The biological activities of 5,15-diaryl-10,20-dihalogeno porphyrins for photodynamic therapy

Abstract

Purpose: Esophageal cancer is the most common gastrointestinal tumor and is difficult to be eradicated with conventional treatment. Porphyrin-based photosensitizers (PSs) mediated photodynamic therapy (PDT) could kill tumor cells with less damage to normal cells. As the most widely used porphyrin-based photosensitizer in clinics, Photofrin II has excellent anti-tumor effect. However, it has some disadvantages such as weak absorption at near infrared region, the complexity of components and prolonged skin photosensitivity. Here series novel 5,15-diaryl-10,20-dihalogeno porphyrin derivatives were afforded and evaluated to develop more effective and safer photosensitizers for tumor therapy.

Methods: The photophysical properties and singlet oxygen generation rates of 5,15-diaryl-10,20-dihalogeno porphyrins (I_1-6, II_1-4) were tested. The cytotoxicity of I_1-6 and II_1-4 were measured by MTT assay. The pathway of cell death was studied by flow cytometry. In vivo photodynamic efficacy of I₃ and II_2-4 in Eca-109 tumor-bearing BABL/c nude mice were measured and histopathological analysis were examined.

Results: 5,15-Diaryl-10,20-dihalogeno porphyrins I_1-6 and II_1-4 were synthesized. The longest absorption wavelength of these halogenated porphyrins (λ_max = 660 nm) displayed a red shift around 30 nm compared to the unhalogenated porphyrins PS₁ (λ_max = 630 nm). The singlet oxygen generation rates of I_1-6 and II_1-4 were significantly higher than PS₁ and HMME. All PSs mediated PDT showed obvious cytotoxic effect against Eca-109 cells compared to HMME in vitro and in vivo. Among these PSs, II₄ exhibited appropriate absorption in the phototherapeutic window, higher ¹O₂ generation rate (k = 0.0061 s^-1), the strongest phototoxicity (IC₅₀ = 0.4 μM), lower dark toxicity, high generation of intracellular ROS in Eca-109 cells and excellent photodynamic anti-tumor efficacy in vivo. Besides, cell necrosis was induced by compound II₄ mediated PDT.

Conclusion: All new compounds have obvious photodynamic anti-esophageal cancer effects. Among them, the photosensitizer II₄ showed excellent efficacy in vitro and in vivo, which has the potential to become a photodynamic anti-tumor drug.

Keywords: Anti-tumor; Halogenated porphyrin; Photodynamic therapy; Photosensitizer.

Fig. 1

The schematic diagram of II₄ mediated PDT

Fig. 2

The structures of 5,15-diaryl-10,20-dihalogeno porphyrins (I_1-6, II_1-4)

Fig. 3

FMOs and ΔE of PS₁, I₂, I₅, I₆

Fig. 4

The spectrum properties of compounds. A UV-Vis absorption spectra of compounds PS₁, I_1-6 and II_1-4 at 10 μM in DMSO. B Fluorescence excitation and emission spectra of compounds PS₁ in DMSO at the concentration of 10 μM

Fig. 5

The singlet oxygen generation of compounds. A Photodegradation of DPBF and compounds I₂, I₆, II₄ under 650 nm laser irradiation (5 mW/cm²) every 10 s. B Photodecomposition of DPBF in the presence of II₄ under 650 nm laser irradiation (5 mW/cm²) every 10 s. C First-order plot for the photodecomposition of DPBF after PDT with the compounds

Fig. 6

The efficacy of compounds against Eca-109 cells in vitro. A Dark cytotoxicity of compound I_1-6, II_1-4 at concentrations ranging from 0 to 30 μM in Eca-109 cells. B The cell viabilities treated by II₄ at concentrations ranging from 2 to 10 μM under different light doses. Data represents mean ± SD

Fig. 7

The fluorescence images of Eca-109 cells incubated with different PSs and probe DCFH-DA under light irradiation. Scale bar: 200 μm

Fig. 8

The extent and mode of cell death induced by different PSs-PDT. A Flow cytometric assay of I₃, II₂, II₃ and II₄ at 4 μM exposed to 2 J/cm² of light. LL: annexin V (−) PI (–), lived cell; LR: annexin V (+) PI (−), early apoptotic cells; UR: annexin V (+) PI (+), late apoptotic cells; UL annexin V (−), necrotic cells. B Histogram of apoptotic cells, necrosis cells and live cells after the treatment. Data represents mean ± SD

Fig. 9

Evaluation of tumor growth inhibition in the Eca-109-tumor-bearing model mediated by PDT. A Tumor images after 14 days. B Tumor growth curves after different treatments. *P < 0.05, **P < 0.001, ***P < 0.0001 vs HMME-PDT group. C Tumor weight. D Histological sections of tumor tissues stained with hematoxylin and eosin. Scale bar = 50 μm