J. Photo&em.


B: Biol., 12 (1992)



Intracellular localization of meso-tetraphenylporphine tetrasulphonate probed by time-resolved and microscopic fluorescence spectroscopy+ Jurina M. Wessels GSF-Forschungszentrum fiir Umwelt W-8042 N-berg (FRG)

und Gesundheit

GwzbH, Ingolstiidter



Wolfgang StrauB Institut j?ir Lawrtechnologien W-7900 Ulm (FRG)

in der Medizin

an o!er Universit(it

Ulm, Postfach


Harald K. Seidlitz GSF-Forschungszentrum fiir Urnwelt und Gesundheit W-8042 N-berg (FRG)

GmbH Ingolstdidter



Angelika Riick and Herbert Schneckenburger Institti fiir Lasertechrwlagien W-7900 Ulm (FRG) (Received

April 23,


in der Medizin


July 29,

an der Universittit

Ulm, Postfach



Abstract The effects of solvent pH on spectral properties and fluorescence decay kinetics were investigated in order to characterize the microenvironment of meso-tetraphenylporphine tetrasulphonate (TPPS,) taken up by cells. Steady-state absorption and fluorescence spectra of TPPS, in buffer solutions of different pH were used to identify a ring protonated species at pH < 4. This dication could also be distinguished from the unprotonated form by its altered fluorescence decay time (3.5 VS. 11.4 ns). In addition, time-resolved spectroscopy gave some evidence of a monocationic species existing at pH 6-9. This was concluded from the occurrence of another component with a decay time of 5 ns. Measurements of the spectral and kinetic properties of the fluorescence emission of single epithelial cells (RR1022) incubated with TPP& indicated that the sensitizer was mainly localized in a microenvironment with a pH of 5, a value which occurs lntracelhrlarly only within lysosomes. Cells kept ln the dark exhibited the characteristic spectra of both the dication and the neutral form. The fluorescence decay showed two components with decay times of 2.6 ns and 10.6 11s.Irradiation of the cells changed the decay times to 4.6 ns and 13.4 ns and the dication fluorescence emission peak vanished, which is in accordance with the results obtained from buffer solutions at pH > 6. Therefore, we deduce that the photodynamic action leads to a rupture of the lysosomes and that the sensitizer is released into the surrounding cytoplasm. Keqworok: photosensitization, meso-tetraphenylporphine tetrasulphonate, fluorescence lifetimes, microspectrofluorometry, pH dependence. +Thispaper is dedicated to Professor Kurt Schafner


on the occasion of his 60th birthday.

0 1992 - Elsevier Sequoia. Ah rights reserved

1. Introduction

Sulphonated derivatives of tetraphenylporphine (TPPS,) have caused considerable interest as photosensitizing dyes used in photodynamic therapy. Compared with the widely used Photofrin II, which is a mixture of several porphyrins with different tumour-localizing properties [ 11, the derivatives of TPPS, are pure drugs. Studies by Wtielman et al. [ 2, 31 and Hiinerbein et al. [ 41 have shown that they exhibit promising tumour-localizing properties and that owing to their high triplet yield they are potent singlet oxygen generators [5]. Several investigations by Kessel et al. [S], Berg et al. [ 71 and Peng et al. [8] on the various derivatives TPPS, TPPS,,, TPP&,, TPPSa and TPPS, (which exhibit different water solubility) indicate that derivatives with different degrees of sulphonation are localized at different sites in cells and in tumour tissue. This has a large effect on the efficiency of the individual photosensitizer. In viva measurements of Peng et al. [8] have shown that the more lipophilic TPPSi and TPP&, mainly localize intracellularly, while the more hydrophilic TPPSe, and TPPS, tend to migrate into the tumour stroma. The cellular localization of a sensitizer depends on the uptake mechanism. Roberts and Berns [9] have shown that water-soluble sensitizers such as mono-L-aspartyl chlorin e6 (MACE) are taken up by endocytosis and finally accumulated in the lysosomes. According to the studies of Berg et al. [7] using fluorescence microscopy, TPPS;?,, TPP$, and TPPS, are mainly localized in extranuclear granules which are supposed to be lysosomes, whereas TPPS, is associated withintracellular membranes. This was concluded from the correlation of the TPPS, emission and the fluorescence of the lysosome marker acridine orange. Porphyrins are capable of accepting protons at the (-N=) nitrogens, thus forming positively charged ionic species. Barrett et al. [lo] and Brault et aE. [ll] have extensively investigated the acid-base properties of haematoporphyrin, protoporphyrin and related compounds. In aqueous solution porphyrins exist mainly as neutral zwitterionic species over a wide pH range (approximately 2-6). Only for pH values less than or equal to 1.5 is the dominating component a dication. Meso-tetraphenylporphins are better electron pair donors than porphyrins without phenyl-substituents in meso positions; accordingly the acid-base equilibria of these two types of porphyrins are different. In addition, the electronic properties of TPPS, are distinctively altered as the pH value changes [12, 131. TPPS, dissolved in buffer solution of pH 4 has a green colour, representing the corresponding diacid, whereas in buffer solutions of pH 2 6 the red colour of the free base is observed. The pH optimum of lysosomes (5.0) [14] is considerably lower than the pH value of the surrounding cytoplasm (7.4). This fact was used to localize TPPS, in individual cells by means of time-resolved fluorescence spectroscopy and microspectrofluorometry.


