Co-staining with DAPI and CellMaskTM green using fluorescence microscope imaging revealed accumulation of hypericin in cell membranes of Caco-2 cells

Hypericin is a natural polycyclic quinone found in Hypericum perforatum (HP). Hypericin is considered as one of the constituents in HP responsible for its antidepressant activity. Although hypericin reportedly has numerous pharmacological activities, only a limited number of studies have been performed on the absorption and transport characteristics of this compound, presumably, because hypericin is a highly lipophilic compound which is sparingly soluble in water. This apparent water insolubility makes the formulation of hypericin for clinical applications difficult. Previous in vitro data using Caco-2 cells proposed that passive diffusion is important for the delivery of hypericin into cells [1]. However, several investigations were conducted to examine hypericin transport in Caco-2 cells, and contradictory results were obtained [2, 3]. We here used for the first time fluorescence imaging to study the cellular uptake mechanism of hypericin in Caco-2 cells. Cells were grown on glass bottom dishes and incubated with 5µM hypericin for 1h. The cell membrane was stained with CellMask™ green plasma membrane stain, and the nucleus with DAPI. Fluorescence microscopy and imaging analysis suggest that hypericin was not co-localized in the nucleus but rather accumulated in the cell membrane (Figure 1G). Due to the lipophilicity of the compound and its high molecular weight (MW = 504.4) it seems that hypericin is not transported across cell membranes via passive diffusion but instead interacts with the phospholipid bilayer. The mechanism through which hypericin might overcome the hydrophobic barrier of cell membranes remains to be elucidated.

Fig. 1: Fluorescence images showing the accumulation of hypericin in cell membranes of Caco-2 cells. A) cells stained with CellMask™ green plasma membrane stain (green fluorescence), B) with DAPI (blue fluorescence) and C) after incubation with hypericin (5µM) for 1h (red fluorescence). D-G show colocalisation experiments with CellMask™ green plasma membrane stain + DAPI (D), DAPI + hypericin (E) CellMask™ green plasma membrane stain + hypericin (yellow fluorescence) (F) and CellMask™ green plasma membrane stain + DAPI + hypericin (G).

Keywords: Hypericin, membrane adhesion, Caco-2 cells, uptake mechanism, fluorescence micrsoscopy

References:

1) Sattler, S., Schäfer, U., Schneider, W., Hoelz, J. Lehr, C.M. Binding, Uptake and Transport of Hypericin by Caco-2 Cell Monolayers. J Pharm Sci. 1997; 86: 1120 – 1126

2) Saw, C.L., Olivo, M., Chin, W.W., Soo, K.C., Heng, P.W. Enhanced photodynamic activity of hypericin by penetration enhancer N-methyl pyrrolidone formulations in the chick chorioallantoic membrane model. Cancer Letters 2006; 238: 104 – 110

3) Weber, N.D., Murray, J.A., Wood, S.G. The antiviral agent hypericin has in vitro activity against Hsv1 through nonspecific association with viral and cellular membranes. Antivir Chem Chemother 1994; 5: 83 – 90