Direct Near-infrared Luminescence Detection of Singlet Oxygen Generated by Photodynamic Therapy in Cells In Vitro and Tissues In Vivo

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1 Photochemistry and Photobiology, 2002, 75(4): Direct Near-infrared Luminescence Detection of Singlet Oxygen Generated by Photodynamic Therapy in Cells In Vitro and Tissues In Vivo Mark Niedre, Michael S. Patterson 2 and Brian C. Wilson* Department of Medical Biophysics, Ontario Cancer Institute/University of Toronto, Toronto, Canada and 2 Hamilton Regional Cancer Center/McMaster University, Hamilton, Canada Received 26 June 200; accepted 9 January 2002 ABSTRACT Singlet oxygen ( O 2 ) is believed to be the major cytotoxic agent involved in photodynamic therapy (PDT). Measurement of O 2 near-infrared (NIR) luminescence at 270 nm in biological environments is confounded by the strongly reduced O 2 lifetime and probably has never been achieved. We present evidence that this is now possible, using a new NIR-sensitive photomultiplier tube. Time-resolved O 2 luminescence measurements were made in various solutions of aluminum tetrasulphonated phthalocyanine (AlS 4 Pc) and Photofrin. Measurements were also performed on suspensions of leukemia cells incubated with AlS 4 Pc, and a true intracellular component of the O 2 signal was clearly identified. Time-resolved analysis showed a strongly reduced O 2 lifetime and an increased photosensitizer triplet-state lifetime in the intracellular component. In vivo measurements were performed on normal skin and liver of Wistar rats sensitized with 50 mg/kg AlS 4 Pc. In each case, a small but statistically significant spectral peak was observed at 270 nm. The O 2 lifetime based on photon count rate measurements at 270 nm was s, consistent with published upper limits. We believe that these are the first direct observations of PDT-generated intracellular and in vivo O 2. The detector technology provides a new tool for PDT research and possibly clinical use. INTRODUCTION Photodynamic Therapy (PDT) is a minimally invasive treatment modality for cancer and other conditions (,2). The Posted on the web site on January 28, *To whom correspondence should be addressed at: Department of Medical Biophysics, Ontario Cancer Institute, 60 University Avenue, Toronto, ON M5G 2M9, Canada. wilson@oci. utoronto.ca Abbreviations: AlS 4 Pc, tetrasulfonated aluminum phthalocyanine; BSA, bovine serum albumin; D 2 O, deuterium oxide; FWHM, full width at half maximum; HSA, human serum albumin; MCS, multichannel scaler; NaN 3, sodium azide; NIR, near infrared; O 2, singlet oxygen; OD, optical density; OPO, optical parametric oscillator; PDT, photodynamic therapy; PMT, photomultiplier tube; PS, photosensitizer American Society for Photobiology /02 $ therapy utilizes a photosensitizing drug, usually administered systemically or topically, which may be preferentially localized in, for example, solid tumors. The photosensitizer is then irradiated with a light source tuned to a wavelength to match the absorption spectrum of the drug. The subsequent photochemical reaction results in oxygen-mediated destruction or modification of the target tissue. The main cytotoxic agent in PDT is widely believed (3) to be singlet oxygen [ O 2 ( g )], a highly reactive oxygen species that oxidizes biological substrates. Critical sites of action for O 2 in PDT include mitochondria, DNA and lipid membranes (4,5). Sustained exposure of the treated tissue to O 2 results in breakdown of cellular microstructures and cell death. O 2 is produced by the following Type-II photochemical pathway (6): S h S 0 0 S T T 3O S 2 0 O 2 (a) (b) (c) where S 0,S and T are the photosensitizer ground state, first excited singlet state and first excited triplet state, respectively and 3 O 2 and O 2 are the ground-state triplet and excited singlet states of oxygen, respectively. Once produced, a molecule of O 2 can undergo nonradiative decay, oxidize surrounding biomolecules or undergo radiative decay at around 270 nm. The time-resolved measurement of this near-infrared (NIR) emission is a commonly used method for determining O 2 lifetimes and quantum yields in solution (7). Measurement of the NIR luminescence during PDT treatment is potentially of value as a direct dosimetry metric (8), so that the ability to detect O 2 luminescence in true biological environments has been attempted previously. Several investigators have reported positive results from cells in suspension (9,0) or red cell ghosts (). However, these have either required the use of deuterium oxide (D 2 O) to increase the lifetime of O 2 and eliminate absorption of the 270 nm luminescence by H 2 O or did not adequately distinguish between intracellular and extracellular O 2. Hence, the results do not reflect optical and photophysical conditions present in vitro or in vivo and so should not be interpreted as successful O 2 measurements in biological environments. 382

2 Photochemistry and Photobiology, 2002, 75(4) 383 Attempts to measure O 2 luminescence have also been made in vivo by several investigators. These have either produced outright negative results (6) or in one case (2) produced apparently positive results in a single animal that were not subsequently reproduced. Hence, the direct measurement of O 2 luminescence in any biological environment has probably never been reliably achieved. These failures have been attributed to the strongly decreased lifetime of O 2 in cells and tissues caused by rapid quenching by biomolecules, combined with a lack of adequately sensitive detectors at NIR wavelengths, because, as will be discussed subsequently (see Eq. 5), the total O 2 luminescence emission is proportional to the lifetime. The lifetime of O 2 in vivo has been estimated by various methods. We previously placed an upper limit of 500 ns on the lifetime, based on the known sensitivity of a germanium photodetector in an NIR luminescence instrument (6). Moan and Berg estimated the lifetime as approximately 0 40 ns, on the basis of the diffusion distance of O 