A Comparative Evaluation of the Photosensitizing Efficiency of Porphyrins, Chlorins and Isobacteriochlorins toward Melanoma Cancer Cells.

Skin cancer is one of the cancers that registers the highest number of new cases annually. Among all forms of skin cancer, melanoma is the most invasive and deadliest. The resistance of this form of cancer to conventional treatments has led to the employment of alternative/complementary therapeutic approaches. Photodynamic therapy (PDT) appears to be a promising alternative to overcome the resistance of melanoma to conventional therapies. PDT is a non-invasive therapeutic procedure in which highly reactive oxygen species (ROS) are generated upon excitation of a photosensitizer (PS) when subjected to visible light of an adequate wavelength, resulting in the death of cancer cells. In this work, inspired by the efficacy of tetrapyrrolic macrocycles to act as PS against tumor cells, we report the photophysical characterization and biological assays of isobacteriochlorins and their corresponding chlorins and porphyrins against melanoma cancer cells through a photodynamic process. The non-tumoral L929 fibroblast murine cell line was used as the control. The results show that the choice of adequate tetrapyrrolic macrocycle-based PS can be modulated to improve the performance of PDT.

Influence of nitro ruthenium isomerization on photochemically induced nitric oxide release: Vasorelaxant activities.

We have synthesized cis-[Ru(bpy)2(NO2-κN)Ln-](n-1) and cis-[Ru(bpy)2(NO2-κO)L n-](n-1) (bpy = 2,2'-bipyridine; k = indication of the coordinated center to Ruthenium; L = pyridine type ligand) by reacting cis-[Ru(bpy)2(H2O)Ln-](n-2) with sodium nitrite or conducting basic cis-[Ru(bpy)2NO(Ln-)](n-3) hydrolysis. Photolysis at the metal-ligand charge transfer band (MLCT) of the isomers yielded nitric oxide (NO) as determined by NO measurement. The NO photorelease rates obtained upon 447 nm laser irradiation of the ruthenium complexes showed that cis-[Ru(bpy)2(NO2-κO)Ln-](n-1) released NO three times faster than cis-[Ru(bpy)2(NO2-κN)Ln-](n-1). We investigated endothelium-dependent vasodilation induced by cis-[Ru(bpy)2(4-pic)(NO2-κN)]+ and cis-[Ru(bpy)2(4-pic)(NO2-κO)]+ (4-pic = 4-picoline) in isolated 3 mm aortic rings precontracted with L-phenylephrine. Maximum vasodilation was achieved under 447 nm laser irradiation of 0.5 µMol.L-1 ruthenium complexes for 100 s.

New Bis-Cyclometalated Iridium(III) Complexes with ß-Substituted Porphyrin-Arylbipyridine as the Ancillary Ligand: Electrochemical and Photophysical Insights.

An efficient synthetic access to new cationic porphyrin-bipyridine iridium(III) bis-cyclometalated complexes was developed. These porphyrins bearing arylbipyridine moieties at ß-pyrrolic positions coordinated with iridium(III), and the corresponding Zn(II) porphyrin complexes were spectroscopically, electrochemically, and electronically characterized. The features displayed by the new cyclometalated porphyrin-bipyridine iridium(III) complexes, namely photoinduced electron transfer process (PET), and a remarkable efficiency to generate 1O2, allowing us to envisage new challenges and opportunities for their applications in several fields, such as photo(catalysis) and photodynamic therapies.

Thiopyridinium phthalocyanine for improved photodynamic efficiency against pathogenic fungi.

The emergence of opportunistic pathogens and the selection of resistant strains have created a grim scenario for conventional antimicrobials. Consequently, there is an ongoing search for alternative techniques to control these microorganisms. One such technique is antimicrobial photodynamic therapy (aPDT), which combines photosensitizers, light, and molecular oxygen to produce reactive oxygen species and kill the target pathogen. Here, the in vitro susceptibilities of three fungal pathogens, namely Candida albicans, Aspergillus nidulans, and Colletotrichum abscissum to aPDT with zinc(II) phthalocyanine (ZnPc) derivative complexes were investigated. Three ZnPc bearing thiopyridinium substituents were synthesized and characterized by several spectroscopic techniques. The Q-band showed sensitivity to the substituent with high absorptivity coefficient in the 680-720 nm region. Derivatization and position of the rings with thiopyridinium units led to high antifungal efficiency of the cationic phthalocyanines, which could be correlated with singlet oxygen quantum yield, subcellular localization, and cellular uptake. The minimum inhibitory concentrations (MIC) of the investigated ZnPc-R complexes against the studied microorganisms were 2.5 µM (C. albicans) and 5 µM (A. nidulans and C. abscissum). One ZnPc derivative achieved complete photokilling of C. albicans and, furthermore, yielded low MIC values when used against the tolerant plant-pathogen C. abscissum. Our results show that chemical modification is an important step in producing better photosensitizers for aPDT against fungal pathogens.

