Photosensitizers
5,10,15,20-tetrakis(1-methylpiridinium-4-yl)porphyrin tetra-iodide (Tetra-Py+-Me), 5-(pentafluorophenyl)-10,15,20-tris(1-methylpiridinium-4-yl)porphyrin tri-iodide (Tri-Py+-Me-PF), 5-(4-methoxicarbonylphenyl)-10,15,20-tris(1-methylpiridinium-4-yl)porphyrin tri-iodide (Tri-Py+-Me-CO2Me), 5-(4-carboxyphenyl)-10,15,20-tris(1-methylpiridinium-4-yl)porphyrin tri-iodide (Tri-Py+-Me-CO2H), 5,10-bis(4-carboxyphenyl)-15,20-bis(1-methylpiridinium-4-yl)porphyrin di-iodide (Di-Py+-Me-Di-CO2H adj), 5,15-bis(4-carboxyphenyl)-10,20-bis(1-methylpiridinium-4-yl)porphyrin di-iodide (Di-Py+-Me-Di-CO2H opp) and 5-(1-methylpiridinium-4-yl)-10,15,20-tris(4-carboxyphenyl)porphyrin iodide (Mono-Py+-Me-Tri-CO2H) (Fig. 1) were prepared in two steps. First, the neutral porphyrins were obtained from the Rothemund and crossed Rothemund reactions using pyrrole and the appropriate benzaldehydes (pyridine-4-carbaldehyde and pentafluorophenylbenzaldehyde or 4-formylbenzoic acid) at reflux in acetic acid and nitrobenzene ([38–40]. After being separated by column chromatography (silica), the pyridyl groups of each porphyrin were quaternized by reaction with methyl iodide. Porphyrin Tri-Py+-Me-CO2Me was obtained by esterification of the corresponding acid derivative with methanol/sulphuric acid followed by quaternization with methyl iodide. Porphyrins were purified by crystallization from chloroform-methanol-petroleum ether and their purities were confirmed by thin layer chromatography and by 1H NMR spectroscopy. The spectroscopic data was in accordance with the literature [38–40]. Stock solutions (500 μM) of each porphyrin in dimethyl sulfoxide were prepared by dissolving the adequate amount of the desired porphyrin in a known volume. The absorption spectral features of the PS were the following: [porphyrin] λmax nm (log ε); [Tetra-Py+-Me] in DMSO 425 (5.43), 516 (4.29), 549 (3.77), 588 (3.84), 642 (3.30); [Tri-Py+-Me-PF] in DMSO 422 (5.48), 485 (3.85), 513 (4.30), 545 (3.70), 640 (3.14); [Tri-Py+-Me-CO2Me] in H2O 420 (5.54), 518 (4.12), 556 (3.74), 583 (3.78), 640 (3.27); [Tri-Py+-Me-CO2H] in H2O 425 (5.40), 520 (4.24), 555 (3.90), 588 (3.82), 646 (3.34); [Di-Py+-Me-Di-CO2H adj] in H2O 425 (5.21), 521 (4.06), 557 (3.78), 590 (3.64), 648 (3.04); [Di-Py+-Me-Di-CO2H opp] in H2O 424 (5.40), 518 (4.16), 558 (3.94), 589 (3.69), 648 (3.58); [Mono-Py+-Me-Tri-CO2H] in butan-1-ol 425 (5.35), 520 (4.25), 553 (4.01), 591 (3.87), 649 (3.74). Selected data: [Di-Py+-Me-Di-CO2H opp] 1H-NMR: (300 MHz, DMSO-d6) δ 9.46 (4H, d, J 6.6 Hz, 10,20-Ar-m-H), 8.99 – 9.05 (12H, m, 10,20-Ar-o- and β-H), 8.41 (4H, d, J 8.0 Hz, 5,15-Ar-m-H), 8.30 (4H, d, J 8.0 Hz, 5,15-Ar-o-H), 4.70 (6H, s, 2 × CH3), -2.99 (2H, s, NH). MS (MALDI-TOF) m/z: 734.2 (M-2I)+; [Di-Py+-Me-Di-CO2H adj] 1H-NMR: (300 MHz, DMSO-d6) δ 9.46 (4H, d, J 6.7 Hz, 15,20-Ar-m-H), 8.92 – 9.12 (12H, m, 15,20-Ar-o- and β-H), 8.40 (4H, d, J 8.2 Hz, 5,10-Ar-m-H), 8.30 (4H, d, J 8.2 Hz, 5,10-Ar-o-H), 4.70 (6H, s, 2xCH3), -2.96 (2H, s, NH). MS (MALDI-TOF) m/z: 734.2 (M-2I)+; [Mono-Py+-Me-Tri-CO2H] 1H-NMR: (300 MHz, DMSO-d6) δ 9.44 (2H, d, J 6.4 Hz, 20-Ar-m-H), 8.90 – 9.03 (10H, m, 20-Ar-o- and β-H), 8.30 – 8.40 (12H, m, 5,10,15-Ar-H), 4.69 (3H, s, CH3), -2.94 (2H, s, NH). MS (MALDI-TOF) m/z: 762.2 (M-I)+.
