br The accumulation and cellular assay
The accumulation and cellular assay was prepared for preincubation with phthalocyanines time. Two maximas, after 4 and 72 h, enables for minimalization of preincubation time – for our studies the shorter one (4 h) was chosen. Microscopic observations in visible channel con-firmed also ZnPcOC cellular accumulation after preincubation times, both 4 and 72 h. During performed experiments, microscopic evalua-tion for ZnPcOC presence in the Me45 USP7/USP47 inhibitor was prepared, and typically green coloured cytoplasm of cells was observed (Fig. 5). Characteristic of UV–Vis spectra of ZnPcOC in DMSO solution dis-played two maxima, first in UV and the second at 690 nm (Fig. 2). For that reason, first we tried to induce activity of ZnPcOC with UV A, B and C with dose of 100 J/m2 for each wavelength. The biological activity was estimated followed by antiproliferative 24 h MTS assay (Fig. 6; A, B and C), after 4 h of preincubation with dose-range of ZnPcOC 0.065–2 μM. Obtained results showed rather on lack of biological ac-tivity after phthalocyanine and UV treatments, and the viability of Me45 cells was similar to the untreated controls. In the next step, we
Effect of the radiation dose on the intensity of ZnPcOC Q band in DMSO.
used led system for cells irradiation with light source (λ = 685 nm), what should resulted with ZnPcOC activation. For Me45 and NHDF cells viability estimation, the 24 h MTT assay confirm activity of used photosensitizer, used at dose of 30 μM, with 4 h of preincubation and light (λ = 685 nm) irradiation. According to the references , and our first results, the 7.5; 4.5 and 2.5 J/cm2 dose of light were tested against Me45 and NHDF cell lines. Dose-dependent irradiation, together with ZnPcOC presence, decreased both cell lines viability about 50%, without selectivity against melanoma cancer cells (Fig. 6; D). In com-parison to the untreated controls, the absorbance decreased, what could be a prove for a photodynamic reaction after compounds, together with irradiation treatments.
Promising results from MTT viability assay, pushed us to ROS and ROS-induced apoptosis evaluations, followed by cytometric measure-ments. The photodynamic mechanism of action, for ZnPcOC, should be confirmed with ROS induction, and then with the apoptotic/necrotic cellular death. ROS measurements were performed directly after irra-diation, so in first 15 min we observed free radicals overproduction after 2.5; 4.5 J/cm2 irradiation in presence of 30 μM of ZnPcOC in Me45 cells (Fig. 7; A). Surprisingly, the highest dose of light did not induce the highest level of ROS, it accursed only after 24 h (Fig. 7; C). In the apoptosis estimation, we discriminated together early with late apop-tosis, and we observed dos-dependent cellular death activation (Fig. 7; B). Late apoptosis was observed even after 24 h after irradiation, what correlated with secondary wave of ROS (Fig. 7; B), and confirmed well biological long-term activity of used photosensitizer (Fig. 7; D). The 4 h of preincubation time of ZnPcOC concentrated at 30 μM, and used doses of light, seemed to be well combined, and could be used in in vitro assays for further photodynamic protocols estimation. Promising PDT activity of used photosensitizer should be also improved after next-step in vivo studies.
According to the recent finding, the phthalocyanines are the second-generation photosensitizers, with long-term and locally displayed ac-tivities . They characterize also with tissue specific activity, what
Complex Radiation dose [J/cm2] λmax (nm), Absorbance of the Q band (692 nm)
Before After 2 h after 4 after 24 h after
irradiation irradiation irradiation irradiation irradiation
Fig. 4. ZnPcOC cellular time-depended uptake (30 μM), absorbance from HaCaT, Me45 and NHDF cells (left) and typically spectra for ZnPcOC full-filled cells and empty control (right).