2. Materials

and Methods

2.1. Chemicals

TPPS, was obtained from Porphyrin Products, Logan, UT and used without further purification. Stock solutions of the sensitizer with a concentration of 5 x 10e5 M were made up in pH-buffer solution and kept in the dark. For time-resolved solution measurements we used a concentration of lop5 M, absorption spectra were made at lop5 M, 0.5~ lop5 M and 0.1 X 10m5 M and fluorescence spectra at 0.1 X 10 -5 M. Only freshly prepared solutions were used in this study. 2.2. Cell cultivatim Cultures of epithelial cells from rats (RR 1022) were grown in Petri dishes in Earl’s MEM with 5% foetal calf serum (FCS). 125 000 cells were seeded 36 h before incubation with the sensitizer. The cells were incubated for 12 h at 37 “C and 5% COz with 1O-5 M TPPS, in Earl’s MEM without FCS. For the microspectrofluorometric measurements the incubation time was 24 h and RPM1was used as the culture medium under the same conditions. All measurements were performed immediately after washing with Hank’s solution. 2.3. Absorption and steady state jkorescence wwcmmmwnts Absorption spectra were obtained with a Uvikon 8 10 two-beam spectrometer (Kontron, FRG). Fluorescence and excitation spectra of solutions were registered by a spectrofluorometer (SFM 25, Kontron, FRG) with a resolution of 5 nm. 2.4. Time-resolved meclsurements A tandem system (Spectra Physics, USA) with a dye laser synchronously pumped by a mode-locked argon ion laser was used to provide excitation pulses of approximately 20 ps duration. The pulse repetition frequency of the dye laser was reduced with a cavity dumper to 400 kHz. Stilbene 3 was used as the laser dye and the wavelength was set at 420 nm. The pulses were directed into a epifluorescence microscope (Leitz, Orthoplan, FRG). Fluorescence emission was detected with an R 928 photomultiplier (Hamamatsu, Japan) and a time-correlated single-photon counting apparatus [ 151. The emission band (610-685 mn) was selected by an appropriate filter combination. The fluorescence data were analysed using a non-linear least-squares deconvolution algorithm [ 161. Data from cells and solutions scatter and therefore we averaged ten cell measurements and four solution measurements in each case. 2.5. Microspectro@4n-ometric measurements Fluorescence spectra of single cells were detected by a microspectrofluorometer (Zeiss, UMSP 80) with variable excitation and emission wavelengths, as described earlier [ 171. As an excitation source a 75 W xenon


high pressure lamp in combination with a monochromator and a 40 x /0.95 objective lens was used. Power densities below 30 mW cmw2 were adjusted for the fluorescence excitation and emission spectra to avoid light-induced reactions during the recording time of about 10 s. A spectral resolution of 10 nm was selected. Photodynamic action was induced by applying increasing light doses at 420 nm ranging from 3 J cmV2 to 60 J cmp2. Only the emission spectra and their specific changes during irradiation are shown in the following.