The Surprisingly Positive Effect of Zinc-Phthalocyanines With High Photodynamic Therapy Efficacy of Melanoma Cancer.

Phthalocyanine (Pc) dyes are photoactive molecules that can absorb and emit light in the visible spectrum, especially in the red region of the spectrum, with great potential for biological scopes. For this target, it is important to guarantee a high Pc solubility, and the use of suitable pyridinium units on their structure can be a good strategy to use effective photosensitizers (PSs) for photodynamic therapy (PDT) against cancer cells. Zn(II) phthalocyanines (ZnPcs) conjugated with thiopyridinium units (1-3) were evaluated as PS drugs against B16F10 melanoma cells, and their photophysical, photochemical, and in vitro photobiological properties were determined. The photodynamic efficiency of the tetra- and octa-cationic ZnPcs 1-3 was studied and compared at 1, 2, 5, 10, and 20 µM. The different number of charge units, and the presence/absence of a-F atoms on the Pc structure, contributes for their PDT efficacy. The 3-(4',5'-dimethylthiazol-2'-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays on B16F10 melanoma cells show a moderate to high capacity to be photoinactivated by ZnPcs 1-3 (ZnPc 1 > ZnPc 2 > ZnPc 3). The best PDT conditions were found at a Pc concentration of 20 µM, under red light (λ = 660 ± 20 nm) at an irradiance of 4.5 mW/cm2 for 667 s (light dose of 3 J/cm2). In these conditions, it is noteworthy that the cationic ZnPc 1 shows a promising photoinactivation ratio, reaching the detection limit of the MTT method. Moreover, these results are comparable to the better ones in the literature.

Non-destructive molecular FTIR spectromicroscopy for real time assessment of redox metallodrugs.

Recent emergence of FTIR spectromicroscopy (micro-FTIR) as a dynamic spectroscopy for imaging to study biological chemistry has opened new possibilities for investigating in situ drug release, redox chemistry effects on biological molecules, DNA and drug interactions, membrane dynamics, and redox reactions with proteins at the single cell level. Micro-FTIR applied to metallodrugs has been playing an important role since the last decade because of its great potential to achieve more robust and controlled pharmacological effects against several diseases, including cancer. An important aspect in the development of these drugs is to understand their cellular properties, such as uptake, accumulation, activity, and toxicity. In this review, we present the potential application of micro-FTIR and its importance for studying metal-based drugs, highlighting the perspectives of chemistry of living cells. We also emphasise bioimaging, which is of high importance to localize the cellular processes, for a proper understanding of the mechanism of action.

Hybrid Nanoparticles as an Efficient Porphyrin Delivery System for Cancer Cells to Enhance Photodynamic Therapy.

Photodynamic therapy (PDT) is a potential non-invasive approach for application in oncological diseases, based on the activation of a photosensitizer (PS) by light at a specific wavelength in the presence of molecular oxygen to produce reactive oxygen species (ROS) that trigger the death tumor cells. In this context, porphyrins are interesting PS because they are robust, have high chemical, photo, thermal, and oxidative stability, and can generate singlet oxygen (1O2). However, porphyrins exhibit low solubility and a strong tendency to aggregate in a biological environment which limits their clinical application. To overcome these challenges, we developed hybrid nanostructures to immobilize 5,10,15,20-tetrakis[(4-carboxyphenyl) thio-2,3,5,6-tetrafluorophenyl] (P), a new third-generation PS. The biological effect of this system was evaluated against bladder cancer (BC) cells with or without light exposition. The nanostructure composed of lipid carriers coated by porphyrin-chitosan (P-HNP), presented a size of ca. 130 nm and low polydispersity (ca. 0.25). The presence of the porphyrin-chitosan (P-chitosan) on lipid nanoparticle surfaces increased the nanoparticle size, changed the zeta potential to positive, decreased the recrystallization index, and increased the thermal stability of nanoparticles. Furthermore, P-chitosan incorporation on nanoparticles increased the stability and enhanced the self-organization of the system and the formation of spherical structures, as observed by small-angle X-ray scattering (SAXS) analysis. Furthermore, the immobilization process maintained the P photoactivity and improved the photophysical properties of PS, minimizing its aggregation in the cell culture medium. In the photoinduction assays, the P-HNP displayed high phototoxicity with IC50 3.2-folds lower than free porphyrin. This higher cytotoxic effect can be correlated to the high cellular uptake of porphyrin immobilized, as observed by confocal images. Moreover, the coated nanoparticles showed mucoadhesive properties interesting to its application in vivo. Therefore, the physical and chemical properties of nanoparticles may be relevant to improve the porphyrin photodynamic activity in BC cells.

