Physico-chemical properties of meso-tetrakis(p-methoxyphenyl)porphyrin (TMPP) incorporated into pluronicTM p-123 and f-127 polymeric micelles

The physicochemical properties (solubilization, structural organization and stability) of meso-tetrakis(p-methoxyphenyl)porphyrin (TMPP), a promising photosensitizer for photodynamic therapy, solubilized in polymeric micelles of tri-block copolymers PluronicTM P-123 and F-127, were studied. The formulations obtained by the solid dispersion method led to monomerization of TMPP in these copolymers. Solubility studies showed that P-123 solubilizes double the photosensitizer than F-127. The self-aggregation phenomenon was affected by the [TMPP]/[poloxamer] ratio and medium temperature. The decrease in the temperature of these systems promoted the formation of different kinds of TMPP aggregates intrinsically connected with the structural changes occurring in the micelles.

Nitric oxide release from the S-nitrosothiol zinc phthalocyanine complex by flash photolysis

The photogeneration of nitric oxide (NO) using laser flash photolysis was investigated for S-nitroso-glutathione (GSNO) and S-nitroso-N-acetylcysteine (NacySNO) at pH 6.4 (PBS/HCl) and 7.4 (PBS). Irradiation of S-nitrosothiol with light (lambda = 355 nm followed by absorption spectroscopy) resulted in the homolytic decomposition of NacySNO and GSNO to generate radicals (GS· and NacyS·) and NO. The release of NO from donor compounds measured with an ISO-Nometer apparatus was larger at pH 7.4 than pH 6.4. NacySNO was also incorporated into dipalmitoyl-phosphatidylcholine liposomes in the presence and absence of zinc phthalocyanine (ZnPC), a well-known photosensitizer useful for photodynamic therapy. Liposomes are usually used as carriers for hydrophobic compounds such as ZnPC. Inclusion of ZnPC resulted in a decrease in NO liberation in liposomal medium. However, there was a synergistic action of both photosensitizers and S-nitrosothiols resulting in the formation of other reactive species such as peroxynitrite, which is a potent oxidizing agent. These data show that NO release depends on pH and the medium, as well as on the laser energy applied to the system. Changes in the absorption spectrum were monitored as a function of light exposure.

Photocytotoxicity of a 5-nitrofuran-ethenyl-quinoline antiseptic (Quinifuryl) to P388 mouse leukemia cells

Quinifuryl (MW 449.52), 2-(5'-nitro-2'-furanyl)ethenyl-4-{N-[4'-(N,N-diethylamino)-1'-methylbutyl]carbamoyl} quinoline, is a water soluble representative of a family of 5-nitrofuran-ethenyl-quinoline drugs which has been shown to be highly toxic to various lines of transformed cells in the dark. In the present study, the toxicity of Quinifuryl to P388 mouse leukemia cells was compared in the dark and under illumination with visible light (390-500 nm). Illumination of water solutions of Quinifuryl (at concentrations ranging from 0.09 to 9.0 µg/ml) in the presence of P388 cells resulted in its photodecomposition and was accompanied by elevated cytotoxicity. A significant capacity to kill P388 cells was detected at a drug concentration as low as 0.09 µg/ml. The toxic effect detected at this drug concentration under illumination exceeded the effect observed in the dark by more than three times. Moreover, the general toxic effect of Quinifuryl, which included cell proliferation arrest, was nearly 100%. Both dose- and time-dependent toxic effects were measured under illumination. The LC50 value of Quinifuryl during incubation with P388 cells was ~0.45 µg/ml under illumination for 60 min and >12 µg/ml in the dark. We have demonstrated that the final products of the Quinifuryl photolysis are not toxic, which means that the short-lived intermediates of Quinifuryl photodecomposition are responsible for the phototoxicity of this compound. The data obtained in the present study are the first to indicate photocytotoxicity of a nitroheterocyclic compound and demonstrate the possibility of its application as a photosensitizer drug for photochemotherapy.