2 in cell membranes as determined by the photobleaching rate of one photosensitizer because of O 2 photogenerated in a second (3), whereas Baker and Kanofsky estimated the lifetime in cells to be approximately 200 ns using the values determined in detergent-dispersed cells of increasing concentration and extrapolating to in vivo cell density (4). In this paper, we report on the use of a novel photomultiplier tube (PMT) in detecting O 2 luminescence in cells in vitro and in tissues in vivo. This PMT is uniquely sensitive in the NIR region and, we believe, for the first time has allowed detection of O 2 luminescence in true biological media during PDT. MATERIALS AND METHODS Theory. As described by Patterson et al. (6), three coupled differential equations can be written describing the kinetics of the aforementioned PDT photochemical reactions: d[s ] [S 0] [S ] (2a) dt S d[t ] T [S ] [T ] dt S T (2b) d[ O 2] D [T ] [ O 2] dt (2c) T T where is the local fluence rate (photons per second per square centimeter), is the photosensitizer ground state absorption cross section (cm 2 ), T is the photosensitizer triplet state quantum yield, D is the O 2 quantum yield, S is the photosensitizer singlet-state lifetime, T is the photosensitizer triplet-state lifetime and D is the O 2 lifetime. The concentrations of each of the photosensitizer states ([S 0 ], [S ], [T ]) and of O 2 ([ O 2 ]) are expressed in molecules per cubic centimeter. For an excitation pulse, N (t), where N is number of photons per square centimeter incident on the sample at time t 0, and assuming that the triplet-state molecules are created instantaneously after excitation (valid for s K T ), it can be shown that the O 2 concentration at time t is: D t t [O](t) N [S ] exp exp (3) 2 0 D D [ ] T D T D Hence, for a sufficiently short excitation pulse width, such that pulse K T and pulse K D, this equation approximates the O 2 luminescence time decay curve. The O 2 luminescence emission (photons per cubic centimeter per second) at time t is then: Figure. Schematic of the experimental system used for O 2 luminescence detection. Inset: purpose built animal holder used for in vivo experiments. Excitation light was delivered through the circular port (a), and measurements made through a second port at 90 (not seen). The animal was held in the hemispheric cylinder (b). [O](t) 2 R L (t) (4) 270 where R is the O 2 radiative lifetime in a given solvent. The total number of photons emitted after a single excitation pulse is the integral of Eq. (4) over all t: N [S 0] D D L (t) dt (5) 270 Note that the luminescence signal decreases with the O 2 lifetime, creating the fundamental challenge in detecting the emission in biological media. As will be shown experimentally, there are other potential sources of light emission in the 270 nm region besides O 2 luminescence, including photosensitizer fluorescence and phosphorescence, autofluorescence from the biological medium and fluorescence from optical components in the system. Here, time-resolved or time-gated detection was used to reduce this fluorescence background signal, exploiting the fact that this is generally prompt compared with the O 2 emission. Any residual fluorescence, although minimal in most cases, was subtracted as described subsequently. Apparatus. The optical excitation and detection system is shown schematically in Fig.. A tunable pulsed laser system (OPO Rainbow 355, OPOTEK Inc., Carlsbad, CA) comprising a nonlinear optical parametric oscillator (OPO) pumped by the second (532 nm) and third (355 nm) harmonics of a Q-switched Nd:YAG laser was used as the excitation source, tuned to the appropriate wavelength to excite the photosensitizer. The laser light was passed through a bandpass filter centered at the excitation wavelength (630 nm, 0 nm bandpass or 670 nm, 0 nm bandpass, OD4 blocking filters, CVI Laser Corp., Albuquerque, NM) and focused onto the sample using an f/ lens (PLCX UV, CVI). The pulse duration was 20 ns, the pulse repetition frequency 0 Hz and the pulse energy at the sample mj. This resulted in an average power at the sample of 0 2 mw over a 3 mm diameter spot and an instantaneous power of 50 kw during the laser pulse. Light from the sample was collected using an f/ lens (BICX UV, CVI) set at 90 to the excitation beam. A 000 nm longpass filter (model 58867, OD3 blocking, Oriel, Stratford, CT) and an 800 nm longpass filter (5736, OD2 blocking, Oriel) were used to remove unwanted scattered excitation light and fluorescence from the sample. Four bandpass filters at 20 nm (9 nm bandpass, OD6 blocking, Andover Corp., Lawrence, MA), 272 nm (8 nm bandpass, OD6 blocking, Andover), 30 nm (0 nm bandpass, OD4 blocking, CVI) and 329 nm (0 nm bandpass, OD4 blocking, CVI) were mounted side by side on a sliding stage in front of the detector. For simplicity, these will be referred to as the 200, 270, 300 and 330 nm filters, R

3 384 Mark Niedre et al. respectively. The 270 nm filter corresponds to the peak of the O 2 luminescence spectrum, whereas the 300 nm filter is off peak but still within the O 2 emission band ( 5% of peak). The 200 nm and 330 nm filters, which lie outside the O 2 band, were used to determine the background fluorescence. The latter also served to check for a potential water-absorption artifact (see subsequent discussion). For some later experiments, two additional filters at 250 nm (0 nm bandpass, OD4 blocking, CVI) and 285 nm (0 nm bandpass, OD4 blocking, CVI) were added to provide further spectral resolution. The overall numerical aperture of the detection system was approximately. The detector was a liquid nitrogen cooled PMT (model R , Hamamatsu Corp., Bridgewater, NJ). This has a uniquely broad spectral response from 300 to 400 nm and therefore enabled extremely sensitive detection in the nm range. Its rapid temporal response (3 ns) allowed photon counting of the O 2 luminescence. The operating voltage was set at 500 V, at which the dark current was na, resulting in negligible dark counts. The PMT output was amplified and converted to