Red versus blue light illumination in hexyl 5-aminolevulinate photodynamic therapy: the influence of light color and irradiance on the treatment outcome in vitro

Abstract. Hexyl 5-aminolevulinate (HAL) is a lipophilic derivative of 5-aminolevulinate, a key intermediate in biosynthesis of the photosensitizer protoporphyrin IX (PpIX). The photodynamic efficacy and cell death mode after red versus blue light illumination of HAL-induced PpIX have been examined and compared using five different cancer cell lines. LED arrays emitting at 410 and 624 nm served as homogenous and adjustable light sources. Our results show that the response after HAL-PDT is cell line specific, both regarding the shape of the dose-survival curve, the overall dose required for efficient cell killing, and the relative amount of apoptosis. The ratio between 410 and 624 nm in absorption coefficient correlates well with the difference in cell killing at the same wavelengths. In general, the PDT efficacy was several folds higher for blue light as compared with red light, as expected. However, HAL-PDT624 induced more apoptosis than HAL-PDT410 and illumination with low irradiance resulted in more apoptosis than high irradiance at the same lethal dose. This indicates differences in death modes after low and high irradiance after similar total light doses. From a treatment perspective, these differences may be important.


Introduction
Presently, 5-aminolevulinate (ALA) and two of its derivatives [methyl 5-aminolevulinate and hexyl 5-aminolevulinate (HAL)] are in routine clinical use as photosensitizer precursors in photodynamic therapy (PDT) and photodiagnosis. 1,2HAL shows improved tissue penetration [3][4][5] and cellular uptake [6][7][8] compared to ALA.Following conversion of HAL to ALA by cellular esterases, the molecule enters the heme synthesis pathway and induces increased intracellular levels of the photosensitizer protoporphyrin IX (PpIX), the direct precursor of heme.PpIX features a typical absorption spectrum for porphyrins with a high Soret peak around 410 nm and Q-bands in the green and red wavelength ranges. 9For PDT or photodiagnosis based on ALA or its esters, both blue and red light lamps are used.1][12] By contrast, blue light penetrates only about 1 mm into tissue, 13 but has much higher efficiency for PpIX activation.
Protocols for PDT treatment in research and in the clinic vary widely, probably at least in part because there is no strong scientific basis for choosing one protocol over another.A solution to this problem will require comparative analyses of biological effects of different photophysical parameters, such as light wavelength, irradiance, and total dose.In addition, general conclusions can only be made by analyzing the effects of the same protocol applied to different tissues and cancer cells.
0][21] Interestingly, the irradiance also modulates immunological responses which are likely to affect treatment outcome.Thus, high irradiance was found to be immunosuppressive, whereas a similar total dose administered using lower irradiance was not immunosuppressive. 22In the present study, we have used five different human cancer cell lines and compared the overall PDT efficiency when using HAL-induced PpIX as photosensitizer activated by light of either 410 or 624 nm.We observed substantial differences among the cell types in their sensitivities to light.More importantly, the death mode, here measured as apoptosis, was not only influenced by the light source and total light dose but also by irradiance.From a perspective of treatment efficacy, our results may contribute to a better fundament for understanding the biological effects of the photophysical parameters in PDT.
2 Materials and Methods

Cell Lines
Five human cancer cell lines were used in this study, all of them representative of a cancer type suitable for clinical HAL-PDT.The cell line A431 (ATCC CRL-1555) is an epidermoid carcinoma and serves as a model system for skin cancer.The A549 cell line (ATCC CCL-185) is a lung carcinoma and HeLa S3 (ATCC CCL-2.2) is a cervical adenocarcinoma.WiDr (ATCC CCL-218) is a colorectal adenocarcinoma and T24 (ATCC HTB-4) is a transitional cell carcinoma from urinary bladder.

