3 April 2013 Ion-induced stacking of photosensitizer molecules can remarkably affect the luminescence detection of singlet oxygen in Candida albicans cells
Author Affiliations +
J. of Biomedical Optics, 18(4), 045002 (2013). doi:10.1117/1.JBO.18.4.045002
Abstract
Singlet oxygen (1O2 ) is an important reactive intermediate in photodynamic reactions, particularly in antimicrobial PDT (aPDT). The detection of 1O2 luminescence is frequently used to elucidate the role of 1O2 in various environments, particularly in microorganisms and human cells. When incubating the fungus, Candida albicans, with porphyrins XF73 (5,15-bis-[4-(3-Trimethylammonio-propyloxy)-phenyl]-porphyrin) or TMPyP (5,10,15,20-Tetrakis(1-methyl-4-pyridinio)-porphyrin tetra(p-toluenesulfonate)), the 1O[sub]2 luminescence signals were excellent for TMPyP. In case of XF73, the signals showed strange rise and decay times. Thus,1O2 generation of XF73 was investigated and compared with TMPyP. Absorption spectroscopy of XF73 showed a change in absorption cross section when there was a change in the concentration from 1×10 −6   M to 1×10 −3   M indicating an aggregation process. The addition of phosphate buffered saline (PBS) substantially changed 1O2 luminescence in XF73 solution. Detailed experiments provided evidence that the PBS constituents NaCl and KCl caused the change of 1O2 luminescence. The results also indicate that Cl ions may cause aggregation of XF73 molecules, which in turn enhances self-quenching of 1O2 via photosensitizer molecules. These results show that some ions, e.g., those present in cells in vitro or added by PBS, can considerably affect the detection and the interpretation of time-resolved luminescence signals of 1O2 , particularly in in vitro and in vivo. These effects should be considered for any other photosensitizer used in photodynamic processes.
Felgenträger, Gonzales, Maisch, and Bäumler: Ion-induced stacking of photosensitizer molecules can remarkably affect the luminescence detection of singlet oxygen in Candida albicans cells

1.

Introduction

The fast development of multiresistant patterns against antibiotics of many species of bacteria has led to novel antibacterial strategies like the antibacterial photodynamic therapy (aPDT).1,2 A lot of work has been done to develop molecular structures and their derivatives that are able to generate reactive oxygen species (ROS), which are the active agents for killing microorganisms.34.5.6.7 The search for photosensitizers (PSs) for aPDT has caused the synthesis of various porphyrin molecules, which have been investigated regarding their photophysics and antimicrobial activity.4,8,9 Naturally occurring porphyrins can be found endogenously, e.g., the protoporphyrin IX that is in the prosthetic group of the hemoglobin or the chlorophylls based on the chlorine structure. Some endogenous porphyrins in bacteria are used to treat acne, where Propionibacterium acnes is a causative of the inflammatory processes.10 The porphyrin TMPyP has been frequently used for cell staining in order to investigate generation and decay of O21.1112.13

Different PSs are considered to localize in different compartments or regions in the eukaryotic or prokaryotic cell due to their number of positive charges and structure of the side chain. In order to determine the subcellular localization of PS and hence the site of O21 generation, fluorescence microscopy is applied by exciting the respective PSs. Since the resolution of light microscopy is limited, this procedure should fail with small bacteria and fungus cells with a diameter of about 1 μm. The direct measurement of O21 luminescence at 1270 nm might be an alternative candidate to elucidate the cellular action of O21 because the rise and decay time of O21 luminescence depend critically on its adjacency.14,15 In addition, singlet oxygen luminescence can provide information about the photodynamic process in bacteria during irradiation.

XF73 is a newly synthesized porphyrin molecule that already showed a high potential in antimicrobial PDT against gram-negative and gram-positive bacteria.16,17 However, principal data are lacking regarding its use in O21 detection in vitro. Thus, it is the goal of the present study to investigate the photophysical properties of XF73 and its potential to monitor photodynamic action in microorganisms. Exemplarily O21 luminescence detection was analyzed in vitro in Candida albicans cells. The well-known TMPyP was used for reference experiments.

2.

Material and Methods

2.1.

