidase inhibitor) was added to the perfusate before preservation of untreated rats. Lipid peroxidation and lactate dehydrogenase release significantly (P < .05) deteriorated in the SPG group, while they ameliorated in the Gd group after 24 hours of preservation. These results demonstrate that Kupffer cells primarily generate superoxide anions and modulate the organ injury in the initial phase of reperfusion after cold preservation.(Hepatology 1997 Feb;25(2):356-60)

Chemiluminescence detection of hydrazine vapor

An efficient, real-time chemiluminescence detector for hydrazine vapor, N 2H 4(g), is described, capable of monitoring sub part-per-billion levels of hydrazine in air. The catalytic oxidation of hydrazine by colloidal platinum forms an intermediate, oxidizing agent ( e.g. OH or OOH) which subsequently oxidizes luminol, generating a chemiluminescence signal that is proportional to the hydrazine concentration. Major components of the instrument include a photomultiplier tube (PMT), a short length of glass tubing coiled directly in front of the PMT cathode surface, a vacuum pump for sampling the air, and a peristaltic pump for circulating the liquid reagent. The liquid reagent, a basic solution (pH 13) of luminol and colloidal platinum, is continuously recycled. The detection sequence is initiated by pumping the hydrazine vapor through a short length of teflon tubing that is concurrently transporting the liquid reagent. The liquid is separated from the gas stream in an impinger and quickly pumped to the PMT. We have evaluated the effect of solution pH, luminol and platinum concentrations, and air and liquid flow rates on the analytical characteristics of this system. A linear, dynamic detection range for hydrazine has been obtained from 1 to 2000 ppb in air, with an instrument response that is fully reversible and achieves plateau response in less than 2 min.

Long-term administration of L-arginine improves nitric oxide release from kidney in deoxycorticosterone acetate-salt hypertensive rats.

To examine the effects of L-arginine (L-Arg) on endothelial function, we administered 0.5 g/L L-Arg in drinking water to deoxycorticosterone acetate (DOCA)-salt rats for 8 weeks and then measured nitric oxide (NO) release from isolated kidneys using a newly developed real-time chemiluminescence method. Renal pathology was also analyzed. Acetylcholine caused much smaller declines in renal perfusion pressure (10(-7) mol/L acetylcholine: -24 +/- 2% [SEM] versus -50 +/- 2%, P < .001) and NO release in DOCA-salt rats (+3 +/- 1 versus +33 +/- 3 fmol/min per gram kidney weight, P < .001) compared with control rats. L-Arg did not influence the time course of systolic blood pressure elevation in DOCA-salt rats (211 +/- 5 versus 208 +/- 6 mmHg, DOCA versus L-Arg/DOCA, P = NS). However, oral administration of L-Arg improved acetylcholine-induced declines in renal perfusion pressure (10(-7) mol/L acetylcholine: L-Arg/DOCA, -39 +/- 3%, P < .01 versus DOCA). This change was associated with an increase in NO release by acetylcholine (10(-7) mol/L acetylcholine: L-Arg/DOCA, +10 +/- 1 fmol/min per gram kidney weight, P < .05 versus DOCA). However, morphological changes in renal vessels and glomeruli were similar between DOCA and L-Arg/DOCA rats. These results suggest that L-Arg administration partially reverses renal endothelial function with respect to vasorelaxation and NO release independent of blood pressure changes, indicating that hypertensive vessels seem to be depleted of L-Arg and/or have defects in the availability of L-Arg for NO synthesis.

A comparison of filter, denuder, and real-time chemiluminescence techniques for nitric acid determination in ambient air

Measurements of gas-phase nitric acid were made by four separate techniques during a 7-day summertime period at a near-coastal site on Long Island, NY. Results from methods intercomparison data for HNO 3, and their relationship to particulate NO 3 − and other odd N and oxidant species show the following: (a) high-volume filter pack HNO 3 concentrations are well correlated with diffusion denuder difference (DD) results, except for small absolute losses with the former; (b) daytime real-time two-channel chemiluminescence HNO 3 levels correlated well with DD results, but were higher during night-time periods; (c) results by a new Al 2(SO 4) 3 denuder/thermal evolution technique were not in agreement with other techniques. Based on HNO 3 and paniculate NO 3 results reported herein, it appears that negative errors in HNO 3filter-sampling techniques resulting from HNO 3 loss by sorption generally exceed positive errors NH 4NO 3 volatilization at this site.