Conjugation of chlorin p₆ to histamine enhances its cellular uptake and phototoxicity in oral cancer cells

Abstract

Purpose Our previous studies in hamster cheek pouch model have shown that chlorin p₆ (Cp₆), a chlorophyll derivative is a suitable photosensitizer for photodynamic treatment (PDT) of small tumors (<5 mm). However, for bigger tumors, the accumulation of Cp₆ was inadequate, which compromised the effectiveness of PDT. The purpose of present study was to investigate the possibility of improving the cellular uptake of Cp₆ by conjugating it to histamine, a biogenic amine that is known to modulate tumor growth and development via cell surface receptors. Methods The conjugate of Cp₆ and histamine (Cp₆-his) was prepared by carbodiimide coupling reaction. Cellular uptake, intracellular localization and cytotoxicity of both Cp₆ and its conjugate were investigated in two human oral cancer cell lines (4451 and NT8e). The percentage of necrotic and apoptotic cells after PDT were also estimated using Hoechst 33342-propidium iodide staining. Results In both the cell line, the cellular uptake of Cp₆-his was found to be ~10 times higher when compared to Cp₆. Histamine led to a slight increase in intracellular uptake of Cp₆-his, whereas ranitidine, a histamine H2 receptor antagonist, and incubation at lower temperature (~15°C) led to its inhibition, suggesting that uptake of Cp₆-his is receptor mediated. Results on western blot confirmed the presence of H2 receptor in both the cell line. Observations on intracellular localization revealed that unlike Cp₆, which localized on multiple sites, Cp₆-his showed localization on the cell membrane and around the perinuclear region. Moreover, the phototoxicity induced by Cp₆-his was ~4 times higher when compared to Cp₆ in both the cell lines. There was, however, no significant difference in the mode of cell death. Conclusion Results suggest that conjugating Cp₆ with histamine can help improve the effectiveness of PDT in oral cancer cells by enhancing its intracellular delivery.