Chemicals

The cationic diporphyrin-based 5,15-bis-[4-(3-Trimethylammonio-propyloxy)-phenyl]-porphyrin (also referred to herein as XF73) with a molar mass of M=765.81g/mol, including the counter ion, was synthesized by Xiangdong Feng (Solvias Company, Basel, Switzerland) and kindly provided by Destiny Pharma Ltd. (Brighton, United Kingdom).

The 5,10,15,20-Tetrakis(1-methyl-4-pyridinio)-porphyrin tetra(p-toluenesulfonate) (also referred to herein as TMPyP) with a molar mass of M=1363.63g/mol, purity 97%, NaN3 sodium azide, Mannitol, NaCl, KCl, Na2HPO4, KH2PO4, and D2O have been purchased by Sigma Aldrich (Taufkirchen, Germany), and were used as received. The photosensitizers (PSs) were dissolved in bi-distilled water at a stock concentration of 1 mM and stored at 4°C until use. Figure 1(a) shows the chemical structure of XF73 and TMPyP.

Fig. 1

(a) Chemical structures of the porphyrins XF73 and TMPyP. (b) Normalized emission spectrum of the Waldmann-UV236 lamp. Absorption spectrum of XF73 and TMPyP with a concentration of 105M each.

JBO_18_4_045002_f001.png

Phosphate-buffered saline (PBS; PAA Laboratories GmbH, Pasching, Austria) at pH 7.4 has been used for aggregation experiments and contains NaCl (0.14 M), KCl (2.7×103M), Na2HPO4 (1.0×102M), and KH2PO4 (1.8×103M). For the NMR spectroscopy, a parent solution of the PSs dissolved in D2O was made and a PBS solution for dilution containing D2O has been prepared by adding NaCl, KCl, Na2HPO4, and KH2PO4 with the accordant concentrations.

2.2.

Absorption Spectrum

Absorption spectra were recorded at room temperature with a spectrophotometer (DU640, Beckman Instruments GmbH, Munich, Germany) in a concentration range of 1×106M to 2×103M. The percentaged transmission has been measured and the absorption cross-section σ(cm2) was calculated according to Eq. (1):

(1)

σ=ln(T/100)c·l·NA,
where c the concentration of PS, l the length of light path through the solution, T the transmission in percentage, and NA the Avogadro constant.

2.3.

Photostability

The PSs were irradiated with an incoherent broadband lamp (UV236; emission λ=380 to 480 nm) provided by Waldmann Medizintechnik (Villingen-Schwenningen, Germany). The maximal light intensity was 15.2mWcm2 at the level of the irradiated samples. The samples were irradiated for either 15 min (13.7Jcm2) or 60 min (54.8Jcm2). The emitted spectrum of the light source was recorded with a spectrometer (270 M, Jobin Yvon, Longjumeau, France) with 300 grid lines/mm and a spectral resolution of approximately 0.4 nm [Fig. 1(b)]. The detection range was 350 to 650 nm. The recorded spectral data were corrected regarding the spectral sensitivity of the spectrometer. The emission spectrum of the Waldmann UV lamp was normalized to its maximum between 400 and 450 nm.

2.4.

Cell Experiments

The C. albicans strain ATCC-MYA-273 was used for the experiments. The planktonic cells of C. albicans were diluted to a number of 106. For the incubation of C. albicans, the PSs stock solution has been diluted with H2O. The cells were incubated with a PS concentration of 104M in the dark for 15 min in H2O plus 50% PBS in falcons at slow rotation. The cells were rinsed twice with PBS to remove the not included or nonadherent PSs and afterward dissolved in pure H2O. For the singlet oxygen luminescence experiments, the planktonic cells were excited with a frequency doubled Nd:YAG-Laser (PhotonEnergy, Ottensoos, Germany).

2.5.

Fluorescence Spectrophotometer

The localization of XF73 in C. albicans was examined by fluorescence microscopy (Zeiss Vario-AxioTech, Goettingen, Germany) with an appropriate dual-band filter set for excitation and emission (Omega Optical, Brattleboro, Vermont) and a 63× magnification. Planktonic C. albicans were incubated 2 h with 104M XF73 in PBS and were rinsed twice with PBS.

2.6.