a voltage pulse using a high-speed current preamplifier (model SR445, Stanford Research Systems, Sunnyvale, CA). A multichannel scaler (MCS; model SR430, Stanford) connected to a personal computer was used for time-resolved single photon counting, with a typical temporal resolution of 80 ns. For some experiments, a dual-channel photon counter (SR-400, Stanford) was used instead of the MCS to give the time-integrated luminescence signal. The PMT has a quantum efficiency of 0.9% at 270 nm and, when operated in photon-counting mode, approximately dark counts per second. For a continuous source, the minimum detectable signal (SNR )withasintegration time is therefore W. This is approximately an order of magnitude lower than the signal detectable with a liquid nitrogen cooled germanium detector (6) under comparable conditions. It is primarily this improvement in sensitivity that has enabled the successful detection of O 2 luminescence from photodynamic sensitizers in cells and tissues, as reported subsequently. Data collection. At each of the NIR detection wavelengths the signal was summed over many laser pulses (at 0 Hz), typically , giving data collection times of s per wavelength. Measurements were made in either time-integrated or time-resolved mode. In the former, the delayed luminescence in the time interval 5 s in solution and 0 s in vivo after the laser pulse was summed, thereby removing the fast fluorescence component. This yielded the time-integrated spectrum of the light emitted from the sample. Because the absolute luminescence intensities varied from sample to sample (particularly in vivo), each spectrum was corrected for the system response at each wavelength and normalized to the signal at 200 nm. The mean spectrum for each control solution, cell suspension or tissue without photosensitizer was then subtracted from that of the corresponding photosensitized sample. For time-resolved measurements, the complete time curve was measured at 270 nm, and the background (i.e. the time curve from a control sample at 270 nm) was subtracted from it. Equation (6), a simplified version of Eq. (3), was then chi squared fitted to the data using commercial software (mmnlfit.m, Matlab 5, The Mathworks Inc., Natick, MA), with T, D anda( N [S 0 ] D ) as free parameters. D t t [O] A exp exp (6) ( ) [ ] 2 T D T D Experiments in solution. Solutions of photosensitizer were measured in quartz cuvettes ( 4 cm), firstly for 2.5 M tetrasulfonated aluminum phthalocyanine (AlS 4 Pc; Porphyrin Products, UT) in methanol and water, and Photofrin (QLT Phototherapeutics, Vancouver, BC, Canada) in methanol to confirm that the lifetimes were in agreement with literature values. Subsequently, all solution studies were done with 6 M AlS 4 Pc. The biophysical complexity was then increased by adding bovine serum albumin (BSA; Sigma Chemical Co., St. Louis, MO) in water up to a molar ratio of [BSA]/ [AlS 4 Pc] 400, to provide a protein-rich environment to bind the photosensitizer and possibly quench O 2. In order to confirm that the 270 nm signal was due to O 2,a known O 2 quencher (5), sodium azide (NaN 3 ; Sigma), was added to AlS 4 Pc in water up to a concentration of 2 M. As a final check that the background corrected signal was caused only by O 2 luminescence, a solution of PS and BSA ([BSA]/[AlS 4 Pc] 200) was prepared. NaN 3 was then added to the solution so that the final concentration of NaN 3 in the cuvette was 0.5 M, and the full time signal was measured. Because the NaN 3 completely quenches the O 2 luminescence but has little effect on the photosensitizer triplet state, the intent was to rule out the (unlikely) possibility that the triplet-state phosphorescence has a spectral peak in the 270 nm region and so could be mistaken for the O 2 signal, for which the kinetics would be similar. The rationale was that the NaN 3 would completely quench the O 2 luminescence but not affect any photosensitizer (PS) triplet-state phosphorescence. Experiments in cell suspensions. AML5 or P388 leukemia cells were grown to confluence in suspension (in -mem or RPMI media, respectively, with 0% fetal bovine serum (GIBCO, Life Technologies Inc., Rockville, MD) and incubated with 3 or 6 M AlS 4 Pc for 24 h. Immediately before measurements, the cells were spun at 5000 rpm for 5 min, resuspended in fresh medium, agitated for 0 min, spun a second time and resuspended in fresh media, in order to minimize the residual photosensitizer in the media. It should be noted that a large amount of the original PS used for incubation was washed out during this step. The suspension was then rapidly transferred to the measurement cuvette at a concentration of cells per milliliter. This will be referred to as the washed cell suspension. Two checks were made to discriminate between AlS 4 Pc in the cells and photosensitizer that may have leaked from the washed cells into the media before or during the luminescence measurements (typically 30 min). Firstly, the washed suspension was spun down immediately after the luminescence measurement, and cells that had not been incubated with AlS 4 Pc were added to the supernatant at the same concentration and a second scan performed. This procedure re-created the same light scattering conditions in the sample. The difference between these two sets of scans then represented the fraction of the O 2 signal originating from the cells in the washed suspension. Secondly, NaN 3 was added to the washed cell suspension after the initial scan so that the final concentration of NaN 3 in the cuvette was 0.5 M. The suspension was then rescanned immediately at 270 nm only (completed within 2 min). Assuming that this time delay was not sufficient to allow significant NaN 3 diffusion into the cells but was long enough to completely quench the O 2 in the media, this determined the fraction of O 2 signal originating from the cells only. Subsequent scans were performed 7 and 5 min later to look for loss of signal as the NaN 3 diffused into the cells. For each set of cell suspension experiments, time-resolved