Effect of photodynamic therapy on the extracellular matrix and associated components

In many countries, photodynamic therapy (PDT) has been recognized as a standard treatment for malignant conditions (for example, esophageal and lung cancers) and non-malignant ones such as age-related macular degeneration and actinic keratoses. The administration of a non-toxic photosensitizer, its selective retention in highly proliferating cells and the later activation of this molecule by light to form reactive oxygen species that cause cell death is the principle of PDT. Three important mechanisms are responsible for the PDT effectiveness: a) direct tumor cell kill; b) damage of the tumor vasculature; c) post-treatment immunological response associated with the leukocyte stimulation and release of many inflammatory mediators like cytokines, growth factors, components of the complement system, acute phase proteins, and other immunoregulators. Due to the potential applications of this therapy, many studies have been reported regarding the effect of the treatment on cell survival/death, cell proliferation, matrix assembly, proteases and inhibitors, among others. Studies have demonstrated that PDT alters the extracellular matrix profoundly. For example, PDT induces collagen matrix changes, including cross-linking. The extracellular matrix is vital for tissue organization in multicellular organisms. In cooperation with growth factors and cytokines, it provides cells with key signals in a variety of physiological and pathological processes, for example, adhesion/migration and cell proliferation/differentiation/death. Thus, the focus of the present paper is related to the effects of PDT observed on the extracellular matrix and on the molecules associated with it, such as, adhesion molecules, matrix metalloproteinases, growth factors, and immunological mediators.

The inhibitory effect of photodynamic therapy and of an anti-VCAM-1 monoclonal antibody on the in vivo growth of C6 glioma xenografts

We investigated the effect of photodynamic therapy (PDT) and of an anti-vascular cell adhesion molecule-1 (VCAM-1) monoclonal antibody on the in vivo growth of C6 glioma. Seven days after inoculation with C6 cells, adult male Wistar rats weighing 280-300 g with MRI-confirmed glioma were randomly assigned to 4 groups (N = 15 per group): PDT + VCAM-1 antibody group; PDT group; VCAM-1 antibody group; control group. Eight days after inoculation, hematoporphyrin monomethyl ether (HMME) was administered as a photosensitizer and PDT was performed at 630 nm (illumination intensity: 360 J/cm²) for 10 min. VCAM-1 antibody (50 µg/mL) was then administered (0.5 mL) through the tail vein every other day from day 8 to day 16. At day 21, 5 rats in each group were sacrificed and cancers were harvested for immunohistochemistry and Western blot assay for the detection of VCAM-1, and TUNEL assay was used to detect apoptosis. Survival and tumor volume were recorded in the remaining 10 rats in each group. In the PDT group, tumor growth was significantly suppressed (67.2%) and survival prolonged (89.3%), accompanied by an increase in apoptosis (369.5%), when compared to control. Furthermore, these changes were more pronounced in the PDT + VCAM-1 antibody group. After PDT, VCAM-1 expression was markedly increased (121.8%) and after VCAM-1 monoclonal antibody treatment, VCAM-1 expression was significantly reduced (58.2%). PDT in combination with VCAM-1 antibody can significantly inhibit the growth of C6 glioma and prolong survival. This approach may represent a promising strategy in the treatment of glioma.

Liposomal photosensitizers: potential platforms for anticancer photodynamic therapy

Photodynamic therapy is a well-established and clinically approved treatment for several types of cancer. Antineoplastic photodynamic therapy is based on photosensitizers, i.e., drugs that absorb photons translating light energy into a chemical potential that damages tumor tissues. Despite the encouraging clinical results with the approved photosensitizers available today, the prolonged skin phototoxicity, poor selectivity for diseased tissues, hydrophobic nature, and extended retention in the host organism shown by these drugs have stimulated researchers to develop new formulations for photodynamic therapy. In this context, due to their amphiphilic characteristic (compatibility with both hydrophobic and hydrophilic substances), liposomes have proven to be suitable carriers for photosensitizers, improving the photophysical properties of the photosensitizers. Moreover, as nanostructured drug delivery systems, liposomes improve the efficiency and safety of antineoplastic photodynamic therapy, mainly by the classical phenomenon of extended permeation and retention. Therefore, the association of photosensitizers with liposomes has been extensively studied. In this review, both current knowledge and future perspectives on liposomal carriers for antineoplastic photodynamic therapy are critically discussed.