Singlet Oxygen Luminescence and Quantum Yield of O21 Formation (ΦΔ)

Solutions with PSs were filled in a cuvette (QS-101, Hellma Optik, Jena, Germany) and solutions of the planktonic cell suspension were investigated in acrylic cuvettes (SARSTEDT, Nümbrecht, Germany), both during magnetic stirring. The PSs were excited with a frequency doubled Nd:YAG-laser (PhotonEnergy, Ottensoos, Germany) with a wavelength λ=532nm, power output P=50mW, frequency of f=2kHz, and therefore, energy per pulse of E=2.5×105J. Every sample was irradiated with 40,000 pulses. The C. albicans planktonic cells were excited with a frequency doubled Nd:YAG-laser (PhotonEnergy, Ottensoos, Germany) with a wavelength λ=532nm, power output P=60mW, frequency of f=5kHz, and therefore, energy per pulse of E=1.2×105J. Every sample was irradiated with 100,000 pulses.

Direct detection as described in previous papers1819.20 was done by time resolved measurements at 1270 nm (30 nm full width half maximum filter) in near-backward direction with respect to the exciting beam using an infrared-sensitive photomultiplier (R5509-42, Hamamatsu Photonics Deutschland GmbH, Herrsching, Germany) with using an additional 950 nm cut-off-filter. The luminescence intensity is given by

(2)

I(t)=CtR1tD1[exp(ttD)exp(ttR)],
where C=[T1]t=0 kT1Δ [O23] was used to fit the singlet oxygen luminescence signal, describing the deactivation of the excited triplet state T1 of the photosensitizer by oxygen in its ground state (O23).20 tR and tD are the rise and decay times, which is the excited triplet state decay time τT1 of the photosensitizer and the decay time of singlet oxygen τΔ. The attribution of τT1 and τΔ depends on the oxygen concentration in the system; at high oxygen concentrations, usually the decay time τD of the signal describes the decay time of singlet oxygen τΔ. In order to determine the rise and decay times, the Levenberg-Marquardt-algorithm of Mathematica (Wolfram Research, Champaign) was used. The luminescence signal was spectrally resolved using interference filters in front of the photomultiplier tube at wavelengths ranging from 1150 to 1400 nm or a monochromator (Horiba, Yobin Yvon Inc., USA) from 1200 to 1350 nm at 10 nm regular steps (XF73 in pure H2O). The values show the integrated luminescence signals detected at a certain wavelength and are normalized to the maximal value. A Lorentz-shaped curve has been fitted through the measurement points, with the maximum at λ=1270nm, referring to the maximal value in H2O.

For the determination of ΦΔ of XF73 in H2O, it is compared with the ΦΔ of TMPyP, which is reported in literature being 0.7421 and 0.77±0.0412 in aqueous solution. Therefore, five probes of each PS of different concentrations (between 30% and 70% absorption at a wavelength of λ=532nm) are irradiated and the emitted O21-photons are determined with the integral over the luminescence curve, given with the fit routine mentioned.

3.

Results and Discussion

As a first experiment, cells of C. albicans were incubated with XF73 or TMPyP for 15 min using a concentration of 100 μM. The cells were washed twice, suspended in H2O solution, and subsequently excited with the laser at 532 nm. TMPyP in the cells produced a clear O21 luminescence signal with a rise time of tR=(1.77±0.2)μs and a decay time of tD=(6.74±0.7)μs [Fig. 2(a)]. In contrast to that, XF73 in C. albicans produced completely different O21 luminescence signals showing no or a very short rise time, whereas the signal decayed in a multiexponentially manner. When starting the fit at 2 μs, the decay time was tD=(5.33±0.5)μs [Fig. 2(b)].

Fig. 2

Singlet oxygen luminescence signal of planktonic solution of C. albicans cells incubated with 104M of TMPyP (a) and XF73 (b) for 15 min in the dark. The cells were washed and are surrounded by pure H2O with a cell concentration of 106 cells per mL.

JBO_18_4_045002_f002.png

On one hand, XF73 molecules were possibly localized at subcellular sites, where high quencher concentrations or low oxygen concentration affected the rise and decay of O21 luminescence. On the other hand, the photophysical properties of XF73 could have been altered after the uptake of C. albicans cells. It is known for many porphyrin species that PS molecules can show stacking to J- (edge-to-edge) and H-aggregates (face-to-face) under certain conditions.22,23 Aggregation of porphyrin derivatives is influenced by concentration of inorganic salts, the polarity of the solvents, or the side chains of the porphyrins,2425.26 whereas the results are still controversially discussed. Aggregation of PSs like TMPyP should not occur for concentrations of less than 104M.2728.29.30 An overview of the discussions related to the aggregation of TMPyP is given by Vergeldt et al., who described adsorption onto surfaces or aggregation effects due to the impurity of the solvent.31

Stacking of porphyrin molecules could occur at high photosensitizer concentrations or could be mediated by inorganic salts, which were particularly added with PBS to cells. Photosensitizer stacking may change the rate and rate constants for XF73 molecules and thereby affect the generation and decay of O21, which could be detected by time resolved detection of its luminescence.

