Hypericin incorporation and localization in fixed HeLa cells for various conditions of fixation and incubation

Hypericin is a photosensitizer expressing high affinity for cancerous cells in vivo. Diagnosis of cancer based on hypericin fluorescence imaging has been successfully assessed in several clinical trials. Our final objective will be to evaluate the potential of hypericin fluorescence imaging to improve the efficacy of cervical cancer diagnosis performed on fixed cell smears obtained from liquid-based cytology. For this purpose, the mechanism of hypericin incorporation and localization in fixed HeLa cells using different incubation media and fixation conditions was investigated. Since the duration of fixation may play an important role, the influence of fixation time on hypericin incorporation in fixed HeLa cells was studied. The uptake and distribution of hypericin in fixed HeLa cells were found to be strongly dependent on the hypericin incubation medium: for a polar organic solvent such as the alcohol-based fixative, the localization was essentially perinuclear and nuclear; for cell culture medium supplemented with serum, the localization was cytoplasmic and non-specific; the highest incorporation was observed for the serum-free culture medium but mainly as non-fluorescent aggregates. The hypericin aggregation in the incubation medium, the passive diffusion and the partitioning between the cells and hypericin carriers seemed to be the major factors accounting for these results. The localization was found to be weakly dependent on fixation time, whereas fluctuations of hypericin fluorescence at short fixation time and stabilization after two days of fixation were observed. These results suggest that the fixed cells reached a steady state after two days of fixation.