Absence Of Photoactivation Research Articles

Photodynamic Inactivation of Human Coronaviruses.

Photodynamic inactivation (PDI) employs a photosensitizer, light, and oxygen to create a local burst of reactive oxygen species (ROS) that can inactivate microorganisms. The botanical extract PhytoQuinTM is a powerful photosensitizer with antimicrobial properties. We previously demonstrated that photoactivated PhytoQuin also has antiviral properties against herpes simplex viruses and adenoviruses in a dose-dependent manner across a broad range of sub-cytotoxic concentrations. Here, we report that human coronaviruses (HCoVs) are also susceptible to photodynamic inactivation. Photoactivated-PhytoQuin inhibited the replication of the alphacoronavirus HCoV-229E and the betacoronavirus HCoV-OC43 in cultured cells across a range of sub-cytotoxic doses. This antiviral effect was light-dependent, as we observed minimal antiviral effect of PhytoQuin in the absence of photoactivation. Using RNase protection assays, we observed that PDI disrupted HCoV particle integrity allowing for the digestion of viral RNA by exogenous ribonucleases. Using lentiviruses pseudotyped with the SARS-CoV-2 Spike (S) protein, we once again observed a strong, light-dependent antiviral effect of PhytoQuin, which prevented S-mediated entry into human cells. We also observed that PhytoQuin PDI altered S protein electrophoretic mobility. The PhytoQuin constituent emodin displayed equivalent light-dependent antiviral activity to PhytoQuin in matched-dose experiments, indicating that it plays a central role in PhytoQuin PDI against CoVs. Together, these findings demonstrate that HCoV lipid envelopes and proteins are damaged by PhytoQuin PDI and expands the list of susceptible viruses.

Fluorinated TiO2-doped, glycine-functionalized MWCNTs for high-performance antibacterial agents

Perennial need for bactericides requires cost-effective nanomaterials with strong antibacterial activities even in the absence of external irradiation. Hence, in this work, we have synthesized f-CFT (fluorinated TiO2-doped, glycine-functionalized MWCNTs) and have studied its antibacterial activities. Both gram-negative and gram-positive bacteria were analyzed in the absence of photo-activation. Zone of inhibition of 15 mm for S. aureus, and 11 mm for P. aeruginosa was observed for f-CFT owing to the synergistic bactericidal properties of functionalized MWCNTs, fluorine and nano-TiO2. Anatase phase TiO2 with average crystallite size as 35 nm was observed in XRD. Scanning electron microscopic images showed uniform mixed cubic structures. Hydroxyl groups observed in FT-IR along with the glycine MWCNTs interface aid the inhibition of bacterial growth even in the absence of photo activation. A desired higher gram-positive bacterial inhibition opens new gates for better antibacterial agents.

Pro- and anti-oxidant properties of near-infrared (NIR) light responsive carbon nanoparticles

Elemental carbon nanomaterials (ECNMs) are redox active agents that can be exploited to purposely modify the redox balance of cells. Both pro- or antioxidant properties have been reported. However, to the best of our knowledge, there are not comprehensive studies exploring both properties on the same material in view of a potential application in medicine. At the same time, the effect of the bulk structure on the pro/antioxidant properties is poorly known. Here, carbon nanoparticles (CNPs) derived by glucose with definite size and shape have been prepared, and their redox properties evaluated in cell free systems in the dark or following activation with a Near Infrared (NIR) laser beam (945 nm, 1.3 W/cm2). We found that, when irradiated with NIR, CNPs efficiently generate heat and singlet oxygen (1O2), a property that can be exploited for dual photo-thermal (PT)/photodynamic (PD) therapy in cancer. On the other hand, in the absence of photo-activation, CNPs react with both oxidant (hydroxyl radicals) and antioxidant (glutathione) species. When tested on a murine macrophages cell line (RAW 264.7) CNPs were clearly antioxidant. Furthermore, albeit efficiently internalized, CNPs do not exert cytotoxic effect up to 80 µg/ml and do not exacerbate TNF-α-mediated inflammation. Overall, the results reported herein suggest that CNPs may represent a new class of safe nanomaterials with potential applications in medicine.

