Subcellular distributions and excited-state processes of hypericin in neurons

Photochemistry and Photobiology, Mar 1999 by English, Doug S, Doyle, Robert T, Petrich, Jacob W, Haydon, Philip G

ABSTRACT

The photodynamic drug, hypericin, is studied in fetal rat neurons using fluorescence microscopy. Hypericin has an extremely high affinity for the cell membrane and is found to a smaller extent in the nucleus. Fluorescent excitation of hypericin is shown to cause irreversible damage to the cell membranes of living neurons. Fixed cells were used to make ultrafast time-resolved measurements to avoid the deleterious effects of long-term exposure to intense light and room temperatures. To our knowledge, these are the first ultrafast time-resolved measurements of the fluorescence lifetime of hypericin in a subcellular environment. Nonexponential fluorescence decay is observed in hypericin in the neurons. This nonexponential decay is discussed in terms of other examples where nonexponential decay is induced in hypericin upon its binding to biomolecules. The nonradiative processes giving rise to the nonexponential hypericin decay are attributed to excited-state electron transfer, excited-state proton transfer or both.

'Abbreviations: DAPI, 4',6-diaidino-2-phenylindole dihydrochloride; DMSO, dimethyl sulfoxide; EBSS, Earl's balanced salt solution; HSA, human serum albumin; MMEM, modified minimum essential medium; NA, numerical aperture; PBS, phosphate-buffered saline.

INTRODUCTION

Hypericin is a naturally occurring polycyclic aromatic dione that has long been recognized as a powerful photodynamic agent. It is well known for its light-induced antiviral (1-5) and antitumor (6-9) activities. In addition, hypericin has also attracted attention for its antidepressant functions (10-12). Hypericin has been shown to have a remarkably fast uptake by cancer cells, strong photopotentiation against EMT-6/ED neoplastic cells (13) and has been used in clinical trials for the detection of stomach cancers (14). Fluorescence measurements using confocal microscopy have located hypericin in the membrane, cytoplasm and nucleus of T47D mammary tumor cells (15). Hypericin has been shown to effect a local pH drop when suspended in phospholipid vesicles (16), and recent measurements have confirmed an intracellular photoinduced pH decrease in 3T3 mouse fibroblasts (17,18). We have furthered our study of the interaction of hypericin with cellular components by examining the subcellular distribution in fetal rat neurons and investigating the effects of the cellular environment on the photophysics of hypericin. Here we report, based on measurements using time-resolved microscopy, how the fluorescence lifetime is affected by the cellular environment. We also examine the remarkable photodynamic action of hypericin on living cells using standard fluorescence microscopy and living fetal rat neurons.

MATERIALS AND METHODS

Cell culture. Hippocampi were dissected from E18 Sprague-Dawley rat embryos and incubated for IS min at room temperature in Earl's balanced salt solution (EBSS,t pH 7.35; Gibco) containing trypsin (0.25%; Sigma). The tissue was washed in fresh EBSS and placed in EBSS containing trypsin inhibitor (1.0%; Sigma) for 5 min. Hippocampi were rinsed and then mechanically dissociated by trituation with a glass pipet in minimal essential medium (MEM; Gibco) containing 10% fetal bovine serum, 20 mM L-glutamine, 10 mM Na pyruvate, 1000 IU/mL penicillin and 10000 (mu)g/mL streptomycin (Penn/Strep Gibco) (modified MEM, MMEM). Cells were plated onto 12 mm glass coverslips coated with poly-L-lysine (1 mg/mL; MW 10000; Sigma). Cultures were maintained at 36 deg C in a 5% CO^sub 2^/ 95% room air environment. The media was replaced 1 h after plating with serum-substituted MMEM containing N^sub 2^ (Gibco). This media results in cultures highly enriched in neurons (>95%) as shown by immunocytochemistry for glial fibrillary-associated protein and the synaptic vesicle protein, synaptotagmin (19). Additionally, electrophysiology studies confirm that cells with the structure shown in this investigation are indeed neuronal.

Hypericin loading. One microliter of hypericin (Calbiochem) was added to 1 mL of culture media (final concentration 10 (mu)g/mL) containing hippocampal cells 1 day in vitro. The cells were incubated in this mixture at 36 degC in 5% CO^sub 2^/95% room air for 30 min. Cells were then rinsed three times with normal saline (in mM: 140 NaCl, 5 KCI, 2 MgCI^sub 2^, 2 CaC1^sub 2^, 10 HEPES, pH 7.35) to remove excess hypericin. For experiments involving living cells the coverslips were mounted in a viewing chamber that allowed a flow of saline to maintain the cells. Other experiments used cells that were fixed in 4% formaldehyde in phosphate-buffered saline (PBS), pH 7.4 for 30 min at room temperature after loading with hypericin. The fixed cells were subsequently rinsed three times with PBS (5 min each rinse) and stored in PBS containing 0.02% NaN^sub 3^.

Hypericin localization. Hippocampal cells loaded with hypericin were imaged using a Nikon Diaphot TMD inverted fluorescence microscope using a 60x 1.4 numerical aperture (NA) oil immersion objective. Fluorescent images were captured using a Photometrics CH250 cooled charge-coupled device camera controlled by Metafluor (Universal Imaging Corporation) software. Hypericin was imaged with an XF34 filter set from Omega Optical (exciter 535DF35, emitter 0G590). Time lapse images of living cells were captured every 15 s with a 10 ms capture time. Single images and confocal sections were collected from fixed cells to determine subcellular location of hypericin. 4',6-Diaidino-2-phenylindole dihydrochloride (DAPI; Molecular Probes) was used to define the nucleus of cells. Hypericin-loaded cells were fixed in 4% formaldehyde, rinsed with PBS and incubated in PBS-DAPI (final concentration 2(mu)g/mL) for 30 min. The cells were washed three times with PBS, twice with doubly deionized H20 and mounted in n-propyl-gallate glycerol for imaging. The lack of crossover in signal from these two dyes was verified by imaging cells stained only with a single dye (e.g. DAPI [absorption 364 nm/emission 460 nm]) using the optical setting for detection of the second dye (e.g. hypericin [absorption 591 nm/emission 594 nm]). The Golgi apparatus was labeled with NBD C^usb 6^ ceramide (Molecular Probes N-1154). Cells were incubated for 30 min at 4 deg C in a 5 (mu)M solution of NBD C^sub 6^ ceramide/bovine serum albumin complexes. The cells were washed three times with normal saline and returned to culture media in the incubator for several hours before imaging with fluorescein optics. Because of spectral overlap between hypericin and NBD C^sub 6^ ceramide it was not possible to perform double labeling with these fluorescent probes. However, side-by-side comparisons indicate that hypericin does concentrate in an intracellular location adjacent to the nucleus, consistent with the position of the Golgi apparatus.