Visible-light-mediated synthesis of helicenes in batch and continuous flow systems

Le présent mémoire décrit le développement d’une méthode de synthèse des hélicènes catalysée par la lumière visible. Les conditions pour la formation de [5]hélicène ont été établies par une optimisation du photocatalyseur, du solvant, du système d’oxydation et du temps réactionnel. Suite aux études mécanistiques préliminaires, un mécanisme oxydatif est proposé. Les conditions optimisées ont été appliquées à la synthèse de [6]hélicènes pour laquelle la régiosélectivité a été améliorée en ajoutant des substituants sur la colonne hélicale. La synthèse de thiohélicènes a aussi été testée en utilisant les mêmes conditions sous irradiation par la lumière visible. La méthode a été inefficace pour la formation de benzodithiophènes et de naphtothiophènes, par contre elle permet la formation du phenanthro[3,4-b]thiophène avec un rendement acceptable. En prolongeant la surface-π de la colonne hélicale, le pyrène a été fusionné aux motifs de [4]- et [5]hélicène. Trois dérivés de pyrène-hélicène ont été synthétisés en utilisant les conditions optimisées pour la photocyclisation et leurs caractéristiques physiques ont été étudiées. La méthode de cyclisation sous l’action de la lumière visible a aussi été étudiée en flux continu. Une optimisation du montage expérimental ainsi que de la source lumineuse a été effectuée et les meilleures conditions ont été appliquées à la formation de [5]hélicène et des trois dérivés du pyrène-hélicène. Une amélioration ou conservation des rendements a été observée pour la plupart des produits formés en flux continu comparativement à la synthèse en batch. La concentration de la réaction a aussi été conservée et le temps réactionnel a été réduit par un facteur de dix toujours en comparaison avec la synthèse en batch.

Monitoring one-electron photo-oxidation of guanine in DNA crystals using ultrafast infrared spectroscopy

To understand the molecular origins of diseases caused by ultraviolet and visible light, and also to develop photodynamic therapy, it is important to resolve the mechanism of photoinduced DNA damage. Damage to DNA bound to a photosensitizer molecule frequently proceeds by one-electron photo-oxidation of guanine, but the precise dynamics of this process are sensitive to the location and the orientation of the photosensitizer, which are very difficult to define in solution. To overcome this, ultrafast time-resolved infrared (TRIR) spectroscopy was performed on photoexcited ruthenium polypyridyl–DNA crystals, the atomic structure of which was determined by X-ray crystallography. By combining the X-ray and TRIR data we are able to define both the geometry of the reaction site and the rates of individual steps in a reversible photoinduced electron-transfer process. This allows us to propose an individual guanine as the reaction site and, intriguingly, reveals that the dynamics in the crystal state are quite similar to those observed in the solvent medium.

Antimicrobial Photodynamic Therapy in the Non-Surgical Treatment of Aggressive Periodontitis: Cytokine Profile in Gingival Crevicular Fluid, Preliminary Results

Background: Aggressive periodontitis is a specific form of periodontal disease that is characterized by rapid attachment loss and bone destruction. Cytokine profiles are of considerable value when studying disease course during treatment. The aim of this trial was to investigate cytokine levels in the gingival crevicular fluid (GCF) of patients with aggressive periodontitis, after treatment with photodynamic therapy (PDT) or scaling and root planing (SRP), in a split-mouth design on -7, 0, +1, +7, +30, and +90 days. Methods: Ten patients were randomly treated with PDT using a laser source associated with a photosensitizer or SRP with hand instruments. GCF samples were collected, and the concentrations of tumor necrosis factor-alpha (TNF-alpha) and receptor activator of nuclear factor-kappa B ligand (RANKL) were determined by enzyme-linked immunosorbent assays. The data were analyzed using generalized estimating equations to test the associations among treatments, evaluated parameters, and experimental times (alpha = 0.05). Results: Non-surgical periodontal treatment with PDT or SRP led to statistically significant reductions in TNF-alpha level 30 days following treatment. There were similar levels of TNF-alpha and RANKL at the different time points in both groups, with no statistically significant differences. Conclusion: SRP and PDT had similar effects on crevicular TNF-alpha and RANKL levels in patients with aggressive periodontitis. J Periodontol 2009;80:98-105.

