Could the Length of the Alkyl Chain Affect the Photodynamic Activity of 5,10,15,20-Tetrakis(1-alkylpyridinium-4-yl)porphyrins?

Abstract

Photodynamic therapy (PDT) is a minimally invasive treatment that uses the combination of a photosensitizing agent (PS) and light to selectively target solid tumors, as well as several non-neoplastic proliferating cell diseases. After systemic administration, PSs are activated by localized irradiation with visible light; in the presence of adequate concentrations of molecular oxygen, this causes the formation of reactive oxygen species (ROS) and subsequent tissue damage. In this study, two series of tetrakis(N-alkylpyridinium-4-yl)porphyrins were synthesized, differing in the presence or absence of a zinc ion in the tetrapyrrole nucleus, as well as in the N-alkyl chain length (from one to twelve carbon atoms). The compounds were chemically characterized, and their effect on cell viability was evaluated using a panel of three tumor cell lines to determine a possible relationship between photodynamic activity and Zn presence/alkyl chain length. The types of cell death mechanisms involved in the effect of the various PSs were also evaluated. The obtained results indicate that the most effective porphyrin is the Zn-porphyrin, with a pendant made up of eight carbon atoms (Zn-C8).

Keywords: ROS; cell death mechanisms; cellular uptake; cytotoxicity; photodynamic therapy (PDT); tetrakis(N-alkylpyridinium-4-yl)porphyrins; tumor cells.

Figure 1

Illustrative scheme of the main phases of photodynamic treatment. Reprinted from Ref. [11].

Figure 2

Illustrative scheme of the reaction to obtain the tetrakis(N-alkylpyridinium-4-yl)porphyrins, the substituents, and the yield of the eight PSs.

Figure 3

Illustrative scheme of Zn-C8 photodegradation. A 10 μM solution in 1× PBS was illuminated using a tungsten halogen lamp for 2 h. Every 30 min, a sample was subjected to spectrophotometric analysis. The percentage of photodegradation was calculated as the ratio between the intensity of absorption and the absorption at t₀.

Figure 4

IC₅₀ values for HCT116 (A), SKOV3 (B), and MCF7 (C) (2H: solid bars; Zn: striped bars) and statistical significance. Mean ± SD of five independent experiments. Statistical analyses were performed using two-way ANOVA, followed by Duncan’s post hoc test. *** p < 0.001; ** p < 0.01; * p < 0.05.

Figure 4

IC₅₀ values for HCT116 (A), SKOV3 (B), and MCF7 (C) (2H: solid bars; Zn: striped bars) and statistical significance. Mean ± SD of five independent experiments. Statistical analyses were performed using two-way ANOVA, followed by Duncan’s post hoc test. *** p < 0.001; ** p < 0.01; * p < 0.05.

Figure 5

Uptake, obtained by cytometric analysis, expressed as mean fluorescence intensity (MFI) values obtained after treatment with 100 nM of each PS, and the normalized vs respective controls, for HCT116, SKOV3, and MCF7 cells (Ctrl: black; C1: blue; C4: yellow; C8: green; C12: orange), along with the statistical significance. Mean ± SD of five independent experiments. Statistical analyses were performed using two-way ANOVA, followed by Duncan’s post hoc test. *** p < 0.001; ** p < 0.01; * p < 0.05.

Figure 6

Arbitrary fluorescence units of ROS in HCT116, SKOV3, and MCF7 cells after PDT treatment (Ctrl: black; C1: blue; C4: yellow; C8: green; C12: orange), along with statistical significance. In the control samples, PS treatment was omitted. Mean ± SD of five independent experiments. Statistical analysis was performed using two-way ANOVA, followed by Duncan’s post hoc test. *** p < 0.001; ** p < 0.01; * p < 0.05.

Figure 7

Percentage of apoptotic cells, obtained by flow cytometric analysis, after PDT treatment on HCT116, SKOV3, and MCF7 cells (Ctrl: black; C1: blue; C4: yellow, C8: green; C12: orange), along with statistical significance. Mean ± SD of five independent experiments. Statistical analyses were performed using two-way ANOVA, followed by Duncan’s post hoc test. *** p < 0.001; ** p < 0.01; * p < 0.05.

Figure 8

Percentage of necrotic cells, obtained by flow cytometric, after PDT treatment on HCT116, SKOV3, and MCF7 cells (Ctrl: black; C1: blue; C4: yellow; C8: green; C12: orange) and statistical significance. Mean ± SD of 5 independent experiments. Statistical analyses were performed using two-way ANOVA, followed by Duncan’s post hoc test. *** p < 0.001; ** p < 0.01; * p < 0.05.

Figure 9

Western blot and densitometric analysis of LC3-II levels in HCT116 (A), SKOV3 (B), and MCF7 (C) cells (2H: solid bars; Zn: striped bars). In control samples, PS treatment was omitted, and the mean ± SD of three independent experiments was included. Statistical analyses were performed using two-way ANOVA, followed by Duncan’s post hoc test. *** p < 0.001; ** p < 0.01; * p < 0.05.