Time-Resolved Investigations of Singlet Oxygen Luminescence in

Jan 28, 2005 - In the past, red cell ghosts were frequently used for the detection of singlet oxygen luminescence in vitro.24,30 Red cell ghosts consi...
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J. Phys. Chem. B 2005, 109, 3041-3046

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Time-Resolved Investigations of Singlet Oxygen Luminescence in Water, in Phosphatidylcholine, and in Aqueous Suspensions of Phosphatidylcholine or HT29 Cells Ju1 rgen Baier,‡ Max Maier,‡ Roland Engl,‡ Michael Landthaler,† and Wolfgang Ba1 umler*,† Department of Dermatology, UniVersity of Regensburg, Germany, and Institute of Experimental and Applied Physics, UniVersity of Regensburg, Germany ReceiVed: September 30, 2004; In Final Form: NoVember 15, 2004

Singlet oxygen was generated by energy transfer from the photoexcited sensitizer, Photofrin or 9-acetoxy2,7,12,17-tetrakis-(beta-methoxyethyl)-porphycene (ATMPn), to molecular oxygen. Singlet oxygen was detected time-resolved by its luminescence at 1270 nm in an environment of increasing complexity, water (H2O), pure phosphatidylcholine, phosphatidylcholine in water (lipid suspensions), and aqueous suspensions of living cells. In the case of the lipid suspensions, the sensitizers accumulated in the lipids, whereas the localizations in the cells are the membranes containing phosphatidylcholine. By use of Photofrin, the measured luminescence decay times of singlet oxygen were 3.5 ( 0.5 µs in water, 14 ( 2 µs in lipid, 9 ( 2 µs in aqueous suspensions of lipid droplets, and 10 ( 3 µs in aqueous suspensions of human colonic cancer cells (HT29). The decay time in cell suspensions was much longer than in water and was comparable to the value in suspensions of phosphatidylcholine. That luminescence signal might be attributed to singlet oxygen decaying in the lipid areas of cellular membranes. The measured luminescence decay times of singlet oxygen excited by ATMPn in pure lipid and lipid suspensions were the same within the experimental error as for Photofrin. In contrast to experiments with Photofrin, the decay time in aqueous suspension of HT29 cells was 6 ( 2 µs when using ATMPn.

1. Introduction Singlet molecular oxygen is one of the most active intermediates involved in photosensitized oxygenation reaction in chemical and biological systems.1,2 Among others, singlet oxygen can be generated by an energy transfer of a light absorbing photosensitizer. Singlet oxygen is usually detected either by its chemical products (e.g., lipid peroxidation2,3) or by using specific quenchers (sodium azide).4 However, these indirect methods frequently yield no unequivocal results and no detailed insight into the primary mechanisms of action. The nonradiative deactivation of singlet oxygen is accompanied by radiative deactivation leading to IR luminescence at 1270 nm, which is widely employed for singlet oxygen detection.1,2 Usually, the decay time (τ∆) of singlet oxygen luminescence is measured using short laser pulses.5 The decay time is correlated to the respective environment of singlet oxygen which is mostly a solvent containing a photosensitizer. To measure the very weak luminescence intensity, highly sensitive detectors such as IR photomultipliers have to be used.2,6 Singlet oxygen luminescence was detected in the solvents, H2O7-9 or D2O.10,11 The luminescence detection was already performed in more complex environments such as aqueous suspensions of micelles,12 lipid vesicles,13 or in many other solvents.14,15 In H2O, a value of τ∆ ) 3.1 µs7 was reported; in D2O τ∆ ) 68 µs.11,15 In the case of micellar solutions, cetyltrimethylammonium bromide (CTAB)16 or BRIJ 35 micelles17 were added to aqueous * Author to whom correspondence may be addressed. Department of Dermatology, University of Regensburg, 93042 Regensburg, Germany. Phone: 49-941-944-9607. Fax: 49-941-944-8943. E-mail: baeumler.wolf [email protected]. † Department of Dermatology. ‡ Institute of Experimental and Applied Physics.

