Imidazole metalloporphyrins as photosensitizers for photodynamic therapy: role of molecular charge, central metal and hydroxyl radical production.

Synthesis of Imidazole Porphyrins General Procedures

Absorption spectra and fluorescence spectra were collected at room temperature. Porphyrins were analyzed in neat form by laser desorption mass spectrometry (LD-MS) [ 19 ] and by matrix-assisted laser-desorption ionization mass spectrometry (MALDI-MS) using the matrix 4-hydroxy-α-cyanocinnamic acid. In both LD-MS and MALDI-MS analyses, positive ions were detected. Solvents were dried according to standard procedures. All chemicals were used as received from commercial sources. 5-(1,3-Diethylimidazol-2-ium)-10-phenylporphinatoindium(III) chloride (2-InCl) Following a general procedure [ 20 ] porphyrin 3-Zn [ 18 ] (23 mg, 0.035 mmol) in ethanol (EtOH) (7 mL) was treated with concentrated aqueous HCl (7 mL). The resulting mixture was refluxed for 21 h under argon. The reaction mixture was then cooled to room temperature, whereupon the volatile solvent was removed. The resulting suspension was poured into a large volume of ethyl acetate and water. Saturated aqueous NaHCO 3 solution was added slowly to the aqueous-organic mixture. The organic layer was separated, and the aqueous layer was extracted twice with ethyl acetate. The combined organic extract was washed with saturated aqueous NH 4 Cl solution and water. The organic layer was separated, dried (Na 2 SO 4 ) and concentrated to give free base porphyrin 4 as a dark purple solid. Data for 4 : LD-MS obsd 452.2, calcd 452.2 (C 29 H 20 N 6 ); λ abs (methanol, (MeOH)) 402, 500, 534, 574, nm; λ em (MeOH) 625, 700 nm. A solution of porphyrin 4 (∼0.035 mmol) in anhydrous DMF (7 mL) was treated with InCl 3 (0.30 g, 1.4 mmol). The reaction mixture was heated to reflux for 40 h under argon. The crude reaction mixture was concentrated. The residue was chromatographed [silica, CH 2 Cl 2 /MeOH in a gradient of (9:1) to (4:1)] to afford porphyrin 4-InCl as a green solid. Data for 4-InCl : MALDI-MS 595.8; λ abs (MeOH) 410, 598 nm; λ em (MeOH) 620 nm. The entire sample of 4-InCl (∼0.035 mmol) was treated with EtI (0.28 mL, 3.5 mmol) in DMF (2.0 mL), and the resulting mixture was heated at 65 °C for 3 days. The reaction mixture was then concentrated and chromatographed twice [(1) silica, CH 2 Cl 2 /MeOH (9:1); (2) neutral alumina, CH 2 Cl 2 CH 2 Cl 2 /MeOH (9:1)] to afford a solid residue. The solid residue was purified by preparative size-exclusion chromatography (3 × 30 cm) eluted with HPLC-grade tetrahydrofuran (THF). A final chromatographic procedure [alumina, CH 2 Cl 2 /MeOH in a gradient of (9:1) to (3:1)] afforded the title compound as a dark green solid (9.0 mg, 33% overall yield, assuming an iodide counterion): MALDI-MS obsd 656.8, calcd 657.1 (C 33 H 27 ClInN 6 , lacking a counterion); λ abs (MeOH) 411, 601 nm; λ em (MeOH) 620, 670 nm. Photophysics The absorbance and fluorescence spectra, fluorescence quantum yields and excited-state singlet lifetime measurements of 1-Zn , 2-Zn , 2-Pd , and 2-InCl were investigated at room temperature in a 3:1 mixture of THF and MeOH. The lifetimes of the lowest energy triplet excited state were measured at room temperature and at 77 K on compounds in ethanol. Details of the measurements, including determination of molar absorption coefficients utilized herein to prepare solutions of known concentration, are given elsewhere [ 26 ].