Effect of Serum Albumin on Porphyrin-Quantum Dot Complex Formation, Characteristics and Spectroscopic Analysis.

The effect of bovine serum albumin (BSA) upon interaction between CdTe QD functionalized by 3-Mercaptopropionic Acid (CdTe-3-MPA QD) and two water soluble porphyrins: positively charged meso-tetra methyl pyridyl porphyrin (TMPyP) and negatively charged meso-tetrakis(p-sulfonato-phenyl) porphyrin (TPPS4), was studied in function of pH using the steady-state and time resolved optical absorption and fluorescence spectroscopies. It was shown that, depending on the charge state of the components, interaction with albumin could either prevent the formation of the QD…PPh complex, form a mixed QD…PPh…BSA complex or not affect PPh complexation with QD at all. The obtained results may be of interest for application in photomedicine.

Comparison of the Photodynamic Action of Porphyrin, Chlorin, and Isobacteriochlorin Derivatives toward a Melanotic Cell Line.

Melanoma is the most dangerous form of skin cancer, with an abrupt growth of its incidence over the last years. It is extremely resistant to traditional treatments such as chemotherapy and radiotherapy, but therapies for this cancer are gaining attention. Photodynamic therapy (PDT) is considered an effective modality to treat several types of skin cancers and can offer the possibility to treat one of the most aggressive ones: melanoma. In this work, the effect of PDT on a melanotic cell line (B16F10 cells) was assessed by exposing cultured cells to 5,10,15-tris(pentafluorophenyl)-20-(4-pyridyl)porphyrin (PS1) and to its chlorin (PS2) and isobacteriochlorin (PS3) corresponding derivatives and red LED light (λ = 660 ± 20 nm). The PDT effect in the cells' viability was measured using the MTT assay. The cell apoptosis was quantified by flow cytometry, and the subcellular localization of the photosensitizer was determined by fluorescence microscopy. In addition, the ability of PS2 to generate superoxide radicals was qualitatively assessed by tyrosine nitration. The results show that the efficiency of the PDT process is dependent on the structure of the PS and on their ability to produce singlet oxygen. Besides that, the photoactivation efficiency is highly dependent on the cellular sublocalization of the PS and on its cellular uptake and singlet oxygen production. We also found that the resistant cell line B16F10 has distinctive chlorin, isobacteriochlorin, or porphyrin-specific resistance profiles. Furthermore, it is shown that the highly fluorescent chlorin derivative PS2 can also be considered in imaging diagnostics.

The Photosensitizing Efficacy of Micelles Containing a Porphyrinic Photosensitizer and KI against Resistant Melanoma Cells.

Photodynamic therapy (PDT) is a promising alternative to overcome the resistance of melanoma to conventional therapies. Currently applied photosensitizers (PS) are often based on tetrapyrrolic macrocycles like porphyrins. Unfortunately, in some cases the use of this type of derivative is limited due to their poor solubility in the biological environment. Feasible approaches to surpass this drawback are based on lipid formulations. Besides that, and inspired in the efficacy of potassium iodide (KI) for antimicrobial photodynamic therapy (aPDT), the combined effect of singlet oxygen (1 O2 ) with KI was assessed in this work, as an alternative strategy to potentiate the effect of PDT against resistant melanoma cells.

Efficient photodynamic inactivation of Candida albicans by porphyrin and potassium iodide co-encapsulation in micelles.

