br The most promising for
The most promising for that reason are complexes of zinc- and galium-phthalocyanines. They characteristics are better because of high triplet yields and long lifetimes [1,4]. We studied the activity of novel syn-thesized zinc octacarboxyphthalocyanines (ZnPcOC) for biological manner against melanoma Me45 cancer cell line, followed by photo-dynamic therapy protocol, and we discovered a novel ROS-induced pro-apoptotic potential of used sensitizers.
2.1. Material and Methods
2.1.1. Synthesis of Zinc Octacarboxyphthalocyanine
The details of synthesis of ZnPcOC are included in the supplemen-tary materials.
1,2,4,5-tetracyanobenzene, sulfolane, a.p. Sigma-Aldrich, DBU Merck, ethylene glycol, zinc acetate, H2SO4, DMSO, HCl, NaOH, acetone, methanol a.p. grade were purchased from POCH Gliwice, Poland.
Absorption spectra in the UV–Vis region were recorded with Jasco V-650 –.mechanisms, photosensitizers and combinations, spectro-photometer. The spectra were measured in 10 mm quartz MitoPY1 at 37 °C. Journal of Photochemistry & Photobiology, B: Biology 190 (2019) 146–153
A CP-315 M pH-meter from Elmetron Poland was used for pH mea-surements. Julabo F25 thermostat was used for temperature control. The exposition of the samples on visible light (λ = 685 nm) was carried
out using the LED lamp (250 mW) by OPTEl, λmax = 685 nm at 37 °C. The research was carried out for ZnPcOC concentration in the range of
0,065–30 μM. UV treatments were carried out, with usage of with various wavelengths of UVA (365 nm), UVB (302 nm), or UVC (254 nm) at dose of 100 J/m2, generated by UV crosslinkers (CL- 1000 models, UVP, Upland, CA, USA). The UV doses were adjusted after our previous studies and references in Me45 cells . Elemental analysis was done with CHNS vario EL III, Elementar. Infrared spectra were recorded with FT-IR Nexus, Thermo Nicolet. Mass spectra were measured with Bruker Daltionik micrOTOF-Q-II spectrometer. 1H NMR spectra were recorded with a Bruker Avance-400 MHz spectrometer.
2.3. Biological Evaluation
The human malignant melanoma cells, Me45 line established in Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, branch in Gliwice, Poland, from a lymph node metastasis of primary skin melanoma  was a kind gift from Dr. Maria Widel. HaCaT cells were obtained from collections at the Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, branch in Gliwice, Poland, as kindly gift from Dr. Dorota Scieglinska. Neonatal human dermal fibroblasts, NHDF cells were obtained from Lonza (NHDF-Neo, Lonza, Poland). All cell lines were cultivated in monolayer culture in medium DMEM-F12 (PAA) supplemented with 10% of fetal bovine serum (Eurx, Gdansk, Poland), and 1% of antibiotics (10,000 μg/ml streptomycin and 10,000 units/ml penicillin; Sigma-Al-drich, Germany), at 37 °C in humidified atmosphere with 5% CO2, under standard conditions (incubator Heraeus Instruments, Hanau, Germany). For flow cytometric experiments cells were seeded on 6-well plates (Falcon) at 1 × 105 cells/well in 2 ml of fresh medium for 24 h, then washed with 1xPBS solution (PAA) and incubated in fresh one before addition of drugs. After preincubation time (4 h), the cells were irradiated with UV (A, B or C) or light (λ = 685 nm) for photosensitizer activation. For MTT/MTS assays cells were seeded in 96-well plates (Falcon) at 1 × 104 cells/well in 0.2 ml of medium, grown for 24 h, washed and incubated in fresh medium.
The cellular uptake were measured every hour directly from the HaCaT, NHDF and Me45 cells, previously seeded into 96-well plates (Falcon) at confluence of 1 × 104 cells/well in 0.2 ml of medium, contained final concentration of ZnPcOC 10, 20 and 30 μM. The max-imal absorbance for the tested compounds was measured at 690 nm (Epoch; BioTek, Winooski, VT, USA) after cells with PBS washing. Control cells, at the same wave, do not absorbed the light, and the maxima of ZnPcOC inside the cells were measured during long-term 96 h uptake assay, after which for further studies optimal concentration (30 μM) was chosen. To avoid any light-induced phototoxicity, all in-cubations and measurements were performed in completely darkness .