Influence of pH on the phototransformation process of PhotogemA (R)

PhotogemA (R) is a hematoporphyrin derivative that has been used as a photosensitizer in experimental and clinical Photodynamic Therapy (PDT) in Brazil. Photosensitizers are degraded under illumination. This process, usually called photobleaching, can be monitored by decreasing in fluorescence intensities and includes the following photoprocesses: photodegradation, phototransformation, and photorelocalization. Photobleaching of hematoporphyrin-type sensitizers during illumination in aqueous solution is related not only to photodegradation but is also followed by the formation of photoproducts with a new fluorescence band at around 640-650 nm and with increased light absorption in the red spectral region at 640 nm. In this study, the influence of pH on the phototransformation process was investigated. PhotogemA (R) solutions, 40 mu g/ml, were irradiated at 514 nm with intensity of 100 mW/cm(2) for 20 min with different pH environments. The controls were performed with the samples in the absence of light. The PhotogemA (R) photodegradation is dependent on the pH. The behavior of photodegradation and photoproducts formation (monitored at 640 nm) is distinct and depends on the photosensitizer concentration. The processes of degradation and photoproducts formation were monitored with Photogemin the concentration of 40 mu g/mL since that demonstrated the best visualization of both processes. While below pH 5 the photodegradation occurred, there was no detectable presence of photoproducts. The increase of pH led to increase of photoproducts formation rate with photodegradation reaching the highest value at pH 10. The increase of photoproducts formation and instability of PhotogemA (R) from pH 6 to pH 10 are in agreement with the desired properties of an ideal photosensitizer since there are significant differences in pH between normal (7.0 < pH < 8.6) and tumor (5.8 < pH < 7.9) tissues. It is important to know the effect of pH in the process of phototransformation (degradation and photoproduct formation) of the molecule since low pH values promotes increase in the proportion of aggregates species in solution and high pH values promotes increase in the proportion of monomeric species. There must be an ideal pH interval which favors the phototransformation process that is correlated with the singlet oxygen formation responsible by the photodynamic effect. These differences in pH between normal and tumor cells can explain the presence of photosensitizers in target tumor cells, making PDT a selective therapy.

Hematoporphyrin-based photodynamic therapy for cutaneous squamous cell carcinoma in cats

Photodynamic therapy (PDT) using a haematoporphyrin derivative (Photogem (R), General Physics Institute and clustes Ltda) as photosensitizer and light emitting diodes (LEDs) as the light source was evaluated in 12 cats with cutaneous squamous cell carcinoma. Lesions were illuminated with LEDs, (300 J/cm for 30 min) 24 h after the administration of the photosensitizer. Clinical responses were classified as complete disappearance of the tumour with total re-epithelialization; partial response (a reduction greater than 50%); and no response (less than 50% reduction). Tumours localized to the pinna treated with one (n = 3) or two (n = 4) applications of PDT yielded no response. Highly invasive tumours of the nose and nasal planum also showed no response, after two treatments (n = 2). A combination of PDT and surgery was performed in three cases. Two cats showed partial response and one complete response with one application of therapy 30 days after nasal surgery. Small and noninfiltrative lesions (n = 3) of the nasal planum showed a PR with one application (n = 2) and a CR with two applications (n = 1). This study shows that PDT using Photogem (R) and LEDs can provide local control of low-grade feline squamous cell carcinoma. The addition of PDT to surgery in more invasive cases may help prevent recurrence.