solutions, the respective decay times of singlet oxygen in such mixtures were nearly equal to the value of H2O. Only the decay times in D2O suspensions were affected when adding TritonX-10012,18 or cetyltrimethylammonium chloride (CTAC).18 When using phosphatidylcholine liposomes in H2O or D2O, the decay times were 5 or 71 µs, respectively.19 These values are close to the luminescence decay times in the pure solvents. However, the authors extrapolated the decay time of singlet oxygen in their experiments to get a value for singlet oxygen in pure phosphatidylcholine yielding 12.2 µs. In human plasma the decay time of singlet oxygen was also not measured but only extrapolated to be about 1 µs.20 For many years, a large number of experiments have been performed to detect singlet oxygen luminescence in a very complex environment such as cells. Assuming effective singlet oxygen quenching by H2O,7 mostly D2O cell suspensions were used.21-24 The observed decay time of 30-50 µs was attributed to singlet oxygen decaying in D2O and not in the cellular compartments. There, the singlet oxygen is assumed to be quenched very effectively by proteins22 with a very short luminescence decay time ranging between 30 and 180 ns.25 Recently, a direct optical detection of singlet oxygen from a single cell was reported showing for a D2O-equilibrated nerve cell a singlet oxygen lifetime of 2.7 µs.26 The aim of the present investigations was the detection of the singlet oxygen luminescence not only in aqueous solutions but also inside complex environments such as lipids or cellular membranes using a time-resolved detection system. To interpret the luminescence signal of complex structures, a system of increasing complexity based on H2O was applied: water, pure phosphatidylcholine, which is a major fatty acid in cellular membranes, aqueous suspensions of phosphatidylcholine, and aqueous suspensions of living cells. To generate singlet oxygen,

10.1021/jp0455531 CCC: $30.25 © 2005 American Chemical Society Published on Web 01/28/2005

3042 J. Phys. Chem. B, Vol. 109, No. 7, 2005 two different sensitizers were used. First, 9-acetoxy-2,7,12,17tetrakis-(beta-methoxyethyl)-porphycene (ATMPn) was used, which is a chemically pure, lipophilic porphin isomer27 and is not soluble in water. Second, the clinical approved sensitizer Photofrin was used, which tends to accumulate in lipids28 and in the membranes of living cells.29 In the past, red cell ghosts were frequently used for the detection of singlet oxygen luminescence in vitro.24,30 Red cell ghosts consist only of plasma membranes, therefore most of the singlet oxygen molecules escaped the very thin membranes and decayed in the surrounding water. Additionally, red cell ghosts show a very high protein concentration of 580 mg/mL31 with a high quenching rate constant,22 which might be responsible for the unsuccessful detection of the singlet oxygen luminescence in such cells.30 In contrast to that, the human colonic cancer cells (HT29) used in the present investigations are complete cells, i.e., singlet oxygen in the plasma membrane has approximately equal probability to escape to water outside the cell or an adjacent membrane inside the cell. A further advantage of the HT29 cells is that they have a low content of proteins in the plasma membranes (75 mg/mL32), which could lead to a longer decay time of singlet oxygen. 2. Materials and Methods 2.1. Solutions of Photofrin. Photofrin was dissolved in H2O (bi-distilled) at a concentration of 50 µg/mL. 2.2. Pure Phosphatidylcholine and Suspensions of Phosphatidylcholine. L-R-Phosphatidylcholine (Merck KGaA, Darmstadt, Germany) was added to H2O at a concentration of 15 mg/mL yielding a suspension of lipid droplets that is called lipid suspension. Photofrin (Photofrin, Sanofi, New York, NY) was added to the suspension at a concentration of 50 µg/mL in the suspension. The chemically pure, lipophilic ATMPn (gift of Dr. Alex Cross, Cytopharm Inc.) was dissolved in ethanol and added to the aqueous lipid suspension yielding a concentration of 50 µM in the suspension. After that, the ATMPn was only partially dissolved in the lipid droplets. To remove ethanol the suspension was dried at 70 °C and resuspended at a concentration of 15 mg/mL phosphatidylcholine/water. That leads to a homogeneously ATMPn-colored suspension. For experiments with pure phosphatidylcholine, the suspensions containing Photofrin or ATMPn were attached to a quartz plate and dried at room temperature for 24 h. 2.3. Cell Line and Sensitizer Incubation. The immortalized human colon carcinoma cell line HT29 was maintained in Dulbecco’s modified Eagle’s medium and supplemented with 10% fetal calf serum (Sigma Chemie), 1% L-glutamine, and 1% streptomycin/penicillin (Gibco, Eggenstein, Germany) in a humidified atmosphere of 8% carbon dioxide at 37 °C. Cells were washed with phosphate buffered saline (PBS; Biochrom, Berlin, Germany) and harvested by a treatment with 0.05% trypsin/0.02% EDTA (Gibco) in PBS. Cells were incubated with a Photofrin for 1.5 or 24 h with a concentration of 50 µg/mL in the supernatant. By use of ATMPn, cells were incubated for 24 h with a concentration of 50 µM in the supernatant. After washing the cells twice with PBS,33 7.5 × 106 cells were resuspended in 1 mL PBS. Cells for control experiments were processed in the same way. All cell suspensions were carried to experiments within 30 min. 2.4. Quenchers. Sodium azide or histidine (Merck KgaA, Darmstadt, Germany) were added to cell suspensions 1 min prior to irradiation, yielding a concentration of 50 mM of quencher in the respective suspension. 2.5. Subcellular Localization of Photofrin and ATMPn. The cells were seeded on a glass plate and incubated with the

