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Materials 2020, 13, 2646 8 of 24 For biological assays, cells from confluent cultures were detached from the bottom of the culture flasks by trypsinization, counted, and resuspended in fresh culture medium in order to obtain a cell suspension at a density of 5 × 104 cells/mL. Then, 100 μL of this cell suspension was added per well of 96-well culture plates. Cells were cultured for 24 h, after which the medium was removed, and cells were incubated with squaraine dye solutions (0.1, 1.0, 5.0 and 10.0 μM) prepared in FBS-free culture medium. Since the dye stock solutions were prepared in dimethyl sulfoxide, we previously ensured that the dimethyl sulfoxide maximum concentration, in the cell viability experiments, was not higher than 3%, concentrations shown previously to be non-toxic [37]. A negative control was also carried out, exposing the cells only to the cell culture medium. Squaraine dyes’ cytotoxicity was evaluated under absence or presence of radiation (for 7 and 14 min). For the irradiation of the cells, a self-constructed aluminum gallium indium phosphide light-emitting diode array system emitting at λ = 630.2 ± 0.8 nm with a full width of half maximum of ∆λ = 15.6 ± 0.2 nm and radiant flux of P = 4.3 ± 0.5 mW. This light-emitting diode array device was placed over the 96-well plate, with the emitters facing the cells, in a way that each emitter illuminated a single well at approximately 1.5 cm of distance to the wells’ bottom. The system has a ventilation apparatus coupled to the light source to avoid the overheating of the light-emitting diode lamps and limit the influence of the heat generated by them in the in vitro experiments. At 1 h and at 24 h after radiation treatment, cell viability was assessed by means of the Alamar Blue reduction assay. Dye aqueous solutions were replaced by a 10% (v/v) Alamar Blue solution diluted in FBS-free culture medium. Then, the absorbances at 570 and 620 nm were read approximately 5 h after the addition of Alamar Blue solution in a Multiskan EX microplate reader (MTX Labsystems, Vantaa, Finland). Cell viability was calculated by analyzing the percentage of Alamar Blue reduction as previously detailed [50] and the antiproliferative effect results were expressed as a percentage of control. The photocytotoxicity experiments were executed in quadruplicate, and their cell viability data are stated as mean value ± standard deviation. The difference between the dye treatments and the negative control was considered statistically significant for p-values lower than 0.05 and were determined using Student’s t-test. The half inhibitory concentration values, dye concentration that reduces cell viability to 50% of control, were calculated as described [51]. All data related to biological evaluation presented in this work are illustrative of at least three independent experiments. 3. Results 3.1. Synthesis and Photophysicochemical Characterization 3.1.1. Dyes’ Synthesis Strategy The quinoline- and benzoselenazole-derived unsymmetrical squaraine cyanine dyes 9–12 were synthesized according to the multi-step route outlined in Scheme 1. The synthetic route was initiated by the conversion of 2-methylquinoline (1) and 2-methylbenzoselenazole (7) in the corresponding heterocyclic quaternary ammonium salts, 1-hexyl-2-methylquinolinium iodide (2) and 1-hexyl-2-methylbenzoselenazolium iodide (8), respectively, by alkylation with an excess of iodohexane [47]. Next, dibutyl squarate (4), prepared by the reaction between squaric acid (3) and n-butanol, reacted with an equimolar amount of quaternary quinoline-derived salt 2 to prepare the monosubstituted precursor 5. After its hydrolysis by a 40% sodium hydroxide aqueous solution, and the protonation with a 2 M hydrochloric acid aqueous solution of the resulting sodium salt, the intermediate semisquaraine 6 was obtained with a moderate yield. The latter reacted with the benzoselenazole-based quaternary ammonium salt 8 in a n-butanol and pyridine refluxing mixture to afford the zwitterionic unsymmetrical squaraine dye 9. The methylation reaction of this latter dye with the strong methylating agent methyl trifluoromethanesulfonate, in anhydrous conditions, allowed us to obtain the O-methyl ether intermediate dye 10 [39,42] with good yield. In the last step of the synthetic approach, two aminosquaraines dyes, 11 and 12, were prepared through a nucleophilic reaction of the O-methyl derivative methoxy group by ammonia and methylamine solutions, respectively [25,42].PDF Image | Photophysicochemical Light Antiproliferative vs cancer
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