Light Sources
To obtain a homogenous light field at appropriate wavelengths and within a range of irradiance values, light sources based on a light emitting diode (LED) array were used for this study. 23Each lamp consists of an array containing 247 (13 × 19 diodes, 624 nm, illumination field 9 × 14 cm) or 260 (13 × 20 diodes, 410 nm, illumination field 9 × 15 cm) LEDs (Roithner LaserTechnik, Vienna, Austria) connected in parallel.The irradiance of these LEDs is adjusted by the current applied as described by Pieslinger et al. 23 The assembly of the LEDs and the distance from the LEDs to the illumination field are optimized to achieve a homogenous light field (maximal variation 10%).The LED light source for blue light illumination (LED type VL410-5-15, dominant wavelength 410 nm, spectral half-wave width 18 nm) allows for irradiance values up to 7.0 mW∕cm 2 , and the red light illumination source (LED type R5CA5111P, dominant wavelength 624 nm, spectral half-wave width 20 nm) delivers up to 35.0 mW∕cm 2 .The emittance spectra can be found in Ref. 24 and the light viewing angle is 15 deg.Irradiance was measured in a grid pattern (covering the whole light field) by an LI-189 light meter equipped with a PY pyranometer detector (LI-COR, Lincoln, Nebraska, diameter 7.5 mm) and a LabMaster Ultima laser measurement system with a vis-sensor (Coherent Inc., Wilsonville, California, diameter 8 mm).Both light meters had a typical accuracy of AE3% for the total system.The light transmission of the microplates used is reported to be close to 100% at wavelengths >350 nm according to information from the manufacturer.The transmission spectrum should, therefore, be close to that of the lamp itself.

Cell Culture
All growth media were supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, 100 U ml −1 penicillin∕0.1 mg ml −1 streptomycin, amphothericin B (2.5 μg∕ml), and 10 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), all obtained from Sigma (Oslo, Norway).For culture of T24 and A431, 1-mM Na pyruvate (BioWhittaker, Lonza, Belgium) was also added to the media.All cell lines used Dulbecco's modified eagle medium (DMEM) as a basic medium except for WiDr cells which were cultured in RPMI 1640 medium.Subcultivation was done by rinsing the cells twice with phosphate buffered saline (PBS) (37°C) before detachment by trypsinization.For all experiments, cells were seeded in appropriate cell culture dishes the day before treatment.Cell numbers (total, live, and dead) were determined by using the Countess™ system (Invitrogen™, Oslo, Norway) and the number of live cells was used for calculating the seeding.

Hexyl 5-Aminolevulinate Photodynamic Therapy
A stock solution of 10 mM HAL dissolved in PBS (adjusted to pH 6.0) was freshly prepared on the day of the experiment and sterilized by filtration (0.2-μm filter).The growth medium was carefully removed from the dishes and replaced by medium without serum containing 20 μM HAL.After 3 h of incubation (dark conditions, 37°C, 5% CO 2 , humidified atmosphere), the HAL medium was replaced by growth medium without HAL (with serum).The samples were illuminated from below using either of the LED arrays described above immediately after HAL removal and were subsequently placed in the incubator until further processing.All experimental steps after adding of HAL to the cells were done under subdued light conditions.

Dose-Response
Cells were seeded (1 day before treatment) in 96-well cell culture microplates (black walls, clear bottom, Optilux, Falcon, VWR, Norway, see picture in Fig. 1) at the following densities, resulting in 50% to 60% confluency at the day of treatment: A431: 10,000 cells/well, A549: 10,000 cells/well, HeLa S3: 11,000 cells/well, T24: 9000 cells/well and WiDr: 11,000 cells/well.The standard filling volume was 100 μl∕well.Six wells in each row contained cells while the outer ones only contained medium to minimize evaporation.Every experiment was accompanied by a "control" [no Photosensitizer (PS), no light], a "light-only" sample (no PS, maximal light dose), and a "HALonly" sample (PS, kept in the dark).Please notice that all doses and controls necessary for the one dose-response curve were on the same microplate to ensure identical conditions except those of the varied photophysical parameters.

Resazurin Assay
The resazurin assay is based on the conversion of the nonfluorescent resazurin to the fluorescing resorufin in viable cells.Approximately 22 h after illumination, 20-μl resazurin (2.5 mM, Sigma) was added into each well and incubated for  2 h in the dark incubator.Subsequently, resorufin fluorescence was determined using a microplate reader (BMG Labtech FluoStar Omega, Oslo, Norway, excitation wavelength 544 nm, emission wavelength 590 nm).