Baier et al. respective sensitizers. To image simultaneously the cellular organelles, the cells were costained with the respective dye for lysosomes (LysoTrackerGreen, Molecular Probes, Eugene, OR) or for mitochondria (Rhodamine 6G, Sigma Aldrich, Taufkirchen, Germany) and investigated by fluorescence microscopy as previously described.27 2.6. Luminescence Experiments. The aqueous solutions and lipid or cell suspensions were transferred into a cuvette (QS-1000, Hellma Optik, Jena, Germany). The sensitizers were excited using a frequency-doubled Nd:YAG laser (PhotonEnergy, Ottensoos, Germany) with a repetition rate of 1.77 kHz (wavelength 532 nm, pulse duration 135 ns). The laser pulse energy for luminescence experiments was 56 µJ in aqueous solutions and 90 µJ for pure phosphatidylcholine, phosphatidylcholine, or cell suspensions. The singlet oxygen luminescence at 1270 nm was detected in near-backward direction with respect to the excitation beam using an IR-sensitive photomultiplier (R5509-42, Hamamatsu Photonics Deutschland GmbH, Herrsching, Germany) with a rise time of about 3 ns. At the entrance of the photomultiplier, two interference filters were used with a maximum transmission at 1270 nm, a half width of 25 nm (Schott, Mainz, Germany) or 13 nm (L.O.T. Oriel GmbH & Co. KG, Darmstadt, Germany), and a dielectric IR long-pass filter at 1110 nm (L.O.T. Oriel GmbH & Co. KG). The signal of the photomultiplier was amplified by a preamplifier (Model 6954, Philips Scientific, Ramsey, NJ). The signal was processed by a 7886S Dual Input Multiscaler (time-of-flight, Photon Counter, FAST Com Tec GmbH, Oberhaching, Germany) plugged into a personal computer. The channel width of the Dual Input Multiscaler was set to 128 ns. In general, the number of laser pulses for excitation was varied to optimize the signal-to-noise ratio of singlet oxygen luminescence, in particular in experiments of suspensions with lipids or cells. In experiments of solution, the luminescence was measured by using 104 laser pulses. All other experiments were performed by summing up repeated single experiments each at 2 × 104 laser pulses using always a fresh suspension. We used one experiment for phosphatidylcholine (pure or suspension, Photofrin). We added up 3 experiments for phosphatidylcholine (pure or suspension, ATMPn) and 10 for cell suspensions with either Photofrin or ATMPn. When adding sodium azide to the cell suspensions, the luminescence signal of eight (ATMPn) or three (Photofrin) single experiments were summed. To check whether phosphorescence of the photosensitizer is present in the wavelength range of singlet oxygen luminescence, interference filters at 1270 nm were replaced by an interference filter (Coherent, Dieburg, Germany) with a maximum transmission at 1160 nm (half width 18 nm). An aqueous suspension of HT29 cells incubated with 50 µg/mL Photofrin for 1.5 h was irradiated, and the signal at 1160 nm was measured. Except an intense peak restricted to less than 0.5 µs, which is due to stray light, no signal was detected. Thus, no Photofrin phosphorescence was present for wavelengths of 1160 nm and above. We have also measured the time-integrated luminescence as a function of wavelength for Photofrin and ATMPn in airsaturated solution using a monochromator (HORIBA, Jobin Yvon Inc., USA). The spectral resolution was 9 nm. No signal was detected for wavelengths outside the spectral band of singlet oxygen transition. 2.7. Determination of Singlet Oxygen Luminescence Decay Time. The time resolution of the complete detection system (photomultiplier and detection electronics) is excellent (128 ns). However, the error bars of the rise and decay time of the luminescence in the present experiments are much larger

