Photoinactivation of Pseudomonas syringae pv. actinidiae in kiwifruit plants by cationic porphyrins.

MAIN CONCLUSION: The studied cationic porphyrins formulation allows an effective photoinactivation of Pseudomonas syringae pv. actinidiae in kiwifruit leaves under sunlight irradiation, without damaging the plant. Pseudomonas syringae pv. actinidiae (Psa) is a Gram-negative phytopathogenic bacterium responsible for canker on kiwifruit plant. Over the last decade, this bacterium dramatically affected the production of this fruit worldwide, causing significant economic losses. In general, Psa control consists in the application of copper which are toxic and persist in the environment. The application of antimicrobial photodynamic therapy (aPDT) as an alternative to inactivate Psa has already been demonstrated in recent studies that showed a 4 log Psa reduction using the cationic porphyrin Tetra-Py+-Me as photosensitizer (PS) and 3 consecutive cycles of treatment with a light irradiance of 150 mW cm-2. The present work aimed to evaluate the photodynamic efficiency of a new formulation constituted with five cationic porphyrins as PS in Psa inactivation. This new formulation was prepared to have as main component the tri-cationic porphyrin which is considered one of the most efficient photosensitizers in the photoinactivation of microorganisms. The in vitro study with a PS concentration of 5.0 µM and low irradiance, showed a 7.4 log photoinactivation after 60 min. Posteriorly, several assays were performed with the PS at 50 µM on kiwifruit leaves (ex vivo), under different conditions of light and inoculation. The ex vivo assays with artificially contaminated leaves showed a 2.8 and 4.5 log inactivation with low irradiance and sunlight, respectively, after 90 min. After a second treatment with sunlight, a 6.2 log inactivation was achieved. The photoinactivation on naturally contaminated leaves was about 2.3 log after 90 min sunlight irradiation. Ten consecutive cycles of phototreatment in sub-lethal conditions showed that Psa does not develop resistance, nor recover viability. The results suggest that aPDT can be an alternative to the current methods used to control Psa, since it was possible to inactivate this bacterium under sunlight, without damaging the leaves.

An insight on the role of photosensitizer nanocarriers for Photodynamic Therapy.

Photodynamic therapy (PDT) is a modality of cancer treatment in which tumor cells are destroyed by reactive oxygen species (ROS) produced by photosensitizers following its activation with visible or near infrared light. The PDT success is dependent on different factors namely on the efficiency of the photosensitizer deliver and targeting ability. In this review a special attention will be given to the role of some drug delivery systems to improve the efficiency of tetrapyrrolic photosensitizers to this type of treatment.

Photodynamic inactivation of bioluminescent Escherichia coli by neutral and cationic pyrrolidine-fused chlorins and isobacteriochlorins.

Photodynamic inactivation of bioluminescent Escherichia coli in the presence of cationic chlorin and isobacteriochlorin photosensitizers (PSs) obtained from 5,10,15,20-tetrakis(pentafluorophenyl)-porphyrin is described. The spectroscopic data for the neutral and cationic derivatives and their photophysical characterizations, especially fluorescence and singlet oxygen generation capacity are also reported. The results show that there is a direct relation between the inactivation efficiency and the increasing number of charges on the molecules. The combined effect of higher wavelength absorption and number of positive charges on the PS shows a 6.1 log reduction during the inactivation process. Overall this study shows that the cationic isobacteriochlorin has high potential to be used as PS for the inactivation of Gram (-) bacteria.

Photodynamic therapy of prostate cancer using porphyrinic formulations.