scans were also performed on control, unincubated cells to determine the background signal at each wavelength. In all cases, experiments were repeated in triplicate. Experiments in vivo. The feasibility of O 2 luminescence detection from photosensitizer in tissue in vivo was investigated in a small number of Wistar rats (Charles River Laboratories Inc., Wilmington, MA). Four rats were injected i.p. with 50 mg/kg AlS 4 Pc, and four were uninjected controls. One of each was used in each experimental run to minimize the effect of any changes in instrument response. At 24 h after injection, the animals were anesthetized by i.p. injection of 4.8 mg Xylazine (Bayer Inc., Toronto, ON, Canada) and 30 mg Ketalean (MTC Pharmaceuticals, Cambridge, ON, Canada). In six animals (three drugged, three control) the skin was irradiated, for which the abdomen was shaved and depilated (Nair, Carter-Horner Inc., Mississauga, ON, Canada). For the final two of these, measurements were taken also with the two additional filter wavelengths (250 and 285 nm). In the remaining two animals the liver was irradiated, for which an upper abdominal incision was made to expose the liver surface, as described by Patterson et al. (6). For the measurements the animals were placed in a purpose-built holder (see Fig., insert). The laser beam (3 mm diameter) was directed at 45 to the tissue surface and the signal measured at 90 to the incident beam. Measurements were repeated at two to four different points on each tissue. Immediately after completing the measurements, the animals were euthanized by intracardiac injection of T-6 (Hoechst Roussel Vet, Whitby, SK, Canada). Liver and skin samples outside the treated regions were removed for spectrofluorimetric determination of the photosensitizer concentration using a previously established protocol (7).

4 Photochemistry and Photobiology, 2002, 75(4) 385 Figure 2. O 2 luminescence from simple solutions of AlS 4 Pc and Photofrin in solution. A: Time-integrated spectra ( 5 s) for 2.5 M AlS 4 Pc in methanol, 2.5 M AlS 4 Pc in H 2 O and 2.5 M Photofrin in methanol; errors are smaller than the symbol sizes. B: Timeintegrated spectra ( 5 s) for 2.5 M AlS 4 Pc in H 2 O with additional measurements made at 250 and 285 nm; errors are smaller than the symbol sizes. C: Time-resolved measurements at 270 nm for 2.5 M AlS 4 Pc in methanol, 2.5 M AlS 4 Pc in H 2 O and 2.5 M Photofrin in methanol, showing the best fits of Eq. (6). RESULTS Experiments in solution Figure 2a shows the time-integrated spectra for AlS 4 Pc and Photofrin in solution. Figure 2b shows the spectra measured for AlS4Pc in water with the two additional filters added. In each case, a peak was found at 270 nm, consistent with O 2 luminescence. Figure 2c shows the 270 nm time-resolved curves, together with the best fits of Eq. (6) to the data. The derived photosensitizer triplet and O 2 lifetimes are listed in Table, together with the values reported by Patterson et al. (6), obtained by frequency-domain O 2 luminescence spectroscopy, and Krasnovsky (8), obtained by time-resolved O 2 luminescence measurements. Our values generally agree well with the published data, indicating that the time-resolved system performed correctly and supporting the interpretation of the 270 nm signal as caused by O 2 luminescence. The O 2 detection limit was estimated using Eq. (5) for the case of AlS 4 Pc in water. The irradiation volume was taken as a cylinder of length cm and diameter 3 mm. For 2.5 M AlS 4 Pc solutions the optical density was 0., so that the volume was irradiated approximately uniformly throughout. The total number of photosensitizer molecules in this volume was 0 4, which is much less than N a. 0 6, where N is the number of photons per pulse and a ( 0.25 cm ) is the photosensitizer absorption coefficient at the irradiation wavelength, measured on a spectrometer (model UV60V, Shimadzu, Kyoto, Japan). Hence, all photosensitizer molecules are excited during one 20 ns pulse, whereas the probability of more than one excitation cycle per molecule is low because the triplet-state yield is substantial and T k 20 ns. The O 2 quantum yield, D, was taken as 0.38 (9), and its radiative lifetime in water, R, was taken as 5.55 s (20). This yields molecules of O 2 that underwent radiative decay. The total number of photons counted at 270 nm (background subtracted) over 200 laser pulses was , so that each count corresponded, on average, to O 2 molecules undergoing luminescence decay. Hence, for a minimum signal of 25 counts (SNR 5:), the limiting detection sensitivity was molecules. This is of the same order of magnitude as an estimate based on the geometrical efficiency of the detector system ( %), the PMT quantum efficiency at 270 nm ( %), the transmission of 270 nm photons through about 4 mm of water ( 30%) and the optical components ( 20%) and the projection of the cm long irradiation volume onto the 3 mm wide photocathode ( 30% overlap). For these values, luminescence photons generated would yield 40 counts. Figure 3 summarizes the time-resolved measurements when NaN 3 was added in increasing concentration to AlS 4 Pc in water. For [NaN 3 ] 50 mm, the signal was detectable but too noisy to analyze. It was observed that D decreased rapidly at first and then more slowly, whereas T appeared to decrease slightly. The strong reduction in D observed is consistent with quenching of O 2, and D follows a Stern Volmer quenching relationship. Figure 3b shows a linear relationship between the inverse lifetime and the concentration of NaN 3 as expected. The dashed line shows a quenching constant of k q ( ) 0 8 s M for O 2. This is consistent with the quenching constant of s M for eosin Y and s M for Rose Bengal measured by Hall and Chignell (5). In addition, the PS triplet state appeared to be quenched slowly with a quenching constant, k q, of s M. Slow quenching of the PS triplet state by NaN 3 as a secondary effect has been observed previously (5), although a literature search failed to reveal rate constants for comparison.