Singlet Oxygen Luminescence in Water

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than the time resolution because of the poor signal-to-noise ratio of the extremely weak luminescence, in particular for cell suspensions. Singlet oxygen was detected in complex heterogeneous structures such as phosphatidylcholine suspensions and cell suspensions where diffusion between different compartments is possible. Since the respective environment of singlet oxygen determines its luminescence decay time, multiexponential luminescence decay can occur. However, when the diffusion length of singlet oxygen is large enough during its lifetime, singlet oxygen equilibrium in different compartments is established. In this case, the signal decay is monoexponential2 with an intermediate decay time. The main constituents of the investigated suspensions are H2O, lipids and proteins. Proteins yield a very short decay time due to their high quenching rate. In lipids, contributions to singlet oxygen luminescence were expected, showing a decay time of about 12 µs.19 The decay time is 3.1 µs in H2O.7 In the experiments, we observed a biexponential time dependence consisting of a monoexponential rise and a monoexponential decay. The luminescence intensity is then given by

I(t) )

τR

-1

[ ( )

( )]

C t t exp - exp -1 τ τ - τD D R

(1)

The constant C was used to fit the luminescence signal. τD and τR are the decay and rise times, respectively. To determine the rise and decay times of singlet oxygen, the least-squares fit routine of Mathematica 4.2 (Wolfram Research) was used. The experimental error of the fit was estimated to be between 15 and 40% of the value determined by the fit, depending on the investigated sample. It should be noted that rise times shorter than 1 µs cannot be measured accurately because of the low signal-to-noise ratio. 3. Results and Discussion The clinically approved photosensitizer Photofrin has been frequently investigated regarding its photodynamic action over the last years.29,34 Photofrin is a complex mixture of Porphyrin monomers and oligomers that partially aggregates in aqueous solution.35 Therefore, the chemically pure sensitizer ATMPn was additionally used in the present study. The time dependence of the singlet oxygen luminescence at 1270 nm, which was generated by energy transfer from Photofrin, was investigated in four systems with increasing complexity: first, in a homogeneous solution of Photofrin in water (H2O); second, in the pure lipid; third, in a heterogeneous aqueous suspension of phosphatidylcholine droplets; and fourth, in aqueous suspensions of HT29 cells. Since ATMPn is not water soluble, that sensitizer was used for pure phosphatidylcholine, for lipid suspensions, and for HT29 cells. 3.1. Aqueous Solutions. Figure 1a shows the results of the measurement (solid points) of the intensity of the singlet oxygen luminescence in an air-saturated solution of Photofrin (50 µg/ mL) in water as a function of time together with the fit curve (eq 1, solid line). The signal rises with a time constant of 1.5 ( 0.5 µs, which is determined by the nonradiative lifetime of the triplet T1 state of Photofrin in the air-saturated solution. The luminescence decay time was 3.5 ( 0.5 µs, which is the decay time of singlet oxygen in water. That value is in good agreement with the literature (3.1 µs).7 3.2. Pure Phosphatidylcholine and Aqueous Suspensions of Phosphatidylcholine. After adding a sensitizer, the luminescence of singlet oxygen was measured in pure phosphati-