3.1.

Absorption Spectroscopy in Aqueous PS Solution

Changes in the π-electron-system of porphyrin molecules can lead to the change of absorption cross-section σ and hence may affect O21 generation. TMPyP showed a constant absorption cross-section in the range from 106 to 103M (data not shown). In contrast to TMPyP, the absorption spectrum of XF73 in pure H2O clearly depended on XF73 concentration. The absorption cross-section decreased with increasing XF73 concentration from 105 to 2·103M and the absorption maximum (Soret band) shifted to shorter wavelengths (7nm) [Fig. 3(a)]. Both effects indicate aggregation of XF73 molecules.

Fig. 3

(a) Absorption spectrum of XF73 with increasing PS concentration. A blue-shift of the absorption maxima of 7 nm was detected when increasing the concentration from 105 to 103M. (b) Comparison of the influence of the single components of PBS on the absorption spectrum of XF73. A PS concentration of 2×105M has been used and NaCl, KCl, KH2PO4, and Na2HPO4 had each a concentration of 0.1 M. The table shows the wavelengths λmax of the absorption maximum and its value σmax for each component of PBS, for PBS and H2O.

JBO_18_4_045002_f003.png

3.2.

Absorption Spectroscopy in Aqueous XF73 Solution with PBS or PBS Constituents

The PBS and cytosol of living cells contain various ions like K+, Na+, Cl, HCO3, Mg2+, Ca2+, and HPO42. As a first approximation to cellular environment, XF73 was dissolved in PBS solution. As XF73 was not easily soluble in PBS, the maximum concentration of PBS was 50% in H2O. Absorption spectra of XF73 (2×105M) were recorded in pure H2O, in 50% H2O plus 50% PBS, and in 100% H2O adding single constituents of PBS such as KCl, NaCl, NaH2PO4, or KH2PO4, 0.1 M each [Fig. 3(b)].

In the presence of Na2HPO4 or KH2PO4, the absorption cross-section showed no wavelength shift or new absorption maxima within given experimental accuracy (±2nm) when compared with pure H2O. The maximum value of absorption cross-section at (402±2) nm decreased from σmax=0.71×1015cm2 (pure H2O) to σ=0.41×1015cm2 or σ=0.48×1015cm2 when Na2HPO4 or KH2PO4 was added, respectively.

When adding PBS, σmax decreased from 0.71×1015  cm2 to 0.25×1015  cm2 and shifted to longer wavelengths (red shift) of 24±2nm.

When adding NaCl or KCl to XF73 solution, σmax decreased to 0.25×1015cm2 for each. In addition, σmax shifted to the red by about 25±3nm. At the same time, the absorption spectrum showed new absorption maxima within the spectral range of the Soret band. Addition of Cl leads to a fundamental change of the absorption spectrum including a red shift. It is suggested that Cl affects the tetrapyrrol ring system and enhances the aggregation, which was already reported for other porphyrin structures.32

A visible precipitation of the solute started when using >10% PBS+H2O. This effect was shown to be reversible by diluting the solution with pure H2O. As a consequence of this dilution, the absorption spectrum of XF73 in PBS changed back to the absorption spectrum in pure H2O (data not shown). The precipitation does not affect the absorption measurements because the probes are directly used after being diluted and the precipitation effect needs several hours to develop. No light scattering effect in solutions was detectable by checking the absorption spectrum at shorter wavelengths.

3.3.

Photostability

Also, the photostability and hence the change of absorption spectrum during irradiation may affect O21 luminescence. Therefore, the photostability of XF73 in solution containing PBS was investigated when illuminating the samples up to 54.8Jcm2.