PpIX Absorbance and Emission
Approximately 95% confluent 75 cm 2 cell culture bottles of T24 and A431 cells were incubated with 20 μM HAL for 3 h.Cells were then rinsed once with PBS (37°C) and 5 ml of accutase (Sigma) was added.After a few minutes in the incubator, the detached cells were transferred into tubes.From each cell suspension, 10 μl were taken for cell counting by Bürker chamber.The suspensions were then centrifuged at 450 g (4°C, 5 min).The supernatants were removed and the pellets were resuspended in PBS to a final concentration of 4.0 × 10 6 cells∕ml.All samples were kept on ice until spectra were read using a Fluorolog III fluorometer (Jobin Yvon-Spex, Horiba Group, Kjeller, Norway).For the fluorescence-excitation spectra, an LP700 filter was placed in front of the detector, the excitation slit was 3 nm, and the emission slit was 4 nm.Fluorescence emission was measured at 710 nm and excitation was performed in 1-nm steps from 350 to 700 nm in a signal/ reference mode to make it comparable to absorption spectra.Emission spectra (550 to 750 nm, 1-nm step width) were excited at 405 nm with an excitation slit at 4 nm and an emission slit at 3 nm.

Caspase 3/7 Activity
The lethal dose (LD) values as determined from the doseresponse curves for A431 and T24 cells in Figs. 2 and 3 were used for this part of the study.For determination of the caspase activity, cells were seeded the day before treatment in Optilux-microplates (cell density: 9000/well for T24 10,000/ well for A431).HAL-PDT was done as described above, but the cells were covered with only 30 μl medium (with serum) per well after illumination.At 0, 2, 5, and 8 h post illumination, a microplate was allowed to equilibrate to room temperature before 30 μl of Caspase-Glo® 3/7 Reagent (Promega, Oslo, Norway, room temperature, in accordance with the manufacturers' protocol) was added to each well.The samples were mixed on a plate-shaker at 500 rpm for about 30 s and were subsequently incubated at room temperature for 1 h before light dose (J/cm 2 ) rel.survival (% untreated control) Fig. 2 Dose-response curves for five human cancer cell lines treated with blue light (410 nm) HAL-PDT (20 μM, 3 h, serum free).Three different irradiance values (0.7, 3.5 and 7.0 mW∕cm 2 ) were used for all cell lines.Cell viability is relative to an untreated control in the same microplate (no HAL, no light) and the 0 J∕cm 2 point is the "HAL no light" value.Each point is the average of two to five experiments (each in sextuplicate) with standard error of the mean (SEM) indicated by error bars.luminescence was measured in the plate reader (BMG Labtech FluoStar Omega).

Calculations and Statistics
All calculations for LD values are based on Figs. 2 and 3.The LD's are simply read out, such as LD 25 equals the dose needed to obtain 25% cell death, LD 50 equals the dose needed to obtain 50% cell death and so on.Unfortunately, the exact same doses were not used for all experiments.This made it difficult to make a statistical comparison between the curves in Figs. 2 and 3.The reason that doses were sometimes slightly altered between the independent experiments was to improve the measurement points in order to show the expected sigmoidal shape of the curve.Normally one expects an experimental error of 10% to 15% in MTT and resazurin assays.For calculating the mean and standard error of the means (SEMs) in Figs. 2 and 3, the values from sextuplicate measurements in all three to five biological replicates were added together and used to calculate mean values and SEMs.