Toxicity of tryptophan manganese(i) carbonyl (Trypto-CORM), against Neisseria gonorrhoeae.

The potential for carbon monoxide-releasing molecules (CO-RMs) as antimicrobials represents an exciting prospective in the fight against antibiotic resistance. Trypto-CORM, a tryptophan-containing manganese(i) carbonyl, is toxic against E. coli following photo-activation. Here, we demonstrate that Trypto-CORM is toxic against Neisseria gonorrhoeae in the absence of photoactivation. Trypto-CORM toxicity was reversed by the high CO affinity globin leg-haemoglobin (Leg-Hb), indicating that the toxicity is due to CO release. Release of CO from Trypto-CORM in the dark was also detected with Leg-Hb (but not myoglobin) in vitro. N. gonorrhoeae is more sensitive to CO-based toxicity than other model bacterial pathogens, and may serve as a viable candidate for antimicrobial therapy using CO-RMs.

Synergistic effect of MgO/Ag co-doping on TiO2 for efficient antibacterial agents

Pathogenic bacteria that develop multidrug resistance pose extensive health hazards. TiO2 nanoparticles (NPs) generally show strong antibacterial activity under irradiation. Hence, we have analyzed the bactericidal efficiency of TiO2 in the absence of photo-activation by doping and co-doping MgO NPs, Ag NPs to anatase TiO2 NPs. Co-doped TiO2 (TAM) showed a high inhibition zone of 20mm and14mm against Escherichia coli and Bacillus subtilis respectively with reduced band gap energy of 3.27eV. Undoped TiO2 (TU), Ag NPs doped TiO2 (TA), MgO doped TiO2 (TM) showed particle sizes of 95nm, 35nm, 45nm respectively. Smaller particle size of 33nm observed in TAM combined with the synergistic effects of ROS formation effected by MgO and bactericidal Ag+ ions account for the bacterial inhibition.

A new ER-specific photosensitizer unravels (1)O2-driven protein oxidation and inhibition of deubiquitinases as a generic mechanism for cancer PDT.

Photosensitizers (PS) are ideally devoid of any activity in the absence of photoactivation, and rely on molecular oxygen for the formation of singlet oxygen ((1)O2) to produce cellular damage. Off-targets and tumor hypoxia therefore represent obstacles for the use of PS for cancer photodynamic therapy. Herein, we describe the characterization of OR141, a benzophenazine compound identified through a phenotypic screening for its capacity to be strictly activated by light and to kill a large variety of tumor cells under both normoxia and hypoxia. This new class of PS unraveled an unsuspected common mechanism of action for PS that involves the combined inhibition of the mammalian target of rapamycin (mTOR) signaling pathway and proteasomal deubiquitinases (DUBs) USP14 and UCH37. Oxidation of mTOR and other endoplasmic reticulum (ER)-associated proteins drives the early formation of high molecular weight (MW) complexes of multimeric proteins, the concomitant blockade of DUBs preventing their degradation and precipitating cell death. Furthermore, we validated the antitumor effects of OR141 in vivo and documented its highly selective accumulation in the ER, further increasing the ER stress resulting from (1)O2 generation upon light activation.

Antimicrobial photodynamic effect of phenothiazinic photosensitizers in formulations with ethanol on Pseudomonas aeruginosa biofilms