No changes in the absorption spectrum of TMPyP were noticed within irradiation time of upto 60 min (data not shown). The XF73 in H2O and in 50% PBS+H2O showed a decrease in absorption that was mainly detected in the spectral range of the Soret band (Fig. 4). Obviously, the presence of PBS, i.e., its ions, can additionally reduce radiation absorption of XF73. These effects may also affect the use of XF73 when applied for photodynamic inactivation of microorganisms.

Fig. 4

Photostability measurements with XF73 show a decrease of the absorption cross section with the time of illumination and therefore the applied energy. The light source was the Waldmann-UV236 lamp with an applied energy dose of 13.7 or 54.7Jcm2, respectively. XF73 with a concentration of 105M has been investigated in pure H2O and in PBS (50% in H2O).

JBO_18_4_045002_f004.png

In case of O21 experiments (see below), XF73 solutions were irradiated with 1 J of laser energy (532 nm). It is expected that σ values do not significantly change under these experimental conditions.

3.4.

O21 Luminescence Experiments without PBS

Incubation of bacteria or human cells with XF73 and subsequent irradiation yielded effective cell killing by means of O21 generation, which was confirmed by adding O21 quencher NaN3 that significantly reduced the cell toxicity.16 Since detailed studies on O21 generation of the novel porphyrin molecule XF73 were missing, we investigated XF73 in pure aqueous solution according to previous studies on other photosensitizers.27

After dissolving [XF73]=5×105M in air saturated ([O23]=2.7×104M), pure H2O, the rise and decay part of the time resolved signals could be assigned to the decay time τΔ of O21 and the decay time τT1 of PS, respectively. Experiments yielded τT1=1.6±0.2μs and decay time τΔ=3.5±0.3μs [Fig. 5(a)]. The decay time is in good correlation with the lifetime of O21 in pure water.3334.35 The spectrally resolved O21 luminescence revealed a peak at 1270 nm, which clearly confirmed the generation of O21 [Fig. 5(d)]. The O21 quantum yield ΦΔ of XF73 was determined in air saturated, pure H2O, using TMPyP as reference. The ΦΔ values of TMPyP are 0.7421 and 0.77±0.04.13 Using the previously reported technique,21 XF73 showed a value of ΦΔ=0.57±0.06.

Fig. 5

(a)–(c) O21 luminescence signals of [XF73]=5×105M with different PBS concentrations in H2O with an oxygen concentration of [O23]=2.7×104M. (d) Spectroscopically resolved O12 luminescence signal, generated by XF73 in H2O with an oxygen concentration of [O2]=2.7×104M. A Lorentz-shaped curve has been fitted through the measurement points. (e) O12 luminescence generated by XF73 in H2O+20%PBS at 1270 nm with 2×103M NaCl in solution. (f) Spectroscopically resolved O12 luminescence signal, generated by XF73 in 30% PBS+H2O with an oxygen concentration of [O2]=2.7×104M. A Lorentz-shaped curve has been fitted through the measurement points.

JBO_18_4_045002_f005.png

When changing the concentration of O2 in the solution at a constant concentration of [XF73]=5×105M, the meaning of the rates KΔ and KT1 at [O23]=1.1×104M changed according to the decay paths of O21 and T1 [Fig. 6(a)].20 This change occurs at a crossing point of t11 and t21, which was about [O23]=(0.11±0.02) 103M for XF73. By extrapolating t21, KT1 (([O2]=0M)=0.03μs1 was determined yielding a lifetime of the triplet T1-state of (33±5)μs in aqueous solution without oxygen quenching. The quenching rate constant kq for quenching of the excited triplet state of XF73 by oxygen is therefore kq=2.3×109s1M1 resulting from the Stern-Volmer-plot in Fig. 6(a), where the oxygen concentration was varied and the triplet decay of XF73 was determined.

Fig. 6

(a) Rates t11 and t21 of the time resolved O21 signal depending on the concentration of O2. The meaning of the two rates changes at the crossing point of the curves. (b) The rate t21 characterizes the decay time of the triplet-T1-state and changes with the XF73 concentration; here the oxygen concentration is kept constant at [O23]=5.4×105M.