Figure 1. Luminescence of singlet oxygen at 1270 nm generated by 50 µg/mL Photofrin vs time in (a) H2O, (b) suspension of 15 mg/mL phosphatidylcholine in H2O, (c) suspension of HT29 cells in H2O (incubation time 1.5 h) without quencher, (d) suspension of HT29 cells in H2O (incubation time 1.5 h) with 50 mM NaN3. To improve the signal-to-noise ratio of singlet oxygen luminescence, a different number of laser pulses were applied for the different experiments.

dylcholine attached to a glass plate (without water). By use of Photofrin, the rise and decay times of the luminescence were 2.7 ( 0.5 and 14 ( 2 µs, whereas for ATMPn, the rise and decay times were 2.2 ( 0.5 and 13 ( 2 µs. A comparable value of about 12 µs has been extrapolated for singlet oxygen generated in phosphatidylcholine liposomes.19

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Baier et al.

TABLE 1: Decay Times of Singlet Oxygen Luminescence singlet oxygen decay time (µs) solvent (incubation time)

Photofrin

H2O pure lipid lipid/H2O cells/H2O (1.5 h) cells/H2O (24 h) cells/H2O/NaN3 (1.5 h) cells/H2O/NaN3 (24 h) cells/H2O/histidine (1.5 h) cells/H2O/histidine (24 h)

3.5 ( 0.5a 14 ( 2b 9(2 10 ( 3 11 ( 3 5(2 4(2 c c

ATMPn 13 ( 2b 10 ( 2 6(2 2.5 ( 1.0 c

a

Reference 12: singlet oxygen decay time was 3.1 µs. b Reference 19: extrapolated singlet oxygen decay time was 12.2 µs. c Signal-tonoise ratio was too low to determine a decay time.

When adding the phosphatidylcholine to water, droplets of the fatty acid were formed. It has been shown that Photofrin is mainly localized in lipids when added to aqueous suspensions of fatty acids.28 By use of Photofrin, the luminescence intensity of singlet oxygen in the aqueous suspension of phosphatidylcholine droplets together with the fit curve are shown in Figure 1b. Within the experimental error, single biexponential time dependence was observed with rise and decay times of the luminescence of 1.6 ( 0.5 and 9 ( 2 µs, respectively (Table 1). Since ATMPn is not water soluble, it can be assumed that the sensitizer is restricted to the lipid droplets. Thus, singlet oxygen is generated exclusively in the lipid droplets. By use of ATMPn, the decay time of singlet oxygen luminescence (see Figure 2a) was 10 ( 2 µs, which is comparable to Photofrin (Table 1). With both sensitizers, the presence of water shortened the decay time of singlet oxygen luminescence to some extent as compared to the pure lipid (Table 1). In a system with two phases, lipid droplets and water, one may expect in principle 2-fold biexponential time dependence due to the two contributions to the luminescence, one from the lipid droplets and the other from water. However, the observed time dependence can also be single biexponential for the following reasons: The luminescence intensity of singlet oxygen depends on the number of singlet oxygen molecules in each phase. Since singlet oxygen is predominantly (Photofrin) or exclusively (ATMPn) generated in the lipid phase, the contribution of water to the luminescence depends on the rate of exchange between lipid and water (see below) and may be negligibly small. Diffusion of singlet oxygen has been frequently considered,14,16,18,35 in particular when it takes place through the interface between an exterior and an interior phase leading to an exchange of singlet oxygen molecules between both phases. In this case, the important factors are the values of the rates of entry and exit of singlet oxygen in to and out of the different compartments relative to the decay rates in these compartments.35 It can be shown under simplifying assumptions that the singlet oxygen populations in both phases can be coupled exhibiting the same decay time. The value of the common singlet oxygen decay time depends on the rate of exchange of singlet oxygen molecules ranging between that of singlet oxygen in the exterior and interior phase. To estimate the importance of the rate of exchange of singlet oxygen between lipids and water, we have estimated the average length of diffusion l of singlet oxygen during its lifetime. The diffusion coefficient of oxygen in water is DH2O ) 2 × 10-5 cm2/s.30,36-38 The diffusion coefficients DM for the cytoplasm and for membranes given in the literature10,30,36,37 range from 0.7 to 1.4 × 10-5 cm2/s. To get an upper limit for the diffusion