Partition coefficients
The partition coefficients were determined at 22°C in butan-1-ol/water (log PB/W) according to the shake-flask method. Porphyrin derivatives were individually dissolved in water-saturated butan-1-ol to give the stock solution (absorbance ~0.8 at the Soret band). Then, in duplicate test vessels, different volumes of butan-1-ol-saturated water and stock porphyrin solution were added in order to get at least three different butan-1-ol/water volume ratio. Each vessel was vigorously vortexed and then centrifuged to allow phase separation and kept for equilibration at the test temperature for 2 hours before analysis. The absorbance at the Soret band was measured in both phases and the log PB/W determined using the relationship log PB/W = log (AbsB *VW/AbsW *VB), where AbsW and AbsB are the absorbances at the Soret band and VW and VB are the volumes of aqueous and butan-1-ol phases, respectively [35].
Singlet oxygen generation studies
Stock solution of each porphyrin derivative at 0.1 mM in DMF: water (9:1) and a stock solution of 1,3-diphenylisobenzofuran (DPBF) at 10 mM in DMSO were prepared. The reaction mixture of 50 μM of DPBF and 0.5 μM of a porphyrin derivative in DMF water (9:1) in glass cells (2 mL) was irradiated with white light filtered through a cut-off filter of wavelength < 540 nm, at a fluence rate of 9.0 mW cm-2. During the irradiation period, the solutions were stirred at room temperature. The generation of singlet oxygen was followed by its reaction with DPBF. The breakdown of DPBF was monitored by measuring the decreasing of the absorbance at 415 nm at irradiation intervals of 1 min.
Bacterial strains and growth conditions
Escherichia coli ATCC 13706 (USA) and Enterococcus faecalis ATCC 29212 (USA) were stored at 4°C in triptic soy agar (TSA, Merck). Before each assay the strains were grown aerobically for 24 hours at 37°C in 30 mL of triptic soy broth (TSB, Merck). An aliquot of this culture (240 μL) was aseptically transferred to 30 mL of fresh TSB medium and grown overnight at 37°C to reach an optical density (O.D.600) of ~1.3, corresponding to ~108 cells mL-1.
Experimental setup
The efficiency of the cationic porphyrins at different concentrations (0.5, 1.0 and 5.0 μM) was evaluated through quantification of the colonies of bacteria in laboratory conditions. Knowing that the inactivation of bacteria by cationic porphyrins is very sensitive to ionic strength [41], all the experiments were performed using the same conditions. Bacterial suspensions were prepared from bacterial cultures (~108 cells mL-1) which were diluted ten-fold in phosphate buffered saline, pH 7.4, to a concentration of ~107 CFU mL-1(100–1000 times higher than bacterial concentration in wastewater to ensure that when applied to the field most of similar bacteria were inactivated). In all the experiments, 49.5 mL of bacterial suspension were aseptically distributed in 600 mL acid-washed, sterilised glass beakers and the PS was added from the stock solution (500 μM in DMSO) to achieve final concentrations of 0.5, 1.0 and 5.0 μM. After the addition of the appropriate volume of porphyrin, beakers (total volume of 50 mL) were incubated during 10 minutes at 20–25°C, under stirring (100 rpm), covered with aluminium foil to avoid accidental light exposure.
Light and dark control experiments were carried out simultaneously. In the light controls, the bacterial suspension without PS was exposed to light irradiation. In the dark controls, the PS at the higher concentration (5.0 μM), was added to the beaker, containing the bacterial suspension, covered with aluminium foil to protect from light exposure. The controls also followed the pre-irradiation incubation protocol.
This photosensitization procedure was used for each of the seven PS tested and for both bacterial strains under investigation.
Irradiation conditions
Following the pre-irradiation incubation period, all samples were exposed in parallel to white light (PAR radiation, 13 OSRAM 21 lamps of 18 W each, 380–700 nm) with a fluence rate of 40 W m-2 (measured with a light meter LI-COR Model LI-250, Li-Cor Inc., USA), at 20–25°C for 270 minutes, under 100 rpm mechanical stirring.
Bacterial quantification
A standard volume (1 mL) of undiluted and serially diluted of irradiated samples and controls were plated in duplicate in TSA medium at time 0 and after 15, 30, 60, 90, 180 and 270 minutes of light exposure. After 24 hours of incubation at 37°C in the dark, the number of colonies was counted. The dark control Petri plates were kept in the dark immediately after plating and during the incubation period. The assays for each concentration of each porphyrin and for each bacterial strain were done in duplicate and averaged. Data were presented by survival curves plotted as logarithmic bacterial reduction in log CFU mL-1 versus light fluence in J cm-2. As previously stated, bactericidal activity was defined as a ≥ 3 log decrease (≥ 99,9%) in CFU mL-1, while bacteriostatic activity was defined as a <3 log (< 99,9%) decrease in CFU mL-1 [42].
Statistical analysis
Statistical analyses were performed by using SPSS (SPSS 15.0 for Windows, SPSS Inc., USA). Normal distributions were assessed by Kolmogorov-Smirnov test. The significance of both porphyrin derivatives and irradiation time on bacterial inactivation was assessed by two-way univariate analysis of variance (ANOVA) model with the Bonferroni post-hoc test. A value of p < 0.05 was considered significant.