Cell Culture

HeLa (human cervical squamous carcinoma cells) [ 21 ], and CT26 (murine colon adenocarcinoma) [ 22 ] were obtained from ATCC (Manassas, VA) and cultured in RPMI1600 medium (Gibco Invitrogen, Carlsbad, CA) with L-glutamine and NaHCO 3 supplemented with heat inactivated fetal bovine serum 10% (vol/vol), penicillin (100 U/mL) and streptomycin (100 µg/mL) (all from Sigma, St Louis, MO) at 37 °C in 5% CO 2 humidified atmosphere in 75 cm 2 flasks (BD Falcon, San Jose, CA). When the cells reached 80% confluence, they were washed with PBS (Sigma) and harvested with 2 mL of 0.25% Trypsin-EDTA solution (Sigma). Cells were centrifuged and counted in trypan blue (Sigma) and plated at 5000 cells/well in flat-bottomed 96 well plates (Fisher). Cells were allowed to attach for 24 h. Light Source Illumination of cells utilized a non-coherent white light source (Lumacare, Newport Beach, CA) fitted with a light guide containing a band pass filter (λ400–700 nm) adjusted to give a uniform spot of 4 cm in diameter with an irradiance of 100 mW/cm 2 as measured with a power meter (model DMM 199 with 201 Standard head, Coherent, Santa Clara, CA). For the experiments assessing the production of intracellular ROS we used 405 nm laser light (Nichia Corp, Detroit, MI). PDT Experiments The PS were dissolved in DMSO at a concentration of 5 mM and stored in the dark at room temperature. PS were added at different concentrations to cells in fresh complete medium for 24 h incubation periods. The DMSO concentration in the medium was less than 0.5%. After incubation the medium was replaced with 200 µL of fresh medium and PDT with white light was performed. For experiments where the light dose was varied, fluences of 0 (dark toxicity), 2, 4, 8, 10, 12, 16, and 20 J/cm 2 were used, and 9 wells (one group) were illuminated at one time. Controls were cells with no treatment and cells with light alone at the highest fluence. For experiments where the porphyrin concentration was varied (concentrations between 100 nm and 24 µM) a fixed fluence of 10 J/cm 2 delivered at the same irradiance was used. Here additional groups of cells were incubated with all the concentrations of porphyrins but no illumination was used to determine the dark toxicity of the PS. At the completion of the illumination the plates were returned to the incubator for a further 24 h, before further studies. We used a MTT colorimetric assay (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) a tetrazolium salt that measures mitochondrial reductase activity and correlates well with colony-forming assays as a measure of cell viability, as has been described previously [ 23 – 26 ]. The absorbance for MTT assay was read at 560nm. Photosensitizer Uptake 5×10 3 HeLa cells were plated per well in a 96 well plate and allowed overnight to attach. The next day 2-Zn , 1-Zn or 2-Pd was added and incubated for 24 h. After incubation, the medium was removed and 200 µL of 1M NaOH/1%SDS was added and cells were incubated overnight. The fluorescence ( 2-Zn and 1-Zn ) was measured using excitation at 402 nm and detection at 641 nm with a plate-reader (Molecular Devices, Sunnyvale CA), while the absorption was measured at the Soret maximum (λ402 nm). Unfortunately the 2-Pd compound is non-fluorescent therefore the uptake had to be measured by the means of absorption. The protein per sample was measured with bicinchoninic acid protein assay [ 27 ]. Fluorescence (for 1-Zn and 2-Zn) and absorption standard curves (for 1-Zn, 2-Zn and 2-Pd) were prepared by adding known amounts of 2-Zn , 1-Zn or 2-Pd solutions to pre-prepared cell lysates in NaOH/SDS and incubated for 24 h. On the following day the fluorescence and absorption was measured as described above.