Dose-Response Curves of Five Human Cancer Cell Lines after Blue Light HAL-PDT
To address the question of whether the cytotoxicity of blue light illumination of HAL-induced PpIX (abbreviated as HAL-PDT 410 is dependent only on total light dose or on total light dose and irradiance, we measured dose-response curves of five human cancer cell lines using three different irradiance values (0.7, 3.5 or 7.0 mW∕cm 2 ) under otherwise identical PDT procedures.
As shown in Fig. 2, the overall light dose required to kill close to 100% of the cells varies from 0.2 to 1.4 J∕cm 2 , depending on the cell line.The most sensitive cell line is A431 both in terms of the irradiance and light dose required for complete cell killing, whereas the other cell lines are substantially more resistant.The shape of the dose-response curves differs among the different cell lines.The viability of T24 cells drops rapidly with increasing light dose, while the dose-response curve for HeLa S3 cells has a slight slope (depending on the irradiance) with a near complete cell kill at >1.1 J∕cm 2 .WiDr displays a notable similarity to T24 cells regarding curve characteristics, excluding the HAL-only measurement.Unexpectedly, a less steep dose-response curve was observed when using a lower irradiance (0.7 mW∕cm 2 ) compared to a 10-fold higher irradiance (7 mW∕cm 2 ).This was observed for all five cell lines.
The dark toxicity of 20-μM HAL varied from 35% (A549) to negligible (A431, T24).Blue light illumination without PS using the maximal light dose resulted in up to 40% cell death for A549 cells, and almost no cell killing for A431 and WiDr cells.Hyperthermia from illumination could potentially cause some of the differences between high and low irradiance.However, the temperature in growth medium did not change measurably for 410 nm (7 mW∕cm 2 , 30 min) illumination and increased only 2 deg for 624 nm (35 mW∕cm 2 , 30 min), data not shown.Since illumination is carried out at room temperature, the cells will consequently not be exposed to hyperthermia during the PDT protocols employed.

Dose-Response Curves of Five Human Cancer
Cell Lines after Red Light HAL-PDT Red light is commonly used for ALA-based PDT in the clinic.As a result of PpIX's low absorption of red light compared to blue light and the irradiance range of the lamp, only the highest lamp output irradiance (35 mW∕cm 2 ) was used for red light HAL-PDT (HAL-PDT 624 ).An exception is the A431 cells which required the lowest light doses.Therefore it was possible to establish a dose-response curve for 7 mW∕cm 2 illumination as well.For an irradiance of 35 mW∕cm 2 , the illumination time required to achieve near complete cell destruction was below 30 min for all cell lines tested.Except for irradiance and wavelength, the PDT parameters were identical to those in the HAL-PDT 410 experiments.Dose-response curves for all five cell lines after HAL-PDT 624 are presented in Fig. 3(a).Similar to the dose-response curves for HAL-PDT 410 , the dose-response curves of the individual cell lines to HAL-PDT 624 differ in both shape and the light doses needed to achieve close to 100% cell killing.The required light doses for near complete cell kill varied from 12.6 J∕cm 2 for A431 to >70 J∕cm 2 for WiDr and A549.Interestingly, the order of sensitivity of the cell lines to PDT estimated from LD ∼100 values (A431 > HeLa S3 > T24 > WiDr > A549) is comparable for HAL-PDT 410 and HAL-PDT 624 .Furthermore, A549 cells showed a biphasic response to HAL-PDT 624 (and not a biphasic response to HAL-PDT 410 ): a rapid drop in viability below 10 J∕cm 2 is followed by a plateau up to 40 J∕cm 2 .At an even higher light dose (70 J∕cm 2 ), the viability of A549 cells drops to nearly zero.For the cell line with highest sensitivity to HAL-PDT 624 , A431, the influence of irradiance on the overall cytotoxicity was tested.The results from this experiment are presented again in Fig. 3(b), where lower irradiance (7 mW∕cm 2 ) results in less efficient cell destruction for intermediate doses when compared to the fivefold higher irradiance (35 mW∕cm 2 ).
As expected, the dark toxicity of 20 μM HAL in these experiments was about the same as in the HAL-PDT 410 experiments.Furthermore, red light illumination without PS using the maximal light dose for each cell line induced no cell death within the experimental error, except for A549, which showed about a 30% drop in viability.
To make comparison between the different cell lines and photophysical parameters easier, we have assembled the values from Figs. 2 and 3 in Table 1.We have tabulated the values resulting in 25%, 50%, 75%, and 90% cell death, for LD 25 , LD 50 , LD 75 , and LD 90 , respectively.Since the slope of the dose-response curves is very flat when approaching 100% cell death, the definition of LD 100 is somewhat subjective, and we chose to use the term "LD ∼100 " instead.At LD ∼100 , close to 100% of the cells were dead in all experiments when measured by resazurin assay and there were no signs of viability on visual inspection of the cells.