Prostate cancer (PCa) is the second most frequent cancer diagnosed in men worldwide. Among the common treatment options, photodynamic therapy (PDT) is being considered a promising local therapy to treat this cancer. Although PDT is an established treatment modality approved for several types of cancer, the low solubility, the reduced tumor selectivity, the absorption in the therapeutic window and the poor clearance from the body of the currently approved photosensitizers (PS) hampers its wide clinical application. In this regard, herein we synthesized three fluorinated porphyrinoid derivatives and entrapped them into polyvinylpyrrolidone (PVP) to prevent their aggregation and preserve their desirable photophysical properties under the physiological environment. In vitro studies revealed the negligible dark cytotoxicity of all PVP formulations (PS1@PVP, PS2@PVP and PS3@PVP) at the tested concentrations (5.0 to 20 µM), but also confirmed the significant photodynamic effect of PS2@PVP and PS3@PVP towards the PCa cell line PC-3, upon red light irradiation at an irradiance of 17.6 mW.cm-2. To provide insight into the underlying mechanisms of cell death under PDT treatment induced by PS2@PVP and PS3@PVP, their intracellular localization in PC-3 cells was firstly investigated by confocal microscopy. Since both PS2@PVP and PS3@PVP nanoparticles were mainly localized in mitochondria, the involvement of this organelle in PDT-induced apoptosis mediated by both formulations was further explored. Western blot analysis revealed that PDT treatment of PC-3 cells with either PS2@PVP or PS3@PVP resulted in the reduction of the expression level of the anti-apoptotic protein Bcl-2. As the photodamage to Bcl-2 after PDT with PS2@PVP and PS3@PVP was accompanied by the further activation of pro-caspase-3, we assumed that upon irradiation the photogenerated reactive oxygen species (ROS) were able to activate a caspase-dependent apoptotic response as a consequence of a post-mitochondrial event. Taken together, these findings demonstrate that among the tested fluorinated porphyrinoids, PS2@PVP and, particularly, PS3@PVP, are significantly more effective in overall PC-3 cell killing than PS1@PVP, thus highlighting their great potential as therapeutic agents for PCa.

Photodynamic inactivation of methicillin-resistant Staphylococcus aureus on skin using a porphyrinic formulation.

Staphylococcus aureus is responsible for skin and soft tissue infections. Having in mind increased antibiotic resistance, in this study the efficacy of antimicrobial photodynamic therapy (aPDT) with a porphyrinic formulation (FORM) as photosensitizer (PS) to photoinactivate methicillin-resistant Staphylococcus aureus (MRSA) on skin was evaluated. Potassium iodide (KI) and iodopovidone (PVP-I) were also tested in combination with FORM as potentiator agents of FORM efficacy. The aPDT protocol was first developed in Phosphate Buffered Saline (PBS, in vitro). Porcine skin was artificially contaminated with MRSA (ex vivo) and treated with FORM, FORM + KI or FORM + PVP-I under white light. The in vitro results showed that FORM was effective to inactivate MRSA. A substantial reduction in the irradiation time, when compared to FORM alone, was observed for FORM + KI and FORM + PVP-I combinations. On skin, reductions in MRSA survival of 3.1 Log10 colony forming units (CFU) mL-1 were observed with FORM at 50 µM. Although the combined action of FORM + KI and FORM + PVP-I potentiated the aPDT efficacy in vitro, this was not observed ex vivo. Overall, the results showed that aPDT using FORM, even without coadjutants, is a promising approach for MRSA inactivation on skin.

Antimicrobial photodynamic treatment as an alternative approach for Alicyclobacillus acidoterrestris inactivation.