5 386 Mark Niedre et al. Table. data Photosensitizer triplet state ( T ) and O 2 ( D ) lifetimes (microseconds) for photosensitizers in solution, compared with published Photosensitizer and solvent Fits to Eq. 6 Published values T D T D AlS 4 Pc in water AlS 4 Pc in methanol Photofrin in methanol * * * * * *Patterson et al. (6). Krasnovsky et al. (8). Figure 4 shows the results of adding BSA. For [BSA]/ [AlS 4 Pc] 400, T increased from to 20 2 s. Similar increases in triplet-state lifetime have been reported previously and attributed to binding to albumin and consequent shielding of the triplet state from diffusion of oxygen molecules: for example, Foley et al. (2) reported an increase in T in aqueous phosphate buffered saline solutions of sulphonated aluminum phthalocyanine bound to human serum albumin (HSA) and Aveline et al. (22) reported that T increased when HSA was added to aqueous solutions of benzoporphyrin derivative (BPD-MA). Over the same BSA concentration range, our measured value of D decreased from to s, presumably because of O 2 quenching. A literature search failed to reveal another report of this effect, but the decrease in D seems plausible on the basis of the known effects of quenching by proteins in bio- Figure 3. O 2 luminescence for solutions of 6 M AlS 4 Pc in water with increasing NaN 3 concentration. A: Time-resolved measurements at 270 nm, showing the best fits ( 5 s) to 0, 2.5, 2.5 and 00 mm NaN 3 concentrations. B: / T and / D vs NaN 3 concentration. The error bars correspond to variations in the fitted values between repeated experiments (N 3). The dotted lines show the fits to Stern Volmer relationships (for O 2, k q M s and for the PS triplet state k q M s ). Figure 4. O 2 luminescence for solutions of 6 M AlS 4 Pc in water with increasing BSA:AlS 4 Pc molar ratio. A: Time-resolved measurements at 270 nm showing the best fits ( 5 s) to 0:, :3, 2: 3 and 00: molar ratios. B: T and D vs molar ratio. The error bars correspond to variations in the fitted values between repeated experiments (N 3).

6 Photochemistry and Photobiology, 2002, 75(4) 387 logical media. For [BSA]:[AlS 4 Pc] ratios between :3 and :, a three-parameter fit to Eq. (6) yielded poor results (average per degree of freedom). A six-parameter fit (A, D, T and A 2, D2, T2 ), assuming two components with different lifetimes, gave ( per degree of freedom) D s, T s and D s, T s. The value of A decreased monotonically with increasing BSA concentration, whereas A 2 increased. For [BSA]:[AlS 4 Pc] :3 and :, A /A 2 was and , respectively. The lifetimes shown in Fig. 4b are the weighted averages of the two components: (A A 2 2 )/(A A 2 ). Finally, when NaN 3 was added to solutions of BSA and PS, no signal was observed at 270 nm after background subtraction (data not shown). We interpret this to mean that the long-lifetime signal was only caused by O 2 luminescence and not by possible PS triplet-state phosphorescence. Cell suspension experiments A statistically significant spectral peak was observed at 270 nm in cell suspensions, as shown in Fig. 5a. However, despite repeat washing of the cells before these measurements, a small quantity of photosensitizer was always found in the media by the time the luminescence measurements were completed. Comparing the luminescence intensity from the supernatant only (measured after the initial scan) with that from media containing known photosensitizer concentrations, this was approximately 0.25 M for cells incubated with 6 M AlS 4 Pc. Hence, the signal from cell suspensions represents O 2 luminescence from both the cells and the media. Figure 5b shows the 270 nm time-resolved curve for an AML5 cell suspension incubated with 6 M AlS 4 Pc, as well as that for the supernatant with the same concentration of unincubated cells resuspended in it. For the former, a twocomponent (six-parameter) fit gave T s, D s and T2 9 3 s, D s, with a significantly lower 2 (2.3 per degree of freedom or.4 with outliers removed) than a one-component fit ( 2 36 or 6.6 per degree of freedom). For the resuspension of unincubated cells a single component fitted well ( or.5 per degree of freedom), giving T s, D s, comparable to the values for AlS 4 Pc added to media ( and s, respectively) and to the second component in the cell suspension. In order to determine the fraction of signal originating from intracellular photosensitizer, further experiments were performed on P388 cell suspensions incubated with 3 or 6 M AlS 4 Pc. In the washed suspension, the 270 nm signal, after background subtraction, was times higher with a photosensitizer concentration of 6 M than with 3 M. When the supernatant was removed after spinning down the cells and unincubated cells added to it, the 270 nm signal after background correction was reduced by 5 2% and 6 2% for 5 and 2.5 g/ml AlS 4 Pc, respectively. (The time from the last wash to completing the first set of scans was 30 min and between the start of the first and second measurements was 40 min.) We interpret this to mean that only 5% of the O 2 luminescence from the incubated washed cell suspension originated from the cells Figure 5. O 2 luminescence from suspensions of AML5 murine leukemia cells. A: Time-integrated spectra ( 5 s) for (washed) suspensions incubated for 24 h with 6 or 3 M AlS 4 Pc. The error bars correspond to variations observed in repeated experiments. B: Timeresolved measurements at 270 nm for (washed) suspension of incubated cells (6 M AlS 4 Pc) and for the supernatant from this suspension with unincubated cells resuspended in it. The best-fit curves shown are for T s, D s, T2 9 3 s and D s (incubated cells), and T s and D s (supernatant unincubated cells). and 85% from trace photosensitizer in the media. It should be noted that spinning the cells to obtain the supernatant probably caused additional photosensitizer leakage from the cells, so that the 5% is a lower limit for the