Background dataMethylene blue (MB) and toluidine blue (TB) are recognized as safe photosensitizers (Ps) for use in humans. The clinical effectiveness of the antimicrobial photodynamic therapy with MB and TB needs to be optimized, and ethanol can increase their antimicrobial effect. Formulations of MB and TB containing ethanol were evaluated for their ability to produce singlet oxygen and their antibacterial effect on Pseudomonas aeruginosa biofilms. MethodsPhotoactivated formulations were prepared by diluting the Ps (250μM) in buffered water (pH 5.6, sodium acetate/acetic acid), 10% ethanol (buffer: ethanol, 90:10), or 20% ethanol (buffer: ethanol, 80:20). Biofilms also were exposed to the buffer, 10% ethanol, or 20% ethanol without photoactivation. Untreated biofilm was considered the control group. The production of singlet oxygen in the formulations was measured based on the photo-oxidation of 1,3-diphenylisobenzofuran. The photo-oxidation and CFU (log10) data were evaluated by two-way ANOVA and post-hoc Tukey’s tests. ResultsIn all the formulations, compared to TB, MB showed higher production of singlet oxygen. In the absence of photoactivation, neither the buffer nor the 10% ethanol solution showed any antimicrobial effect, while the 20% ethanol solution significantly reduced bacterial viability (P=0.009). With photoactivation, only the formulations containing MB and both 10% and 20% ethanol solutions significantly reduced the viability of P. aeruginosa biofilms when compared with the control. ConclusionsMB formulations containing ethanol enhanced the antimicrobial effect of the photodynamic therapy against P. aeruginosa biofilms in vitro.

Cytotoxicity of different sized TiO2 nanoparticles in mouse macrophages

With large-scale production and wide application of nano-titanium oxide (TiO2), its health hazard has attracted extensive attention worldwide. In this study, mouse macrophages (Ana-1 and MH-S cells) were used to evaluate the cytotoxicity of different sized TiO2 nanoparticles. The results showed that TiO2 nanoparticles caused low toxicity, especially in MH-S cells. There was a difference in the cytotoxicity induced by different sized TiO2 particles. The 25nm anatase particles induced the strongest cytotoxicity and oxidative stress, followed by 5 and 100nm anatase particles; in contrast, 100nm rutile particles induced the lowest toxicity. Although TiO2 nanoparticles induced high levels of intracellular reactive oxygen species (ROS), the determination of ROS demonstrated that the inherent oxidative capacity of TiO2 nanoparticles was lower in the absence of photoactivation. Therefore, the generation of intracellular ROS could not completely depend on inherent oxidative capacity of TiO2 nanoparticles. Toxicity of TiO2 nanoparticles could mainly depend on the structural characteristics.

Comparison of cellular effects of titanium dioxide nanoparticles with different photocatalytic potential in human keratinocyte, HaCaT cells

Titanium dioxide (TiO2) nanoparticle is an important product for nanotechnology because of its high stability, anticorrosion, and photocatalysis. It is also used in cosmetic and skin care products, particularly in sunblocks, where it helps to protect the skin from UV light, especially in the case of nanosized particles (<100 nm). There are extensive concerns on the potential risks of TiO2 nanoparticle to human health and environment. Some forms at least may be genotoxic, photocatalytic, and/or carcinogenic. In this study, we have characterized the physico-chemical properties of commercially available photocatalytic TiO2 nanoparticle and compared to cellular effects in HaCaT Cells with or without photoactivation. The present study has shown that TiO2 nanoparticles are cytotoxic to HaCaT cells even in the absence of photoactivation. This effect became more pronounced in the simultaneous irradiation of UVA dependent on photocatalytic potential of TiO2 nanoparticle. We also found that the cytotoxicity and oxidative stress of TiO2 nanoparticles strongly depends on physico-chemical properties including structure (anantase, rutile, or mixture) or photocatalytic potency of TiO2 nanoparticles.

Oxidative damage and OGG1 expression induced by a combined effect of titanium dioxide nanoparticles and lead acetate in human hepatocytes

