Application of a novel boronated porphyrin (H₂OCP) as a dual sensitizer for both PDT and BNCT

Abstract

Background and objective: Boronated porphyrins have emerged as promising dual sensitizers for use in both photodynamic therapy (PDT) and boron neutron capture therapy (BNCT), by virtue of their known tumor affinity, low cytotoxicity in dark conditions, and easy synthesis with high boron content. Octa-anionic 5,10,15,20-tetra[3,5-(nido-carboranylmethyl)phenyl] porphyrin (H₂OCP) is a boronated porphyrin having eight boron clusters linked to the porphyrin ring. To evaluate H₂OCP's applicability to both PDT and BNCT, we performed an in vitro and ex vivo study using F98 rat glioma cells.

Materials and methods: We examined the time-dependent cellular uptake of H₂OCP by measuring the boron concentration over time, and compared the cellular uptake/clearance of boron after exposure to H₂OCP in conjunction with boronophenylalanine (BPA) and sodium borocaptate (BSH), both of which are currently used in clinical BNCT studies. We evaluated the cytotoxicity of H₂OCP-mediated PDT using a colony-forming assay and assessed the tumorigenicity of the implantation of pre-treated cells using Kaplan-Meier survival curves. Fluorescence microscopy was also performed to evaluate the cellular uptake of H₂OCP.

Results: H₂OCP accumulated within cells to a greater extent than BPA/BSH, and H₂OCP was retained inside the cells to approximately the same extent as BSH. The cell-surviving fraction following laser irradiation (8 J/cm², 18 hours after exposure to 10 µg B/ml H₂OCP) was <0.05. The median survival times of the pre-treated cell-implanted rats were longer than those of the untreated group (P < 0.05). The fluorescence of H₂OCP was clearly demonstrated within the tumor cells by fluorescence microscopy.

Conclusions: H₂OCP has been proven to be a promising photosensitizer for PDT. H₂OCP has also been proposed as a potentially effective replacement of BPA or BSH, or as a replacement of both BPA/BSH. Our study provides more evidence that H₂OCP could be an effective novel dual sensitizing agent for use in both PDT and BNCT.

Fig. 1

Cellular uptake of boron (ng B/ml 10⁶ cells) after 6, 12, and 18 hours of exposure to 20 μg B/ml H₂OCP.

Fig. 2

Comparison of cellular uptake/clearance of boron (ng B/ml 10⁶ cells) after exposure to 20 μg B/ml of either H₂OCP, BPA, or BSH under identical conditions. Left and right Y-axes show the measured value (ng B/ml 10⁶ cells) and percentage of boron concentration, respectively. The cellular uptake of boron using H₂OCP showed values higher than those for BPA and BSH (P < 0.05), and the cellular retention of boron using H₂OCP showed values similar to those obtained using BSH.

Fig. 3

Colony-forming assay using F98 rat glioma cells exposed to 0 (control), 5, and 10 μg B/ml of H₂OCP and irradiated with light doses of 0, 2, 4, and 8 J/cm², respectively. The cell-surviving fraction following laser irradiation (8 J/cm², 18 hours after exposure to 10 μg B/ml H₂OCP) was < 0.05.

Fig. 4

Kaplan–Meier survival curves following in vitro pre-treated F98 cells using H₂OCP-mediated PDT. Rats were implanted i.c. with F98 cells and were either untreated (dotted line) or treated with PDT (continuous line). Cells were exposed to 20 μg B/ml of H₂OCP for 24 hours at 37°C prior to laser irradiation. After laser irradiation (4 J/cm²), the tumor cells were implanted into the rats. Cell viability was determined by trypan blue exclusion staining and 10⁵ viable cells were implanted stereotactically into the caudate nucleus. The median survival times of the untreated control and the treated group were 12 and 14 days, and the mean survival times were 11.8 and 14.6 days after implantation, respectively (P < 0.05).

Fig. 5

Images obtained using an inverted fluorescence microscope. A: Bright field image. Although the cells showed evidence of cytotoxic damage, the cytotoxicity of H₂OCP in dark conditions was not found at twice the concentration of H₂OCP used in this fluorescence microscopy experiment. B: Fluorescence of porphyrin H₂OCP. C: Nuclear fluorescence by Hoechst dye (excitation wavelength was 340–380 nm). D: Merged image. (Magnification of all images: ×600).