JBO_18_4_045002_f006.png

As a next step, XF73 concentration was varied from [XF73]=106 to 5×103M at [[O32]=5.6×105M [Fig. 6(b)]. The value of t21 increased with increasing concentration that indicated a clear self-quenching effect of the excited triplet-T1-state for [XF73] up to about 2×104M. Above this concentration, the quenching effect decreased and reached a plateau at t21=0.205μs1, which is equivalent to a decay time of the triplet-T1-state of tT1=4.9μs [Fig. 6(b)]. According to the absorption spectroscopy data, a stacking of XF73 molecules occurred, which is easily detectable for XF73 concentration higher than 1×104M [Fig. 3(a)]. Obviously, the stacking process had already led to the formation of dimers or oligomers of XF73 molecules at this concentration. Besides a different absorption cross-section, these aggregates also show different deactivation of triplet T1-state as compared with XF73 monomers [Fig. 6(b)].

3.5.

O12 Luminescence Experiments with PBS

In light of the results above, O12 luminescence signals should be affected by molecule stacking, in particular when the photosensitizer in located in C. albicans cells [Fig. 2(b)]. Therefore, we investigated the PBS effect on time-resolved O12 luminescence generated by XF73 in air saturated solution at a concentration of 5×105M, for which stacking due to PS concentration should be still minimal [Fig. 3(a)]. The results clearly show that O12 luminescence substantially changed with increasing PBS concentration [Fig. 5(a)5(c)]. From 0% to 50% PBS in H2O, the rising part of O12 luminescence signal disappeared, whereas the decaying part shortened. Now, the luminescence signals at high PBS concentrations [Fig. 5(c)] were similar to those recorded for XF73 in C. albicans cells [Fig. 2(b)] yielding again a multiexponential decay.

When adding O12 quencher NaN336,37 to the 20% PBS solution up to a high concentration of 2×103M NaN3, the O12 luminescence signal almost disappeared. The residual signal should not originate from O12 luminescence [see Fig. 5(e)]. The same residual signal was detected in solutions without NaN3 and without oxygen (data not shown).

O12 luminescence was also spectrally resolved for PBS 0% and 50% in H2O [Fig. 5(d) and 5(f)]. A Lorentz-shaped curve has been fitted through the measurement points and the values were normalized to the maximal value. Without PBS, the fit shows a clear maximum at 1270 nm that confirms the generated O12.38 At 50% PBS, the maximum at 1270 nm almost disappeared, the baseline moved for wavelengths <1270nm, and the signal-to-noise ratio decreased, which indicates a substantial decrease of O12 generation.

Comparable to absorption spectroscopy, the changes of time- and spectral resolved O12 luminescence signals, induced by PBS, could be simply reversed by diluting the used solutions with H2O and hence reducing the PBS concentration. A high degree of dilution of PBS concentration yielded time- and spectrally resolved O12 luminescence signals comparable with Fig. 5(a) and 5(d).

Scattering of photons within solution might also cause a O12 luminescence signal equal to the one in Fig. 5(e), and might be originating from precipitation due to the stacking of the porphyrins. To exclude any scattering effects, the scattering agent SiO2 was added to aqueous solutions containing 5×105M XF73 or TMPyP. No effect on the shape of the O12 luminescence signal and no change of the rise and decay times were detected for both photosensitizers. Additionally there was no scattering effect visible in the absorption spectrum of XF73 in H2O+50% PBS.

4.

Conclusions

The detection of singlet oxygen by its luminescence is a great tool to show the action of singlet oxygen even in cells or bacteria. In this context it is important to have a detection procedure that provides reliable data from inside such cells, in particular when knowing that cellular constituents can substantially affect singlet oxygen luminescence. The interaction of porphyrins with C. albicans is controversially discussed that ranges from no uptake to tight binding or even internalization.3940.41.42.43 Many porphyrins are lipophilic and hence should accumulate in cellular membranes but the high water-solubility of XF73 suggests localization in the cytoplasm as well. Fluorescence microscopy showed the overall attachment of XF73 to the cell after washing; however, the low spatial resolution of optical microscopy impedes the evaluation of the subcellular photosensitizer localization (Fig. 7). Thus, it would be of importance to gain additional insight by evaluating the O12 luminescence data.

Fig. 7

Fluorescence image of C. albicans; the cells were incubated 2 h with 104M XF73 in PBS and rinsed twice. An attachment of XF73 to the cells can be seen.

JBO_18_4_045002_f007.png

However, XF73 showed substantial stacking of molecules that affected light absorption as well as the generation and decay of O12. Stacking already occurred in pure H2O along with the increase of the PS concentration. The stacking is additionally forced by the ionic pressure of Cl. Such ions are either present in cells or are usually added in cell experiments in vitro via PBS to protect the cells from osmosis. Therefore, it is impossible to exclude such ions when investigating photosensitizers in cell experiments.