Photodynamic inactivation of bacterial and fungal pathogens is a promising alternative to the extensive use of conventional single-target antibiotics and antifungal agents. The combination of photosensitizers and adjuvants can improve the photodynamic inactivation efficiency. In this regard, it has been shown that the use of potassium iodide (KI) as adjuvant increases pathogen killing. Following our interest in this topic, we performed the co-encapsulation of a neutral porphyrin photosensitizer (designated as P1) and KI into micelles and tested the obtained nanoformulations against the human pathogenic fungus Candida albicans. The results of this study showed that the micelles containing P1 and KI displayed a better photodynamic performance towards C. albicans than P1 and KI in solution. It is noteworthy that higher concentrations of KI within the micelles resulted in increased killing of C. albicans. Subcellular localization studies by confocal fluorescence microscopy revealed that P1 was localized in the cell cytoplasm, but not in the nuclei or mitochondria. Overall, our results show that a nanoformulation containing a photosensitizer plus an adjuvant is a promising approach for increasing the efficiency of photodynamic treatment. Actually, the use of this strategy allows a considerable decrease in the amount of both photosensitizer and adjuvant required to achieve pathogen killing.

Photodynamic treatment of melanoma cells using aza-dipyrromethenes as photosensitizers.

In this study, we report for the first time the use of four aza-dipyrromethenes (ADPMs) as photosensitizers for cancer PDT. The synthesis and characterization of the ADPMs and their photodynamic action against B16F10 melanoma cells were assessed. ADPM 2 is the best singlet oxygen generator and the most phototoxic (at 2.5 µM) towards B16F10 cells.

Real-time redox monitoring of a nitrosyl ruthenium complex acting as NO-donor agent in a single A549 cancer cell with multiplex Fourier-transform infrared microscopy.

Multiplex Fourier-transform infrared microscopy (µFT-IR) helped to monitor trans-[Ru(NO) (NH3)4 (isn)]3+(I), uptake by A549 lung carcinoma cell, as well as the generation of its product, nitric oxide (NO), inside the cell. Chronoamperometry with NO-sensor and µFT-IR showed that exogenous NADH and the A549 cell induced the NO release redox mechanism. Chemical imaging confirmed that (I) was taken up by the cell, and that its localization coincided with its consumption in the cellular environment within 15 min of exposure. The Ru-NO absorption band in the IR spectrum shifted from 1932 cm-1, when NO was coordinated to Ru as {RuII-NO+}3+, to 1876 cm-1, due the formation of reduced species {RuII-NO0}2+, a precursor of NO release. Futhermore, the µFT-IR spectral profile demonstrated that, as a result of the NO action on the target, NO interacted with nucleic acids, which provided a biochemical response that is detectable in living cells.

The Influence of Some Axial Ligands on Ruthenium-Phthalocyanine Complexes: Chemical, Photochemical, and Photobiological Properties.

This work presents a new procedure to synthesize ruthenium-phthalocyanine complexes and uses diverse spectroscopic techniques to characterize trans-[RuCl(Pc)DMSO] (I) (Pc = phthalocyanine) and trans-[Ru(Pc)(4-ampy)2] (II) (4-ampy = 4-aminopyridine). The triplet excited-state lifetimes of (I) measured by nanosecond transient absorption showed that two processes occurred, one around 15 ns and the other around 3.8 µs. Axial ligands seemed to affect the singlet oxygen quantum yield. Yields of 0.62 and 0.14 were achieved for (I) and (II), respectively. The lower value obtained for (II) probably resulted from secondary reactions of singlet oxygen in the presence of the ruthenium complex. We also investigate how axial ligands in the ruthenium-phthalocyanine complexes affect their photo-bioactivity in B16F10 murine melanoma cells. In the case of (I) at 1 µmol/L, photosensitization with 5.95 J/cm2 provided B16F10 cell viability of 6%, showing that (I) was more active than (II) at the same concentration. Furthermore, (II) was detected intracellularly in B16F10 cell extracts. The behavior of the evaluated ruthenium-phthalocyanine complexes point to the potential use of (I) as a metal-based drug in clinical therapy. Changes in axial ligands can modulate the photosensitizer activity of the ruthenium phthalocyanine complexes.

Copper-phthalocyanine coordination polymer as a reusable catechol oxidase biomimetic catalyst.