true intracellular fraction of the signal. In the second determination of the intracellular fraction, the signal dropped to 9 3% immediately after adding NaN 3 to the washed cell suspension. After 7 and 5 min, the value decreased further to 0 3% and 2 3%, respectively. In comparison, the 270 nm luminescence signal fell to approximately % of the original value in a solution of 2.5 M AlS 4 Pc in cell-free media immediately upon addition of the same concentration of NaN 3, showing that the time required for complete mixing of the NaN 3 was negligible. We interpret these results to mean that 9 3% of the signal from the washed cell suspension was intracellular and that some time was required for the NaN 3 to diffuse into the cells and quench this remaining signal. The values for the intracellular fraction of the signal were therefore in good agreement between the two independent methods. Figure 6 summarizes these findings.

7 388 Mark Niedre et al. Figure 6. Summary of the time-integrated O 2 luminescence for P388 cells in suspension under different conditions. The values are normalized to that for the corresponding incubated cell suspensions in each case. Figure 8. In vivo time-resolved measurements at 270 nm in the liver of a rat injected with 50 mg/kg AlS 4 Pc. Typical errors ( standard deviation in photon counts) are shown. Also included is the best fit to a single exponential decay, corresponding to T 30 5 s. Figure 7. In vivo time-integrated spectra ( 0 s) in rats injected with 50 mg/kg AlS 4 Pc. A: Delayed luminescence measured in sensitized animals normalized to the spectrum in uninjected controls. The peak values plotted, in individual animals, are , and The error bars correspond to standard deviation in the point-to-point signal variations in each animal. B: Precorrected delayed luminescence spectra (with extra filters at 250 and 285 nm), measured on the skin of a sensitized rat and of an unsensitized rat. The error bars correspond to point-to-point signal variations in each animal. Experiments in vivo As summarized in Fig. 7a,b, a 270 nm peak was seen in all photosensitized tissues. Figure 7b shows the presubtracted signals from the control and sensitized animals as well as the signal measured at the two additional wavelengths. The normalized intensity was, on average, 2 3% higher in skin and 64 2% higher in liver for the sensitized animals compared with uninjected controls. At 300 and 330 nm there was no significant difference between sensitized and control tissues. Hence, the spectral peak at 270 nm appears to be caused by O 2 luminescence from photosensitizer in the tissue. Because the measurements were made 24 h after injection and the plasma half-life for AlS 4 Pc in rodents is.5 h (23), the photosensitizer should be fully tissue bound, rather than be in the circulation. The concentration of photosensitizer in tissue measured in postmortem samples was in skin and g/g in liver. The time-resolved 270 nm luminescence was corrected for background by calculating: [ ] # 270S(t) dt 270 C S 200 S # S(t) dt C L (t) S(t) S (t) (7) where S S (t) and S C (t) are the delayed, time-integrated signals at wavelength in sensitized and control animals, respectively. The rationale here is that the background signal at 270 nm equals that at 200 nm, scaled by the relative system detection efficiency and background luminescence contributions as represented by the ratio in the control animals. Figure 8 shows an example of the corrected signal from liver. Points at 0 s time delay are highlighted, because these early points appear to have a significant contribution from fluorescence, as evidenced by analysis of their spectral composition and kinetics. This was likely because of the tissue surface being at 45 to the laser beam, such that some specularly reflected excitation light entered the detection optics, generating secondary fluorescence. This was confirmed by checking the signal from a cuvette filled with water at an angle of 45 to the incident light. Because the

8 Photochemistry and Photobiology, 2002, 75(4) 389 Table 2. Summary of AlS 4 Pc triplet state ( T ) and O 2 ( D ) lifetimes (microseconds) obtained in solution, cell suspensions and in vivo Medium T D H 2 O MeOH H 2 O BSA ( 2: molar ratio) Component Component 2 Cell suspension Component Component 2 Resuspension of supernatant with unincubated cells In vivo (liver) In vivo (skin) initial rising part of the time-resolved curves was thus obscured, Eq. (6) could not be fitted reliably. Instead, a single exponential decay was fitted to the data above 0 s, giving a time constant of 30 5 s. This can be attributed to the PS triplet state (i.e. T 30 5 s), because it is known that T k D in vivo. Similarly, a PS triplet-state lifetime of 26 5 s was measured in the skin. Table 2 summarizes the various AlS 4 Pc triplet state and O 2 lifetimes obtained in these experiments in solution, cells and tissue. DISCUSSION As in our earlier attempt to measure O 2 luminescence in vitro and in vivo (6), the luminescence spectrum was sampled here using discrete bandpass filters rather than a monochromator, in order to achieve the highest possible signal-to noise-ratio. Although this limited the spectral information, the O 2 luminescence signal should be unambiguous, because it comprises a single peak at around 270 nm with a full width at half maximum (FWHM) of 30 nm (24) that is adequately sampled by the 3 filters at 200, 270 and 300 nm. The 200 nm and 300 nm filters also allow subtraction of the background tissue and photosensitizer fluorescence. The 250 nm and 285 nm filters were added in later experiments to provide further spectral confirmation of the 270 nm peak. The agreement in simple solutions between our measured photosensitizer triplet state and O 2 lifetimes and the published values, and the observed spectral peak at 270 nm, support the interpretation of the signal as O 2 luminescence, as