Alicyclobacillus acidoterrestris is a cause of major concern for the orange juice industry due to its thermal and chemical resistance, as well as its spoilage potential. A. acidoterrestris spoilage of orange juice is due to off-flavor taints from guaiacol production and some halophenols. The present study aimed to evaluate the effectiveness of antimicrobial Photodynamic Treatment (aPDT) as an emerging technology to inactivate the spores of A. acidoterrestris. The aPDT efficiency towards A. acidoterrestris was evaluated using as photosensitizers the tetracationic porphyrin (Tetra-Py+-Me) and the phenothiazinium dye new methylene blue (NMB) in combination with white light-emitting diode (LED; 400-740 nm; 65-140 mW/cm2). The spores of A. acidoterrestris were cultured on YSG agar plates (pH 3.7 ± 0.1) at 45 °C for 28 days and submitted to the aPDT with Tetra-Py+-Me and NMB at 10 µM in phosphate-buffered saline (PBS) in combination with white light (140 mW/cm2). The use of Tetra-Py+-Me at 10 µM resulted in a 7.3 ± 0.04 log reduction of the viability of A. acidoterrestris spores. No reductions in the viability of this bacterium were observed with NMB at 10 µM. Then, the aPDT with Tetra-Py+-Me and NMB at 10 µM in orange juice (UHT; pH 3.9; 11°Brix) alone and combined with potassium iodide (KI) was evaluated. The presence of KI was able to potentiate the aPDT process in orange juice, promoting the inactivation of 5 log CFU/mL of A. acidoterrestris spores after 10 h of white light exposition (140 mW/cm2). However, in the absence of KI, both photosensitizers did not promote a significant reduction in the spore viability. The inactivation of A. acidoterrestris spores artificially inoculated in orange peels (105 spores/mL) was also assessed using Tetra-Py+-Me at 10 and 50 µM in the presence and absence of KI in combination with white light (65 mW/cm2). No significant reductions were observed (p < .05) when Tetra-Py+-Me was used at 10 µM, however at the highest concentration (50 µM) a significant spore reduction (≈ 2.8 log CFU/mL reductions) in orange peels was observed after 6 h of sunlight exposition (65 mW/cm2). Although the color, total phenolic content (TPC), and antioxidant capacity of orange juice and peel (only color evaluation) seem to have been affected by light exposition, the impact on the visual and nutritional characteristics of the products remains inconclusive so far. Besides that, the results found suggest that aPDT can be a potential method for the reduction of A. acidoterrestris spores on orange groves.

An efficient formulation based on cationic porphyrins to photoinactivate Staphylococcus aureus and Escherichia coli.

AIM: Antibiotic resistance is an increasingly serious worldwide problem that needs to be addressed with alternative tools. Antimicrobial photodynamic therapy seems a promising approach but in some cases the synthesis of highly efficient photosensitizers requires laborious processes burdened by extensive chromatographic purifications. In this study, we evaluate the suitability of a formulation (Form-1) containing porphyrins bearing different charges, obtained during the synthesis of the highly efficient photosensitizer 5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin tri-iodide. RESULTS: Form-1 was equally effective in the photoinactivation of Escherichia coli and Staphylococcus aureus (reductions >5 log) as the best stand-alone photosensitizer. CONCLUSION: The effective reduction of bacteria with Form-1 provided promising indications supporting its use, leading to a substantial decrease in costs and production time.

An Insight Into the Potentiation Effect of Potassium Iodide on aPDT Efficacy.

Antimicrobial photodynamic therapy (aPDT) is gaining a special importance as an effective approach against multidrug-resistant strains responsible of fatal infections. The addition of potassium iodide (KI), a non-toxic salt, is recognized to increase the aPDT efficiency of some photosensitizers (PSs) on a broad-spectrum of microorganisms. As the reported cases only refer positive aPDT potentiation results, in this work we selected a broad range of porphyrinic and non-porphyrinic PSs in order to gain a more comprehensive knowledge about this aPDT potentiation by KI. For this evaluation were selected a series of meso-tetraarylporphyrins positively charged at meso positions or at ß-pyrrolic positions and the non-porphyrinic dyes Methylene blue, Rose Bengal, Toluidine Blue O, Malachite Green and Crystal Violet; the assays were performed using a bioluminescent E. coli strain as a model. The results indicate that KI has also the ability to potentiate the aPDT process mediated by some of the cationic PSs [Tri-Py(+)-Me, Tetra-Py(+)-Me, Form, RB, MB, Mono-Py(+)-Me, ß-ImiPhTPP, ß-ImiPyTPP, and ß-BrImiPyTPP] allowing a drastic reduction of the treatment time as well as of the PS concentration. However, the efficacy of some porphyrinic and non-porphyrinic PSs [Di-Py(+)-Me opp , Di-Py(+)-Me adj , Tetra-Py, TBO, CV, and MG] was not improved by the presence of the coadjuvant. For the PSs tested in this study, the ones capable to decompose the peroxyiodide into iodine (easily detectable by spectroscopy or by the visual appearance of a blue color in the presence of amylose) were the most promising ones to be used in combination with KI. Although these studies confirmed that the generation of 1O2 is an important fact in this process, the PS structure (charge number and charge position), aggregation behavior and affinity for the cell membrane are also important features to be taken in account.