The development of selective, efficient, and recoverable/reusable catalysts for oxidation reactions has become one of the main challenges of modern chemistry. Considering our interest in efficient catalytic systems based on porphyrin (Por) and phthalocyanine (Pc) coordination polymers, we report here the synthesis, characterization and catalytic activity of a new Pc coordination polymer (coined hereafter as Cu4CuPcSPy). The new Pc material was obtained in excellent yield, from the reaction of H2PcSPy with an excess amount of copper(ii) acetate. The catalytic efficiency of Cu4CuPcSPy was evaluated in the oxidation of 3,5-di-tert-butylcatechol (3,5-DTBC) in the presence of molecular oxygen from air, with or without the addition of aqueous 30% hydrogen peroxide. The comparison between the homogeneous (CuPcSPy) and the heterogeneous (Cu4CuPcSPy) catalytic systems showed a better performance for the Cu4CuPcSPy material, that can be recovered by filtration and reused, maintaining its activity roughly unchanged during three catalytic cycles. An increment of approximately 30% was also verified in the catalytic activity of the homogeneous and heterogeneous systems by adding aqueous hydrogen peroxide to the catalytic reactions.

Targeting the mitochondrial VDAC in hepatocellular carcinoma using a polyclonal antibody-conjugated to a nitrosyl ruthenium complex.

The rational design of anti-cancer agents includes a new approach based on ruthenium complexes that can act as nitric oxide (NO) donor agents against specific cellular targets. One of the most studied classes of those compounds is based on bis(bipyridine) ruthenium fragment and its derivative species. In this work, we present the chemical and cytotoxicity properties against the liver hepatocellular carcinoma cell line HepG2 of cis-[RuII(NO+)Cl(dcbpy)2]2- conjugated to a polyclonal antibody IgG (anti-VDAC) recognizing a cell surface marker. UV-visible bands of the ruthenium complex were assigned with the aid of density functional theory, which also allowed estimation of the structures that explain the biological effects of the ruthenium complex-IgG conjugate. The interaction of cis-[RuII(NO+)Cl(dcbpy)2]3- with mitochondria was evaluated due to the potential of these organelles as anti-cancer targets, and considering they interact with the anti-VDAC antibody. The cytotoxicity of cis-[RuII(NO+)Cl(dcbpy)2]3--anti-VDAC antibody was up to 80% greater in comparison to the free cis-[RuII(NO+)Cl(dcbpy)2]3- complex. We suggest that this effect is due to site-specific interaction of the complex followed by NO release.

Endothelial modulation of a nitric oxide donor complex-induced relaxation in normotensive and spontaneously hypertensive rats.

We hypothesized that endothelium modulates relaxation induced by a nitric oxide (NO) donor ruthenium complex (TERPY, [Ru(terpy)(bdq)NO]3+) in mesenteric arteries of normotensive and spontaneously hypertensive (SHR) rats in different ways. We analyzed the mechanism involved in TERPY-induced relaxation in the second and third branches of mesenteric arteries and investigated how endothelium contributes to the TERPY vasodilator effect on SHR blood vessels. TERPY induced concentration-dependent relaxation in endothelium-denuded (E-) and endothelium-intact (E+) mesenteric arteries of normotensive rats and SHR. Pretreatment with ODQ (which inhibits soluble guanylyl cyclase) or TEA (tetraethylammonium, which blocks potassium channels) significantly reduced the TERPY vasodilator effect on E- mesenteric arteries of normotensive rats and SHR. The presence of endothelium shifted the concentration-effect curves for TERPY in E+ mesenteric arteries of normotensive rats to the right. Conversely, the presence of endothelium shifted the concentration-effect curves for TERPY in the case of SHR E+ mesenteric arteries to the left, which suggested increased potency. L-NNA, a more selective endothelial NO synthase (eNOS) inhibitor, reduced TERPY potency in SHR. The presence of endothelium and notably of NOS contributed to the TERPY vasodilator action in SHR: TERPY promoted eNOS Ser1177 phosphorylation with consequent NO production and increased soluble guanylyl cyclase activity, which may have directly activated potassium channels.

Nitric oxide donor [Ru(terpy)(bdq)NO]3+ induces uncoupling and phosphorylation of endothelial nitric oxide synthase promoting oxidant production.