Figure 2. Luminescence of singlet oxygen at 1270 nm generated by 50 µM ATMPn vs time in (a) suspension of 15 mg/mL phosphatidylcholine in H2O, (b) suspension of HT29 cells in H2O (incubation time 24 h) without quencher, (c) suspension of HT29 cells in H2O (incubation time 24 h) with 50 mM NaN3. To improve the signal-to-noise ratio of singlet oxygen luminescence, a different number of laser pulses were applied for the different experiments.

length of singlet oxygen, we use the lifetime τD ) 14 µs in the pure lipid and a diffusion coefficient of D ) 2 × 10-5 cm2/s. The diffusion length is calculated2 from ∆x ) (6Dτ)1/2 leading to a value of l ) 0.4 µm. As detected by normal microscopy, the diameter of the lipid droplets ranged from several tens of micrometers down to 0.5 µm (resolution of the microscope). Especially for droplets with a diameter larger than 1 µm, there is a high probability for singlet oxygen to decay within phosphatidylcholine. That would lead to a decay time comparable to pure phosphatidylcholine. However, singlet oxygen can escape into the aqueous environment when generated in the small droplets (diameter of the order of or less than the diffusion length l ) 0.4 µm) or at the margins of the larger droplets. Since both possibilities may take place at the same time, this could explain the measured intermediate decay time of 9 (Photofrin) or 10 µs (ATMPn) in the luminescence experiments of the suspensions, which is

Singlet Oxygen Luminescence in Water shorter than the value for pure phosphatidylcholine (14 µs) and larger than the value of water (3.5 µs). 3.3. HT29 Cell Suspensions. By use of the fluorescence microscopy, the subcellular localization of Photofrin for incubation times of 1.5 h was the plasma membrane. For incubation times of 24 h, the Photofrin fluorescence was mostly restricted to the mitochondria of HT29 cells, in particular to the mitochondrial membranes. After incubation of HT29 cells, ATMPn localizes in the membranes of the lysosomes, which confirms previous experiments.27 The cells without Photofrin or ATMPn (control cells) yielded no luminescence at 1270 nm. For luminescence experiments, HT29 cells were incubated with either ATMPn (24 h) or Photofrin (1.5 or 24 h) and suspended in air-saturated H2O. An example of the time-resolved intensity of the singlet oxygen luminescence from the aqueous HT29 cell suspension incubated with Photofrin for 1.5 h is shown in Figure 1c and for ATMPn in Figure 2b. Within the experimental error, a monoexponential decay was observed. The decay time of the singlet oxygen luminescence at 1270 nm in the cell suspension incubated with photofrin determined from the fit is 10 ( 3 µs, i.e., comparable to the decay time of singlet oxygen in the aqueous phosphatidylcholine suspensions of about 9 µs. After an incubation time of 24 h, the decay time was 11 ( 3 µs (Table 1). No singlet oxygen luminescence contribution with short decay times, e.g., from water or proteins, was observed within the experimental error. When using HT29 cells incubated with ATMPn, the decay time of singlet oxygen luminescence was 6 ( 2 µs, which is shorter as compared to experiments with Photofrin. However, that decay time of singlet oxygen is still clearly longer than for water (3.5 µs). The obvious difference of decay times when using Photofrin or ATMPn remains unclear, so far. However, Photofrin and ATMPn are localized in different compartments in the HT29 cells, which may lead to the following suggestions. First, the protein concentration of the plasma membrane and mitochondrial membranes might be different as compared to the lysosome membranes in HT29 cells. Since proteins and the amino acids therein are very effective quenchers of singlet oxygen, different decay times of singlet oxygen luminescence may occur. Second, the proteins in the compartments may contain different concentrations of amino acids with different quenching rate constants,39 in the case of the lysosomes and ATMPn that may lead to a different quenching of singlet oxygen and therefore to a shortening of the decay time as compared to mitochondria and the plasma membrane containing Photofrin. When incubating the HT29 cells with Photofrin or ATMPn, the photosensitizer molecules were located in the cellular membranes, which are fluid, heterogeneous mosaics of proteins and lipids. After being generated in the respective cellular membranes, singlet oxygen can distribute by diffusion within its respective decay time. But, the estimation of the diffusion length of l ) 0.4 µm presented in section 3.2 shows that, for a cell diameter of about 10 µm, singlet oxygen generated in membranes inside the cells stays mainly within the cells. For singlet oxygen in the plasma membrane (1.5 h incubation time of Photofrin), there is about equal probability for diffusion into the cell (cytoplasm) or out of the cell (surrounding water). When diffusion is important, an intermediate time is expected between the decay times of the lipids, the proteins, and the surrounding H2O, with decay times of 14 µs, less than 0.526,39 and 3.5 µs, respectively. The photomultiplier detected a luminescence signal decaying monoexponentially with a decay