Titanium dioxide (TiO(2)) is a widely used nanomaterial that can cause biological damage through oxidative stress. At low concentrations, TiO(2) can interact with lead acetate (PbAc) to produce different toxic responses, compared with TiO(2) or PbAc alone. In this study, we utilized the following as indicators of toxic responses in human embryo hepatocytes (L02): reactive oxygen species (ROS), reduced glutathione (GSH), superoxide dismutase (SOD), and the DNA adducts 8-hydroxydeoxyguanosine (8-OHdG) and 8-oxoguanine DNA glycosylase homolog 1 (OGG1). These were used to evaluate the oxidative stress of TiO(2) (at 0.001, 0.01, 0.1, 1, and 10 μg mL(-1)) mixed with PbAc (1 μg mL(-1)) on L02 cells without photoactivation. Compared with the negative control (1‰ dimethyl sulfoxide), TiO(2) mixed with PbAc induced increased release of ROS (at 0.001, 0.01, 0.1, 1, 10 μg mL(-1) TiO(2)), intracellular SOD activity (at 0.1 and 0.01 μg mL(-1) TiO(2)), GSH levels (at 0.01-1 μg mL(-1) TiO(2)), 8-OHdG levels (at 1 and 10 μg mL(-1) TiO(2)), OGG1 expression (at 0.001-1 μg mL(-1) TiO(2)), and cytotoxicity (at 0.1, 1, and 10 μg mL(-1) TiO(2)) in L02 cells. There were no significant changes in ROS, GSH, SOD, 8-OHdG, or OGG1 levels when L02 cells were treated with TiO(2) alone or PbAc alone. These findings indicate that TiO(2) and PbAc in combination induce cytotoxicity and oxidative stress in L02 cells in the absence of photoactivation.

Interaction between titanium dioxide nanoparticles and human serum albumin revealed by fluorescence spectroscopy in the absence of photoactivation

Titanium dioxide (TiO 2) nanoparticles (NPs) are widely used as an important kind of biomaterials. The interaction between TiO 2 (P25) at 20 nm in diameter and human serum albumin (HSA) was studied by fluorescence spectroscopy in this work. Under the simulative physiological conditions, fluorescence data revealed the presence of a single class of binding site on HSA and its binding constants ( K a ) were 2.18±0.04×10 4, 0.87±0.05×10 4, 0.68±0.06×10 4 M −1 at 298, 304 and 310 K, respectively. In addition, according to the Van’t Hoff equation, the thermodynamic functions standard enthalpy (Δ H 0) and standard entropy (Δ S 0) for the reaction were calculated to be −75.18±0.15 kJ mol −1 and −170.11±0.38 J mol −1 K −1. These results indicated that TiO 2 NPs bond to HSA mainly by van der Waals force and hydrogen bonding formation in low dielectric media, and the electrostatic interactions cannot be excluded. Furthermore, the effects of common ions on the binding constant of TiO 2 NPs–HSA complex were discussed.

Ultrafine titanium dioxide particles in the absence of photoactivation can induce oxidative damage to human bronchial epithelial cells

Ultrafine titanium dioxide (TiO 2) particles have been shown to exhibit strong cytotoxicity when exposed to UVA radiation, but are regarded as a biocompatible material in the absence of photoactivation. In contrast to this concept, the present results indicate that anatase-sized (10 and 20 nm) TiO 2 particles in the absence of photoactivation induced oxidative DNA damage, lipid peroxidation, and micronuclei formation, and increased hydrogen peroxide and nitric oxide production in BEAS-2B cells, a human bronchial epithelial cell line. However, the treatment with anatase-sized (200 and >200 nm) particles did not induce oxidative stress in the absence of light irradiation; it seems that the smaller the particle, the easier it is for the particle to induce oxidative damage. The photocatalytic activity of the anatase form of TiO 2 was reported to be higher than that of the rutile form. In contrast to this notion, the present results indicate that rutile-sized 200 nm particles induced hydrogen peroxide and oxidative DNA damage in the absence of light but the anatase-sized 200 nm particles did not. In total darkness, a slightly higher level of oxidative DNA damage was also detected with treatment using an anatase–rutile mixture than with treatment using either the anatase or rutile forms alone. These results suggest that intratracheal instillation of ultrafine TiO 2 particles may cause an inflammatory response.