Depending on the uptake mechanisms and the chemical structure, a PS localizes in cellular membranes or in the cytoplasm close to any cellular structures.44,45 Cytoplasm shows a similar concentration of Cl like PBS; therefore, it is very likely that aggregation of XF73 occurs in cells such as C. albicans. The time-resolved detection of the O12 luminescence in a solution of planktonic C. albicans cells incubated with XF73 and surrounded by pure H2O has been done [Fig. 2(b)]. In fact, the luminescence signal is similar to the signal of XF73 generating O12 in 30% PBS [Fig. 5(c)] showing a multiexponential decay. This signal indicates a surrounding of XF73 within C. albicans cells whose ionic concentration is similar to that of >30% PBS. Usually, the rise and decay times of luminescence provides information about the localization of O12 and hence of the photosensitizer applied due to the short diffusion length of O12 in cells. As the molecule XF73 is strongly influenced by the salts of the phosphate buffer PBS, such interpretations could be misleading at the moment. This problem may also occur for any other PS that undergoes stacking in the presence of ions such as Cl.

Despite the results with XF73, the O12 luminescence detection in cells is a great tool to elucidate photodynamic processes. The porphyrin TMPyP showed neither stacking in the investigated range of concentration nor interference with the salts of PBS. After attached to or taken up by C. albicans, the generated O12 could be easily detected by its luminescence with clear rise and decay components. The decay time of the O12 luminescence in Fig. 2(a) of tD=(6.74±0.5)μs, which is clearly longer than in pure water (3.5 μs) and can be most likely attributed to the decay time of the T1-state of TMPyP. If so, a triplet state decay time of 6.74 μs suggests an oxygen concentration of its surrounding of [O2]=8×105M, which is 30% compared with the oxygen concentration of [O2]sat=2.7×104M of air saturated water.

Nevertheless, the striking phototoxic effect of XF73 in bacteria was demonstrated.16 In vitro experiments showed a substantial reduction of bacteria (8log10 steps), which were incubated very small XF73 concentrations (108M) for 5 min and subsequently irradiated with 13.7Jcm2. The action of O12 was proven with the addition of the O12 quencher NaN3; however, the photodynamic effect could not be completely inhibited by the quencher. In addition, the rather small XF73 concentration in the range of 0.01 to 10 μM in those bacteria experiments could have minimized the stacking effect and therefore maximized phototoxicity by an effective singlet oxygen generation.

Aggregation effects influence also the fluorescence of a dye, which has recently been described by López-Chicón et al. with an investigation of Hypericin in different species of Candida.46 The grade of aggregation depends on the surrounding and the fluorescence is low or not existent at a high PS aggregation, which occurs in H2O-environment. Upon incubation of different species of Candida with Hypericin, one can draw a conclusion about the localization of the PS by monitoring the radiative decay, here the fluorescence that depends on the aggregation status.

Recently, with an optimized experimental setup singlet oxygen generation in C. albicans cells was detected by irradiating directly the Soret-band of the porphyrin TMPyP at 420 nm.47 With irradiation of the absorption maximum, it is possible to detect singlet oxygen generation and decay at already very low photosensitizer concentrations in the range of few μM offering a concentration range where aggregation effects are expected to be low and thus the singlet oxygen generation is effective.

Since the phototoxic efficacy depends on the localization and also on the aggregation status of the photosensitizer, which is influenced by ions, further investigations and comparative studies on the change of the singlet oxygen luminescence in different species of microorganisms should lead to better insights about the change of the decay times due to the localization.

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Ariane Felgenträger, Fernanda P. Gonzales, Tim Maisch, Wolfgang Baeumler, "Ion-induced stacking of photosensitizer molecules can remarkably affect the luminescence detection of singlet oxygen in Candida albicans cells," Journal of Biomedical Optics 18(4), 045002 (3 April 2013). http://dx.doi.org/10.1117/1.JBO.18.4.045002
Submission: Received ; Accepted
JOURNAL ARTICLE
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KEYWORDS
Luminescence

Oxygen

Absorption

Molecules

Picosecond phenomena

Ions

Bacteria

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