Spectra of Intracellular HAL-Induced PpIX
In order to identify PpIX and possible its derivatives by spectroscopy, fluorescence excitation [Fig.4(a)] and emission spectra [Fig.4(b)] of HAL-induced intracellular PS were recorded in A431 and T24 cells.
A431 and T24 were chosen as models for a PDT sensitive and a more resistant cell line, respectively.A431 is the most sensitive to HAL-PDT of all tested cell lines for both blue and red light illumination, while T24 is one of the most resistant cell lines when one compares doses required to achieve close to Table 1 Calculated LD values based on Fig. 2 and Fig. 3, LD values are given in J∕cm 2 .In the upper part of the table, LD values for HAL-PDT 410 are presented and the corresponding LD-values for HAL-PDT 624 are presented in the lower part of the table.For a few measurements, the dark toxicity was too high to make a LD 25 -calculation, the missing values are denoted "-".At 624 nm, the red light fluorescence excitation peak has dropped to 55% from its maximum at 633 nm.Therefore, the overall illumination period could be reduced by roughly one half if a light source with a maximum at 633 nm was used for illumination instead of the 624 nm as used in the present experiments.

HAL-PDT
The ratio between the peak heights of 410 and 624 nm is 56.3 for A431 and 56.4 for T24 cells, under the conditions used in this study [Fig.4(a)].
T24 cells had lower fluorescence emission than A431 cells [Fig.4(b)].Red light fluorescence emission peaks at 636 nm for both cell lines, while a smaller maximum was determined at 706 nm.For both peaks, the overall fluorescence from T24 cells is about half of the fluorescence value from A431 cells.

HAL-PDT 410
In order to determine the effect of varying irradiance, HAL-PDT 410 was performed using irradiance values of either 0.7 mW∕cm 2 [Fig.5 Among the light doses tested, sub-full doses generally induced the highest caspase 3/7 activity in A431 cells at all three time points for both irradiance values.The results are similar for T24 cells but the less pronounced and for the lowest irradiance for the sub-low doses seem to induce the highest caspase 3/7 activity.Furthermore, caspase 3/7 activity increases with increasing time post HAL-PDT in the time range tested.A431 generally shows higher caspase 3/7 activity when compared to T24.

HAL-PDT 624
In Fig. 6, a low dose corresponds to about LD 20 (A431) and LD 30 (T24), a sub-low to LD 40 (A431) and LD 50 (T24), a sub-full to LD 65 (A431) and LD 75 (T24), and a full dose close to 100% cell death.Following red light illumination, T24 cells show almost no increased caspase 3/7 activity for any of the light doses and time points tested in this study (Fig. 6).Only a marginal increase was observed at 5 and 8 h for sub-low and sub-full doses.By contrast, A431 displayed increasing caspase 3/7 activity with increasing light doses at 5 and 8 h post HAL-PDT 624 .
Interestingly, when comparing HAL-PDT 410 to HAL-PDT 624 , the most pronounced differences of caspase 3/7 activity are that (i) blue light induces the highest activity at sub-full doses (except for T24 0.7 mW∕cm 2 ) but red light does so at full doses (except for T24 which showed only minor caspase 3/7 activity after HAL-PDT 624 ) and (ii) blue light induces a maximum threefold increase in the activity when compared to untreated controls while red light induces a maximum fourfold increase.

