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.

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.

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.

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.

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.

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.

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.

Release of NO from a nitrosyl ruthenium complex through oxidation of mitochondrial NADH and effects on mitochondria.

Nitrosyl ruthenium complexes are promising NO donor agents with numerous advantages for the biologic applications of NO. We have characterized the NO release from the nitrosyl ruthenium complex [Ru(NO(2))(bpy)(2)(4-pic)](+) (I) and the reactive oxygen/nitrogen species (ROS/RNS)-mediated NO actions on isolated rat liver mitochondria. The results indicated that oxidation of mitochondrial NADH promotes NO release from (I) in a manner mediated by NO(2) formation (at neutral pH) as in mammalian cells, followed by an oxygen atom transfer mechanism (OAT). The NO released from (I) uncoupled mitochondria at low concentrations/incubation times and inhibited the respiratory chain at high concentrations/incubation times. In the presence of ROS generated by mitochondria NO gave rise to peroxynitrite, which, in turn, inhibited the respiratory chain and oxidized membrane protein-thiols to elicit a Ca(2+)-independent mitochondrial permeability transition; this process was only partially inhibited by cyclosporine-A, almost fully inhibited by the thiol reagent N-ethylmaleimide (NEM) and fully inhibited by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). These actions correlated with the release of cytochrome c from isolated mitochondria as detected by Western blotting analysis. These events, typically involved in cell necrosis and/or apoptosis denote a potential specific action of (I) and analogs against tumor cells via mitochondria-mediated processes.

Characterization of the mechanisms of action and nitric oxide species involved in the relaxation induced by the ruthenium complex.

Nitric oxide (NO) plays an important role in the control of vascular tone. NO donors have therapeutic use and the most used NO donors, nitroglycerin and sodium nitroprusside have problems in their use. Thus, new NO donors have been synthesized to minimize these undesirable effects. Nytrosil ruthenium complexes have been studied as a new class of NO donors. trans-[RuCl([15]aneN(4))NO](2+), induces vasorelaxation only in presence of reducing agent. In this study, we characterized the mechanisms of vasorelaxation of trans-[RuCl([15]aneN(4))NO](2+) in denuded rat aorta and identified which NO forms are involved in this relaxation. We also evaluated the effect of this NO donor in decreasing the cytosolic Ca(2+) concentration ([Ca(2+)]c) of the vascular smooth muscle cells. Vasorelaxation to trans-[RuCl([15]aneN(4))NO](2+) (E(max): 101.8 +/- 2.3%, pEC(50): 5.03 +/- 0.15) was almost abolished in the presence of the NO* scavenger hydroxocobalamin (E(max): 4.0 +/- 0.4%; P < 0.001) and it was partially inhibited by the NO(-) scavenger L-cysteine (E(max): 79.9 +/- 6.9%, pEC(50): 4.41 +/- 0.06; P < 0.05). The guanylyl cyclase inhibitor ODQ reduced the E(max) (57.7 +/- 4.0%, P < 0.001) and pEC(50) (4.21 +/- 0.42, P < 0.01) and the combination of ODQ and TEA abolished the response to trans-[RuCl([15]aneN(4))NO](2+). The blockade of voltage-dependent (K(v)), ATP-sensitive (K(ATP)), and Ca(2+)-activated (K(Ca) K(+) channels reduced the vasorelaxation induced by trans-[RuCl([15]aneN(4))NO](2+). This compound significantly reduced [Ca(2+)]c (from 100% to 85.9 +/- 3.5%, n = 4). In conclusion, our data demonstrate that this NO donor induces vascular relaxation involving NO* and NO(-) species, that is associated to a decrease in [Ca(2+)]c. The mechanisms of vasorelaxation involve guanylyl cyclase activation, cGMP production and K(+) channels activation.