Polymerization contraction stress in dual-cure cements and its effect on interfacial integrity of bonded inlays

Objectives. To compare the polymerization contraction stress of resin cements in dual-cure and self-cure modes, and verify its effect on microleakage and gap formation in class I cavities restored with porcelain inlays. Methods. Dual-cure cements (Calibra, Choice and RelyX ARC) were monitored for contraction stress during 30 min in the tensilometer, in the presence or absence of photo-activation. Microleakage was evaluated in porcelain inlays bonded to cavities prepared in bovine incisors, combining the same adhesive (One-Step) with each of the cements in both activation modes. Epoxy replicas of the sectioned specimens were analyzed in the SEM to determine the percentage of discontinuous margins. Results. The stress developed in dual-cure mode was higher than in self-cure mode for all cements. In self-cure mode, no significant difference in stress was found among materials. Choice and RelyX ARC showed higher microleakage in dual-cure mode than in self-cure mode. Calibra did not show a significant difference in microleakage between activation modes. Contraction gap results showed wide variations, and no significant differences were detected among the groups. Gaps occurred more often in dentin, with an overall average of 22±17.5%. Conclusions. The higher stress generated in dual-cure cements subjected to photo-activation may lead to higher marginal leakage. In the confinement condition represented by the class I inlay, even the lower contraction stress of the cements in self-cure mode had enough magnitude to disrupt the bonding to dentin.

A novel method for the study of autophagy: destruction of hepatocytic lysosomes, but not autophagosomes, by the photosensitizing porphyrin tetra(4-sulphonatophenyl)porphine.

A photoactivatable porphyrin, tetra(4-sulphonatophenyl)porphine (TPPS4), was shown to accumulate in rat hepatocytes as a linear function of dose after intravenous injection, and to localize predominantly in hepatocytic lysosomes. A major fraction of the lysosomal enzymes acid phosphatase and N-acetyl-beta-D-glucosaminidase was inactivated by TPPS4 after 20 h of contact with the drug in vivo in the absence of photoactivation. On exposure of isolated hepatocytes to light, photoactivated TPPS4 caused additional inactivation of the lysosomal enzymes as well as inactivation of intralysosomal lactate dehydrogenase (LDH), a cytosolic enzyme that accumulated in lysosomes as a result of autophagy during a 2 h incubation of hepatocytes at 37 degrees C in the dark (in the presence of the proteinase inhibitor leupeptin to prevent degradation of intralysosomal LDH). Photoactivation of TPPS4 also induced lysosomal rupture, with a loss of lysosomal enzymes, autophagocytosed LDH, endocytosed 125I-tyramine-cellobiose-asialo-orosomucoid and TPPS4 from the lysosomes. However, LDH-containing autophagosomes, accumulated in the presence of vinblastine (a microtubule inhibitor used to prevent the fusion of lysosomes with autophagosomes or endosomes), were not affected by TPPS4. TPPS4 may thus be useful as a selective lysosomal (or endosomal) perturbant in the study of autophagic-endocytic-lysosomal interactions.

Evidence for the involvement of a 35-kDa membrane protein in the synthesis of glucosylphosphoryldolichol

Photoactivatable (beta-32P)5-azidoUDPGlc binds to two proteins in rat liver microsomes. As determined by SDS-PAGE electrophoresis, the molecular masses of the 32P-labeled proteins were found to be 62- and 35-kDa. Binding of the photoprobe to both proteins was inhibited by addition of unlabeled UDPGlc. Labeling of the higher molecular weight protein occurred in the absence of photoactivation. In contrast, formation of the 32P-labeled 35-kDa protein was dependent on exposure of the membranes to UV light (250 nm). Moreover, labeling of the 35-kDa protein required the intact sugar nucleotide and divalent cations and was affected by the level of the endogenous and exogenous dolichylphosphate. All of these results are consistent with the possibility that the 35-kDa membrane protein is a component of glucosylphosphoryldolichol synthase.

3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine photolabels a substrate-binding site of rat hepatic cytochrome P-450 form PB-4.