Discussion
Although the basic principle of PDT is simple, a number of physical, chemical and biological parameters may influence its efficacy.Different light sources with diverse characteristics are used both in research and in clinical applications. 25,26By altering the illumination period, different irradiance values can be used to deposit one specific light dose.However, the tumor response may not solely depend on the light dose but may also depend on the irradiance.8][29][30] This effect is assumed not to be the case for in vitro experiments, where the oxygen supply is much better than in tissues.In HAL-PDT, the effective photosensitizer is endogenous PpIX, which has its largest absorption around 410 nm and a smaller peak of absorption around 630 nm.Red light is commonly used in PDT due to its longer penetration depth in tissue.In the present study, all cells were monolayer cells and were irradiated from below.Hence, scattering, depth of penetration, and cell density would not make a large impact on light delivery.The significance of these parameters is, however, of great importance in clinical treatment.In a study from 2011, the efficacies of different light sources used in the clinic to activate PpIX were compared and demonstrated that blue light sources were the most efficient. 31][34][35] We have used two in-house made lamps to examine whether there is a difference in the outcome of HAL-PDT with red and blue light and after illumination with different irradiances in five different human cancer cell lines.The following discussion is divided into three main topics: irradiance and wavelength, cell-line specific response, and apoptosis induction.

Irradiance and Wavelength
Interestingly, our cell survival studies indicate that when using blue light and identical total light doses, high irradiance is more efficient in cell killing than low irradiance.This tendency is most strongly pronounced for A431 and HeLa S3 cells.In addition, at least for A431 cells, high irradiance red light induces a higher cytotoxicity than low irradiance at an identical total light dose.This observation may be (at least partially) ascribed to the counteracting effect of the cellular reactive oxygen species (ROS) defense systems, which have been shown to antagonize PDT when fractionated illumination is applied. 36It seems likely that during longer illumination at lower irradiance, the ROS defense may have sufficient capacity to partially detoxify the PDT-induced oxidants.By contrast, high-irradiance PDT might overwhelm the antioxidative mechanisms.
The absorption ratios between 410 and 624 nm were ∼56 for both cell lines, A431 and T24 [Fig.4(a)].This generally correlates quite well with killing efficiencies (Table 1), although there is some variation at different LD values.Wavelength-dependent light absorption by PpIX is probably a major parameter in PDT, but probably not the only one.Other factors likely to contribute are ROS detoxification, such as superoxide dismutase, as well as DNA repair and autophagy.
A difference in killing dependent on irradiance but with a constant dose has also been studied when using another sensitizer.Thus, a photofrin II PDT study on A549 cells demonstrated lower efficacy at lower irradiance values. 37Although a lower irradiance may reduce killing in PDT treatment, that reduction might then to some extent be compensated by an improved oxygenation. 38

Cell-Line Specific Responses
Essentially, the response to PDT treatment differs notably between cell lines, both regarding sensitivity and shape of survival curves (Figs. 2 and 3).Similar observations were made by others using different cell lines and PDT protocols. 39Obviously, generation of increased amounts of PpIX is essential for efficient PDT.However, T24 has only half the amount of total PpIX when compared to A431 (Fig. 4), but is more than twice less sensitive to light (at least at wavelength 410 nm) than A431, again pointing to a complex basis for sensitivity to PDT treatment.Difference in the survival of different cell lines could possibly be explained by different intracellular distributions of PpIX.However, confocal microscopy of HAL-incubated cells (20 μM, 3 h) co-stained with MitoTracker Green performed in this study indicated that PpIX is mainly localized in mitochondria in all cell lines used here.After another hour of HALincubation, some PpIX is redistributed to the cell membrane, without notable differences between the cell lines (data not shown).To explore the basis for the different sensitivities would require substantial efforts beyond the scope of the present paper, but it is likely that cell-specific differences in response of cancer cell lines also apply to tumors.