does the elimination of the 270 nm signal by NaN 3. The extra filter was added at 330 nm to cross-check for a possible differential light absorption artifact in vivo that could arise as follows. Water has a local absorption maximum at 90 nm and a minimum at around 270 nm (25). Hence, if the PDT treatment caused an increase in tissue hydration, this could give rise to an artifactual increase in the 270 nm signal relative to the 200 nm signal. However, because the water absorption increases from 300 to 330 nm, such altered hydration would also produce a signal at 330 nm that is less than that at 300 nm. This was not observed (Fig. 7), so that we conclude that this hydration artifact does not occur and that the 270 nm peak seen in vivo is the true O 2 luminescence. In simple solutions, it was observed that the PS triplet state lifetime was longer in water than in methanol. This effect has been observed previously (2) and has been attributed to higher solubility of oxygen in methanol than in water. As BSA was added to solutions of AlS 4 Pc in water, T increased from s in the self-aggregated, unbound state (with no BSA) to 20 2 s for the likely completely bound state ([BSA]/[AlS 4 Pc] 2). For molar ratios between :3 and :, curve fitting indicated two distinct sets of kinetics, corresponding approximately to unbound and bound values for T. As the concentration of BSA increased, the signal from the bound (long T ) and unbound (short T ) fractions monotonically increased and decreased, respectively. Hence, the measurement system appears to be sensitive enough to distinguish between two components in a sample with different photophysical kinetics that are at least qualitatively indicative of the microenvironment. Ideally, this would be verified by independently measuring T using a pulsed transient absorption spectroscopy system such as that used by Aveline et al. (22). We are currently planning to add this capability to our experimental system. In the cell suspension experiments, it was critical to demonstrate that the observed O 2 luminescence originated from intracellular photosensitizer in the cells, not in the media. This has been a potential artifact in other published studies in vitro that have reported positive results (9,0). The fact that only some 5 9% of the signal appeared to come from the cells, even using a long photosensitizer incubation time (to allow intracellular binding) and measuring as soon as possible after multiple washing, reinforces this point. The interpretation of this fraction as truly intracellular in origin is supported by the consistency between the resuspension experiments and the change in signal upon adding NaN 3 to quench the O 2 in the media. The kinetics of the O 2 luminescence also supports this conclusion. Thus, in resuspension of the supernatant with unincubated cells, T and D were the same as for solutions of AlS 4 Pc in media alone, but both were substantially different in the incubated cell suspensions. Specifically in the latter, there appeared to be two distinct sets of kinetics. One set ( T s, D s) was comparable to AlS 4 Pc in media, whereas the other was significantly different, with increased triplet lifetime ( T2 9 3 s) consistent with protein binding in the cellular environment and decreased O 2 lifetime ( D s) consistent with increased quenching of O 2. Other authors have reported similar results of increased T in cells (22,26,27). For example, Aveline et al. (22) showed that, for benzoporphyrin derivative in pellets of P388D and NBT-II cells, T increased to approximately 23 s (again, T in solution was lower but was not explicitly stated), whereas Truscott et al. (26) obtained a value of 7.7 s using transient absorption spectroscopy in fibroblast suspensions incubated with hematoporphyrin derivative compared with 2.4 s in water (6). Again, an experiment to independently measure T would be valuable to verify these results. Although we recognize that only a small number of ani-

9 390 Mark Niedre et al. mals were used for the in vivo experiments, a statistically significant peak at 270 nm was observed in all cases and in both tissues (P for liver and 0.05 for skin by Student s t-test) for sensitized animals compared with controls. As would be expected from the much higher AlS 4 Pc uptake in liver (4), the O 2 signal was stronger than that from skin ( approximately three-fold) but was also more variable from point to point, probably because of breathing movement during scanning. Because of the distortion in the early part of the signal, determining D in vivo by directly fitting Eq. (3) to the timeresolved data was not accurate, as discussed previously. However, it could be determined using the total number of photon counts observed, as follows. The reliable part of the time-resolved data ( 0 s) was fitted to a single exponential, and the area under this curve from 0 to 80 s was taken as the total O 2 counts: average values and (after background subtraction) over 200 laser pulses for liver and skin, respectively. A cuvette containing 2.5 M AlS 4 Pc was then positioned in a geometry similar to that used in vivo, i.e. with the face at 45 degrees to the excitation light, and the signal measured. As in the aforementioned estimate of the system sensitivity, it was calculated that each photon count represented (4.9 0.) 