J. Phys. Chem. B, Vol. 109, No. 7, 2005 3045 time of about 11, 10, or 6 µs. In view of the rather long decay time, the singlet oxygen luminescence detected might be attributed to singlet oxygen decaying mainly in the lipid domain of cellular membranes. However, we cannot exclude the possibility that the experimentally observed decay time is an average value corresponding to different microenvironments of singlet oxygen. Sodium azide (quenching rate constant (2.5 ( 0.6) × 108 s-1 M-1, using 8-MOP in D2O)6 and histidine ((4.6 ( 0.4) × 107 s-1 M-1, using 8-MOP in D2O)6 are potential quenchers of singlet oxygen. By addition of 50 mM sodium azide to the cell suspensions, both the luminescence intensity and the decay time of singlet oxygen decreased yielding decay times of 5 ( 2 (Photofrin) or 2.5 ( 1.0 µs (ATMPn) (Figures 1d and 2c, respectively), which additionally confirms that the luminescence signal is due to singlet oxygen. Sodium azide may also quench the triplet state of Photofrin and ATMPn. However, the quenching rate constant of the first triplet state of Photofrin15 by sodium azide is smaller than 107 s-1 M-1, whereas for ATMPn,40 the value is (2.0 ( 0.4) × 107 s-1 M-1. Both values are smaller as compared to singlet oxygen. The plasma membrane is not a coplanar and smooth layer but shows a rough and complex structure being able to transport polar molecules through such a membrane. It is known that sodium azide is taken up by cells, e.g., sodium azide is used for instance as an inhibitor of the mitochondrial respiratory chain.41 This leads to a close contact of transported sodium azide molecules to the membrane constituents. Thus, it can be assumed that part of sodium azide gets into contact with part of singlet oxygen decaying in the membranes. The latter is in accordance with the present results of the luminescence experiments. The measured quenching of the singlet oxygen luminescence is not complete, showing decay times of about 5 or 2.5 µs. The quenching of singlet oxygen was more impressive when adding the quencher histidine to cell suspensions. When using Photofrin or ATMPn, the luminescence at 1270 nm completely disappeared (see Table 1). Conclusions The singlet oxygen luminescence was detected in different environments of increasing complexity. First, the good time resolution and sensitivity of the detection system was clearly demonstrated by measuring not only the decay time of singlet oxygen in water (H2O) but also the rise time of the luminescence signal. Second, the decay time of singlet oxygen was measured for pure phosphatidylcholine, which is a major cellular constituent, as well as for aqueous suspensions of phosphatidylcholine. Third, when singlet oxygen was generated by Photofrin in the plasma membrane or the mitochondrial membranes of living HT29 cells, a luminescence decay time was found comparable to phosphatidylcholine suspensions. The observed luminescence might be attributed to singlet oxygen decaying inside cells, although it is possible that the observed decay time is an average value corresponding to different microenvironments of singlet oxygen. The photosensitizer ATMPn localizes in lysosome membranes in HT29 cells. In this case, the measured luminescence decay time of singlet oxygen was shorter than in the case of photofrin but longer than in water. References and Notes (1) Krasnovsky, A. A., Jr. Membr. Cell. Biol. 1998, 12, 665. (2) Schweitzer, C.; Schmidt, R. Chem. ReV. 2003, 103, 1685. (3) Wassell, J.; Davies, S.; Bardsley, W.; Boulton, M. J. Biol. Chem. 1999, 274, 23828.

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