Hepatic microsomes isolated from untreated male rats or from rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (3-MC) were labeled with the hydrophobic, photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). [125I]TID incorporation into 3-MC- and PB-induced liver microsomal protein was enhanced 5- and 8-fold, respectively, relative to the incorporation of [125I]TID into uninduced liver microsomes. The major hepatic microsomal cytochrome P-450 forms inducible by PB and 3-MC, respectively designated P-450s PB-4 and BNF-B, were shown to be the principal polypeptides labeled by [125I]TID in the correspondingly induced microsomes. Trypsin cleavage of [125I]TID-labeled microsomal P-450 PB-4 yielded several radiolabeled fragments, with a single labeled peptide of Mr approximately 4000 resistant to extensive proteolytic digestion. The following experiments suggested that TID binds to the substrate-binding site of P-450 PB-4. [125I]TID incorporation into microsomal P-450 PB-4 was inhibited in a dose-dependent manner by the P-450 PB-4 substrate benzphetamine. In the absence of photoactivation, TID inhibited competitively about 80% of the cytochrome P-450-dependent 7-ethoxycoumarin O-deethylation catalyzed by PB-induced microsomes with a Ki of 10 microM; TID was a markedly less effective inhibitor of the corresponding activity catalyzed by microsomes isolated from uninduced or beta-naphthoflavone-induced livers.(ABSTRACT TRUNCATED AT 250 WORDS)

Photoaffinity labeling of specific alpha-thrombin binding sites on Chinese hamster lung cells.

Binding sites for alpha-thrombin on cultured Chinese hamster lung cells were identified using photoactivatable cross-linking conjugates of diisopropylphosphorofluoridate-inactivated alpha-thrombin. A series of photoaffinity reagents was synthesized that permitted systematic variation of the extent of thrombin modification and of steric factors affecting the ability of the photoaffinity reagent to contact thrombin receptors. The reagents were synthesized with a tritium label to accurately determine the number of photoaffinity molecules linked to each thrombin. Also, they were synthesized with different length spacer arms between the photoreactive cross-linking group (a nitroarylazide) and the end which linked to alpha-thrombin (a succinimide ester). By calculating the percentage of the thrombin surface that would be accessed by modifying it with a fixed molar excess of each reagent, it was possible to select the photoaffinity reagents that would be most effective for cross-linking 125I-labeled diisopropylphosphorofluoridate-inactivated alpha-thrombin to its cellular binding sites. The validity of this selection procedure was confirmed in experiments in which an Mr = 150,000 cellular component was labeled. This component had the properties of a specific binding site for thrombin since labeling was readily competed for by nonlabeled alpha-thrombin. The cross-linking achieved was due to the photoactivatable reagent since no detectable cross-linked complex was formed in the absence of photoactivation or with 125I-labeled diisopropylphosphorofluoridate-inactivated alpha-thrombin that was not conjugated with the photoaffinity reagent.

Photocatalyzed labeling of adipocyte plasma membranes with an aryl azide derivative of glucose.

The photoreactive glucose derivative, N-(4-azido-2-nitrophenyl)-2-amino-2-deoxy-D-glucose, has been synthesized and used as a site-directed probe to label adipocyte plasma membranes. In the absence of photoactivation, the glucose derivative was shown to inhibit D-glucose and 3-O-methyl-glucose uptake in the isolated adipocyte system. Glucose was also shown to partially inhibit the uptake of the photoreactive probe. Photolysis of this reagent with light of wavelength greater than 300 nm in the presence of intact adipose cells resulted in the covalent incorporation of radioactivity into the plasma membrane fraction. Analysis of this material by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that four membrane components were significantly labeled and that two of these components corresponded in electrophoretic mobility to the major glycoproteins with apparent molecular weights of 100,000 and 81,000. Photolysis of the glucose analog in the presence of the particulate pellet derived from homogenized adipocytes resulted in the incorporation of the radioactive label into most of the membrane proteins. Although the cells appear to be permeable to the photoreactive probe, only a few membrane components are labeled when the cells are intact, suggesting that the probe has a high affinity for these membrane components which may be functionally involved in the glucose transport system.