Apoptosis Assay

The induction of apoptosis by imidazole-porphyrin mediated PDT was measured by a fluorescence assay using Ac-DEVD-AFC (BD Pharmingen, San Jose, CA), a caspase fluorescent substrate [ 28 ]. The results were normalized to the content of protein in the sample. Briefly HeLa cells were treated with PDT sufficient to kill 80% of the cells. Following PDT, samples were collected at 2, 4, 6, 12, and 24 h and centrifuged. The pellet was resuspended in 100 µL of lysis buffer [ 29 ] containing protease inhibitor and subjected to 3–4 cycles of freezing and thawing. Then 50 µL of each sample was transferred to separate wells and 50 µL of 2X reaction buffer was added together with Ac-DEVD-AFC (final concentration 50 µM). Samples were incubated in the dark for 1 h at 37 °C, and fluorescence was measured in a plate reader (λex 400 nm, λem 505 nm). The protein per sample was measured with bicinchoninic acid protein assay. Fluorescence Microscopy – Intracellular Localization 5×10 5 HeLa cells were plated on 35mm dishes and allowed overnight to attach. The next day 5 µM 2-Zn or 1-Zn in culture medium was added and incubated for 24 h. Cells were washed in PBS and 5 µg/mL of lysotracker or mitotracker (LysoTracker Green DND-26, MitoTracker Green FM, Molecular Probes Invitrogen) was added and incubated for 30 min at 37 °C. Cells were again washed in PBS and 5–10 min later a Leica DMR confocal laser fluorescence microscope (Leica Mikroskopie und Systeme GmbH, Wetzler, Germany) with excitation by a λ488 nm argon laser and emission with either a λ525 nm +/− 10-nm bandpass filter, or a λ580-nm longpass filter and a 63×1.20 NA water immersion lens was used to image the cells at a resolution of 1024×1024 pixels. Images were acquired using TCS NT software (Version 1.6.551, Leica Lasertechnik, Heidelberg, Germany). The intracellular localization of 2-Pd compound by confocal microscopy could not be identified due to the lack of fluorescence of this compound.

Detection of photodamage by fluorescent probes

We used the fluorescent probes, acridine orange (AO) and rhodamine 123 (Rho 123) (both from Sigma, St Louis, MO) to detect the location of PDT associated intracellular damage. 5×10 5 HeLa cells were plated on 35mm dishes and allowed overnight to attach. The next day 5 µM 2-Zn or 0.5 µM 2-Pd in culture medium was added and incubated for 24 h. Cells were washed in PBS and 5 J/cm 2 of white light was delivered. Immediately after PDT 0.5 µM of AO or 0.5 µM Rho 123 was added to the cells and incubated for 5 min in complete medium at 37 °C. Next the cells were washed and the intracellular localization of the dye was observed by confocal microscopy. Both probes were excited with Ar-laser 488-nm and emission wavelengths were AO (green fluorescence 525 nm +/− 10-nm, or red fluorescence 580-nm longpass) and Rho 123 (525 nm +/− 10-nm) [ 30 ]. As controls we used HeLa cells that were incubated with either AO or Rho 123 but did not receive PDT. Intracellular ROS HeLa cells were incubated with 5 µM 2-Zn , 1-Zn or 2-Pd for 24 h, and on the next day 5 µg/mL of 5-(and-6)-chloromethyl-2'7'-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA, Molecular Probes Invitrogen) in complete medium was added and incubated for 30 min at 37 °C. Cells were then washed with PBS, and 5 J/cm 2 of 405 nm laser light (Nichia Corp, Detroit, MI) was delivered. Blue light was used to minimize the photoactivation of dichlorodihydrofluorescein by small amounts of contaminating dichlorofluorescein that has been shown to be able to act as a PS when green light is used [ 31 ]. In similar experiments, 10 µM of 3`-(4-hydroxyphenyl)fluorescein (HPF, Molecular Probes Invitrogen) was added and incubated for 1 h in complete medium at 37 °C. After the incubation, the cells were washed with PBS and 5 J/cm 2 of white light was delivered. The cells were imaged immediately after PDT with the confocal microscope (488-nm excitation, 530-nm detection). Cell-free experiments were also performed with HPF in 96 well plates. The dyes were aliquoted at the final concentration of 5 µM per well in PBS, and HPF was added to each well at the final concentration of 10 µM. Four wells formed one experimental group. For hydroxyl radical quenching experiments 100 mM mannitol was added. All groups were illuminated simultaneously and white light was delivered in sequential doses of 5 J/cm 2 . After each dose the fluorescence was measured by fluorescence plate reader (488-nm excitation, 520-nm detection).

General Procedures

Absorption spectra and fluorescence spectra were collected at room temperature. Porphyrins were analyzed in neat form by laser desorption mass spectrometry (LD-MS) [ 19 ] and by matrix-assisted laser-desorption ionization mass spectrometry (MALDI-MS) using the matrix 4-hydroxy-α-cyanocinnamic acid. In both LD-MS and MALDI-MS analyses, positive ions were detected. Solvents were dried according to standard procedures. All chemicals were used as received from commercial sources.

Supplementary Material 01