0 3 molecules of O 2 undergoing radiative decay in the cuvette. Hence, we calculate that approximately and O 2 molecules underwent radiative decay in liver and skin, respectively. Using these data along with Eq. (5), D in vivo could be calculated using the following additional values: photosensitizer uptake g/mg in liver and g/mg in skin, effective irradiation volume equivalent to a sphere of 3 mm radius (based on tissue optical properties) and O 2 quantum yield ( D ) The O 2 radiative lifetime ( R ) is unknown in vivo, but was assumed to be close to its value of 5.55 s in water (20). This gives D 0.7 s in liver and 0.8 s in skin. (The concentration of O 2 produced in vivo was also calculated using Eq. (5) and these values for D, giving an average concentration of O 2 immediately after the laser pulse of approximately 4 nm in the liver and 2 nmin the skin.) It should be noted that this calculation of D is sensitive to the value of R used. For example, it has been shown (20) that R can be estimated using the refractive index (n) of the solvent. In tissue, typical values for n are (28), which yields an estimate of the radiative lifetime of approximately.5 s. This reduces D to 0.04 s in liver and 0.05 s in skin. The range of values for D agrees with estimates on the upper limit of 0.2 s by Baker and Kanofsky (0), the range s by Moan and Berg (3) and our previous upper estimate of 0.5 s (6). To our knowledge, no measurement for triplet-state lifetimes in vivo has been published for any photosensitizer. However, on the basis of the increase found in cell suspensions and protein-rich environments compared with simple solutions, it would be expected to increase in vivo as observed. Gorman and Rogers noted that it would be difficult to differentiate O 2 luminescence from PS triplet-state phosphorescence, given the strongly reduced O 2 lifetime in vivo (29). However, this did not take into account the possibility of spectrally resolving the NIR emission, as done here. Certainly, we observe an increase in the overall background NIR signal in vivo in the presence of photosensitizer: e.g. for the data of Fig. 7B the signal at 200 nm was about four times higher in the sensitized animal than in the uninjected control. However, the 270 nm peak is only seen in the former. It is unlikely, but possible, that the triplet-state phosphorescence also happens to have a peak emission at 270 nm. (This can be investigated in the future using other photosensitizers, because the triplet-state phosphorescence spectrum should be photosensitizer-specific.) Furthermore, we showed that the PS triplet state phosphorescence was negligible for AlS 4 Pc in BSA solutions and therefore would probably be small also in vivo. At a recent conference where we presented this work (30), Hirano et al. also reported successful measurement of O 2 luminescence spectra in a murine tumor model injected with 25 mg/kg Photofrin or ATX-S0Na(a) using the same type of PMT, which supports our conclusion that this detector does enable measurement of the 270 nm O 2 luminescence emission in vivo (3). In conclusion, utilizing this new PMT technology has provided compelling evidence that it is possible to detect and quantify O 2 luminescence from intracellular photosensitizer in cell suspension in H 2 O-based media and from tissues in vivo. Previous attempts to detect O 2 in cells have either required the use of D 2 O to increase the lifetime to detectable levels and increase transmission at 270 nm (9 ) or have not definitively distinguished intra- and extracellular photosensitizer (9,0). For the latter, even with repeated washing of cells after prolonged incubation, the trace quantities of photosensitizer in the external media can contribute a large fraction of the detected luminescence signal, because of the much greater fractional volume ( 98%) and longer O 2 lifetime. It should be noted that this effect may be less significant with non water soluble photosensitizers that are not photodynamically activated in aqueous environments because of self-aggregation. Nevertheless, very careful design of in vitro studies is required, for example in experiments aimed at determining the relationship between O 2 generation and photobiological damage in cells. In vivo, the conditions used here were aimed at maximizing the O 2 luminescence signal and are not necessarily generally applicable. For example, the measurements were performed on well-vascularized and therefore well oxygenated tissues, and the OPO laser used to generate the luminescence signal did not have high enough average power to deliver a therapeutic light dose in a reasonable treatment time. Hence, in order to apply this instrumentation to correlate in vivo O 2 generation with the PDT response, a separate PDT irradiation light source, with switching between this and the OPO during the experiments, or a high repetition-rate and high average power laser will be required. Both options are currently being explored, together with improvements in the optical collection efficiency by increasing the numerical aperture of the detection system. The problem of secondary fluorescence from specular reflection will also be addressed in the next system by adapting the beam tissue detector geometry, using low fluorescence components or by using polarizing optics or both. With these modifications, we intend to start applying this technology to determining the contribution of O 2 to the photobiological effect of PDT in a variety of in vitro and in vivo models, for example to test the hypothesis that photochemical depletion of molecular oxy-

10 Photochemistry and Photobiology, 2002, 75(4) 39 gen in tissue at high fluence rates is responsible for reduced photodynamic efficacy because of decreased O 2 generation (32). At the same time, we will evaluate the utility of O 2 luminescence monitoring as a PDT dosimetry tool in comparison with other direct and indirect dosimetry methods (8). If these studies are encouraging, we then intend to develop this system for clinical use during PDT treatments. Acknowledgements This work was supported by the Canadian Cancer Society under a grant from the National Cancer Institute of Canada. The authors wish to thank Hamamatsu Corp., Japan, and in particular Dr. Ken Kaufmann, Hamamatsu, NJ, for providing the PMT system. Dr. Richard Hill, OCI, provided the AML5 cell line and QLT Phototherapeutics, Vancouver, BC, Canada, kindly supplied Photofrin. 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