Apoptosis Induction
Apoptosis and necrosis are two major modes of cell death.We have specifically examined apoptosis and have assigned cell death that was not clearly apoptotic to most likely be necrotic.We consequently assume that the difference between the resazurin results and the caspase 3/7 activity measurements is caused by necrosis.However, there are additional mechanisms of cell death, including mitotic catastrophe and autophagy, both of which may, in fact, have a role in PDT. 21,40The cell death mode apparently may depend both on the type of sensitizer and properties of the cells being treated.Apoptosis and mitotic catastrophe have some common and some unique properties. 41,42PDT was shown to trigger immune suppression at high irradiances but immune stimulation at low irradiances in normal skin. 22This is interesting from the perspective of different death modes observed at high and low irradiance in our study.4][45][46] Exploration of the effect of different illumination protocols on cell death mode in vitro may help in establishing a better basis for the choice of illumination parameters.
To examine the influence of different illumination parameters on the cell death mode, caspase 3/7 activity was measured at 2, 5 and 8 h post-illumination.These results demonstrate that the cellular response depends on the target cells as well as illumination protocol.The most pronounced difference in caspase 3/7 activity between blue and red light PDT was that HAL-PDT 410 showed maximal caspase 3/7 activity at sub-full doses and HAL-PDT 624 maximum at full doses, which appears to be quite unique.For comparison, an ALA-PDT study on five different cell lines showed that the time course of apoptosis varied between the different cell lines and that the amount of apoptosis and necrosis varied with the cell line and dose, which is in line with the results presented in our study. 47n conclusion, the shape of the dose survival curve, the LD values, and the amount of apoptotic cells vary significantly between the different cell lines examined.When comparing blue and red light illumination, the light absorbance of the respective wavelength corresponds well to the light sensitivity of the cells.Furthermore, there has been some uncertainty as to whether one could make a direct comparison or not between studies when different wavelengths or irradiances have been used.This study shows that if one compares results between studies with either different irradiances or wavelengths, one should be especially aware of the differences at sublethal doses.

Fig. 1 A
Fig. 1 A flow chart of the experimental design including the number of parallels and experiments is presented.

Fig. 3
Fig.3Dose-response curves for five human cancer cell lines treated with red light (624 nm) HAL-PDT (20 μM, 3 h).In (a) all five cell lines were illuminated at 35 mW∕cm 2 and in (b) A431 cells were illuminated with 7 mW∕cm 2 in addition (n ¼ 6, small SEMs on error bars).Cell viability is relative to an untreated control in the same microplate (no HAL, no light) and the 0 J∕cm 2 point is the "HAL no light" value.Each point in (a) is the average of two to five experiments (each in sextuplicate) with standard error of the mean (SEM) indicated by error bars.

Fig. 4
Fig.4Spectra of intracellular HAL-induced (20 μM, 3 h) PpIX and possible derivatives thereof in A431 and T24 cells (4 × 10 6 cells∕ml).The spectra from untreated cells are subtracted from the presented PpIX spectra.(a) Fluorescence at 710 nm was measured for excitation wavelengths from 350 to 700 nm at 1 nm steps in signal/reference mode, making the spectra equivalent to absorption spectra.(b) Cells were excited at 410 nm and emission measured at 1 nm steps from 550 to 750 nm.

3. 4 Fig. 5
Fig. 5 Caspase 3/7 activity of A431 and T24 cells following HAL-PDT with 410-nm light and (a) 0.7 mW∕cm 2 and (b) 7.0 mW∕cm 2 .Values are related to an untreated control, set as 100%.Caspase 3/7 activity was measured for different light doses at 2, 5, and 8 h after completed illumination.Bars represent averages from three independent experiments with triplicates in each experiment and SEMs are indicated.
(a)] or 7.0 mW∕cm 2 [Fig.5(b)].In Fig. 5(a), a low dose corresponds to about LD 35 , a sub-low to LD 65 , a sub-full to LD 85 , and a full dose close to 100% cell death.In Fig. 5(b), the variance in the dose ranges is somewhat larger: the low dose corresponds to about LD 20 (A431) and LD 30 (T24), the sub-low to LD 35 (A431) and LD 60 (T24), the sub-full to LD 85 , and the full dose close to 100% cell death.

Fig. 6
Fig. 6 Caspase 3/7 activity in A431 and T24 cells following HAL-PDT with 624 nm light at 35.0 mW∕cm 2 .Values are related to an untreated control, set as 100%.Caspase 3/7 activity was measured for different light doses at 2, 5, and 8 h after completed illumination.Bars represent averages from three independent experiments with triplicates in each experiment and SEMs are indicated.