Phototransformation of hematoporphyrin in aqueous solution: Anomalous behavior at low oxygen concentration

The photoactivation of a photosensitizer is the initial step in photodynamic therapy (PDT) where photochemical reactions result in the production of reactive oxygen species and eventually cell death. In addition to oxidizing biomolecules, some of these photochemical reactions lead to photosensitizer degradation at a rate dependent on the oxygen concentration among other factors. We investigated photodegradation of Photogem A (R) (28 mu M), a hematoporphyrin derivative, at different oxygen concentrations (9.4 to 625.0 mu M) in aqueous solution. The degradation was monitored by fluorescence spectroscopy. The degradation rate (M/s) increases as the oxygen concentration increases when the molar ratio of oxygen to PhotogemA (R) is greater than 1. At lower oxygen concentrations (< 25 mu M) an inversion of this behavior was observed. The data do not fit a simple kinetic model of first-order dependence on oxygen concentration. This inversion of the degradation rate at low oxygen concentration has not previously been demonstrated and highlights the relationship between photosensitizer and oxygen concentrations in determining the photobleaching mechanism(s). The findings demonstrate that current models for photobleaching are insufficient to explain completely the effects at low oxygen concentration.

Analysis of the combined effect of lasers of different wavelengths for PDT outcome using 600, 630, and 660 nm

We investigated the effects of photodynamic therapy (PDT) outcome when combining three laser systems that produce light in three different wavelengths (600, 630, and 660 nm). Cooperative as well as independent effects can be observed. We compared the results of the combined wavelengths of light with the effect of single laser for the excitation of the photosensitizer. In the current experiment, the used photosensitizer was Photogem (R) (1.5 mg/kg). Combining two wavelengths for PDT, their cumulative dose and different penetrability may change the overall effect of the fluence of light, which can be effective for increasing the depth of necrosis. This evaluation was performed by comparing the depth and specific aspect of necrosis obtained by using single and dual wavelengths for irradiation of healthy liver of male Wistar rats. We used 15 animals and divided them in five groups of three animals. First, Photogem (R) was administered; follow by measurement of the fluorescence spectrum of the liver before PDT to confirm the level of accumulation of photosensitizer in the tissue. After that, an area of 1 cm(2) of the liver was illuminated using different laser combinations. Qualitative analysis of the necrosis was carried out through histological and morphological study. [GRAPHICS] (a) - microscopic images of rat liver cells, (b) - superficial necrosis caused by PDT using dual-wavelength illumination, (c) - neutrophilic infiltration around the vessel inside the necrosis, and (d) - neutrophilic infiltration around the vessel between necrosis and live tissue (C) 2011 by Astro Ltd. Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA

Photostability of different chlorine photosensitizers

In this paper, we report the photodegradation of three different chlorine photosensitizers (Photoditazine (R), Radachlorin (R), and Foscan (R)). The photosensitizer degradation was analyzed by changes in the fluorescence spectrum during illumination. The rate of fluorescence variation was normalized to the solution absorption and the photon energy resulting in the determination of the necessary number of photons to be absorbed to induce photosensitizer photodegradation. The parameter for rate of the molecules decay, the photon fluence rate and optical properties of the solution allow us to determine the photosensitizer stability in solution during illumination. The results show that the order of susceptibility for photodegradation rate is: Radachlorin (R) < Photoditazine (R) < Foscan (R). This difference in the photodegradation rate for Foscan can be explained by the high proportion of aggregates in solution that inhibit the photo-oxidative process that impede the singlet oxygen formation. We hypothesize that there is a correlation between photodegradation rate and photodynamic efficacy witch is governed by the singlet oxygen formation responsible for the most relevant reaction of the cell death photodynamic induction. Then its is important to know the photostability of different types of drugs since the photodegradation rate, the photodegradation as well as the photodynamic efficacy are strong correlated to the oxygen concentration in the tissue.