[Ru(terpy)(bdq)NO]3+ (TERPY) is a nitric oxide (NO) donor that promotes relaxation of the mesenteric artery and aorta in rats. We sought to investigate whether it acts as both an NO donor and endothelial NO synthase (eNOS) activator, as shown previously for nitroglycerin. Human umbilical vein endothelial cells (HUVECs) and human embryonic kidney 293 cells transfected with empty vector (HEK) or eNOS cDNA (HEK-eNOS) were treated with TERPY (1µM) for different lengths of time. eNOS expression, dimerization, and Ser1177 phosphorylation, caveolin-1 (Cav-1) oligomerization, Cav-1 Tyr14 phosphorylation were evaluated by Western blotting. Studies also assessed the production of reactive oxygen/nitrogen species (ROS/RNS) in HUVECs and HEK-eNOS cells. In HEK cells devoid of eNOS, TERPY released NO without additional stimulus indicating that is an NO donor. Moreover, in HEK-eNOS cells, TERPY-induced NO production that was blocked by L-NAME. In addition, TERPY increased ROS and ONOO- production which were blocked by more than 80% by BH4 (essential eNOS co-factor) and eNOS siRNA. These results suggest that TERPY-induced ROS and ONOO- production were originated from eNOS. HUVECs stimulated with TERPY showed increased eNOS Ser1177 and Cav-1 Tyr14 phosphorylation, and decreased eNOS dimerization, Cav-1 oligomerization, and Cav-1/eNOS interaction after 20min. It suggests that TERPY induces eNOS hyperactivation and uncoupling by disrupting Cav-1/eNOS interaction and depleting BH4. Endothelium-dependent vasodilation in response to NO donor TERPY is associated with eNOS activation and uncoupling, and thereby appears to be mediated, at least in part, via eNOS-dependent ROS/RNS production.

The nitric oxide donor RuBPY does not induce in vitro cross-tolerance with acetylcholine.

PURPOSE: We have demonstrated that RuBPY induces hypotensive effect in hypertensive rats, promotes vasodilation at low concentrations, and presents low cytotoxicity. This study aimed to verify whether the NO donor RuBPY synthesized in our laboratory induces in vitro tolerance and cross-tolerance to acetylcholine (ACh) and sodium nitroprusside (SNP) in rat cava vein. METHODS: We compared the maximum relaxing effect (ME) and potency (pD2) of RuBPY and nitroglycerin (GTN) in cava vein rings. Exposure to RuBPY or GTN induced in vitro tolerance. Western Blotting helped to evaluate phosphorylation of endothelial nitric oxide synthase (NOS3/eNOS) at the Ser1177 activation site and at the Thr495 inactivation site and to determine the ratio between active eNOS dimers and inactive eNOS monomers. The NO and ROS ratio was assessed by flow citometry. RESULTS: RuBPY did not induce cross-tolerance with ACh, and this NO donor took longer to induce tolerance than GTN. Only GTN elicited phosphorylation of eNOS at Ser1177 and Thr495. In contrast to results obtained with pre-exposure to GTN, pre-exposure to RuBPY did not reduce the formation of NO. The O2- ratio increased in cells incubated with GTN. CONCLUSIONS: A major contribution of this work has been to evaluate the phenomenon of tolerance induced by GTN and by the new ruthenium complex RuBPY in a venous bed. RuBPY is more advantageous than GTN: RuBPY takes longer to induce tolerance, does not induce endothelial dysfunction or cross-tolerance to ACh, and generates lower amount of ROS.

Light induced cytotoxicity of nitrofurantoin toward murine melanoma.

The cytotoxicity of nitrofurantoin (NFT) in the dark and after light exposure (UVA irradiation, λ = 385 nm) was evaluated in murine melanoma B16F10 cells. NFT induces both cell proliferation and inhibition of cell viability. The dominance of one or the other effect depends on the drug concentration, incubation time (tinc) and irradiation dose. The uptake of NFT in these cells, as well as its photocytotoxicity, reaches saturation after 24 hours of incubation. The mechanism of cell death in the dark is associated with the enzymatic release of nitric oxide (NO). The increase of NFT cytotoxicity under light irradiation is associated with the increase of NO concentration due to photorelease. NO photorelease by NFT in solution was confirmed by chemiluminescence, while NO formation in cells was confirmed by fluorescence microscopy using DAF-2DA, a specific indicator of NO in living cells. The NFT does not enter nuclei, distributing preferentially in the cell cytoplasm, as shown by fluorescence microscopy.