3. Results and Discussion 3.1. Absorption and emission spectra of solutions Figure 1 shows the absorption spectra of TPPS4 measured in buffer solutions of pH 4 and pH 7. The Soret band, which peaks for neutral pH at 413 nm exhibits a pronounced red shift towards 435 nm if the pH value is lowered to 4. The Q bands show the well known etio-type structure at pH 7 with Qy peaks at 515 nm and 552 nm and Q, peaks at 580 nm and 634 nm. If the pH value of the buffer solution decreases to 4 we observe a strong band at 645 nm with a weak vibrational satellite at 593 nm. At pH 5 (spectra not shown) both the Soret band and the Q bands are regarded as a superposition of the structures at pH 4 and pH 7. Spectroscopic properties of TPPS, affect only the conjugated ring structure because of the generally low pk, values for sulphonic acids. The protonation of the nitrogens at low pH forms an almost symmetric non-planar molecule 1.0





---- pH4.0


Fig. 1. Absorption spectra of TPPS, in buffer solution at pH 4.0 and at pH 7.0, concentration 1O-5 M.


(dication) and the Q, and Qq transitions degenerate, thus only one Q band with its vibronic can be detected. The red shift of the complete absorption spectrum, which becomes most obvious at the Soret band, is probably due to an increase in resonance interaction of the phenyl rings with the porphyrin nucleus in going from the free base to the diacid [ 121. In addition, the degeneration of Q, and Qy leads to an intensification of the Q absorption band at pH 4. The fluorescence spectra of TPPS, in buffer solutions of pH 4 and 7 are depicted in Pig. 2(a). At pH 7 an emission with a peak wavelength of 647 nm corresponding to a (O-O) transition and its (O-l) vibronic at 701 run can be seen. The dication shows only one structureless emission band at 672 nm due to a (O-O) transition. The coexistence of both the neutral free base and the dication at pH 5 mentioned above can also be concluded from Fig. 2(b). Excitation at 410 nm gives rise to emission of the unprotonated molecule whereas excitation at 433 nm results in an emission characteristic to the ionic species. Both species can be excited simultaneously at 420 run. Figure 3 shows the pH dependence of the relative fluorescence intensity of the species excited at 433 nm representing the dication and the species excited at 410 nm assigned to the neutral free base. The rapid decrease in the dication fluorescence intensity between pH 4 and 6 illustrates the deprotonation of the diacid with increasing pH values.

_-__ pH 4.0 k_= 433 nm

300 -

pH 5.0 150 -

x 250 z 2 2



fi 8 6 :








50 -

600 (a)


------ &= 433 Nn


! mE p .!50f 5 *










: :

,’ ‘\ \ ‘\ ‘\




Fig.2. Fluorescence spectra of TPPS, in buffer solution: (a) pH 4.0, excitation and pH 7.0, excitation at 410 run, concentration 10d5 M; (b) pH 5.0, excitation and excitation at 410 nm, concentration 10M5 M.



at 433 NII at 433 nrn




Fig. 3. Effect of pH on the relative fluorescence intensity of TPPS.,, excitation at 433 nm, detection at 673 nm and excitation at 410 nm, detection at 648 mn; concentration 10e5 M.



Fig. 4. Effect of pH on the fluorescence decay time, excitation at 420 nm, detection at 610-685 nm, concentration 10S6 M: X, short component, 0, long component.

3.2. Time-resolved rneasur~ of solutim The results of the fluorescence decay measurements are given in Fig. 4. Excitation was performed at 420 nm, and fluorescence was detected in the spectral range from 610 nm to 685 nm in order to obtain decay data


from all species simultaneously. At pH 4 only one decay component of 3.5 ns can be deduced, which is attributed to the emission of the dication. This decay time is in excellent agreement with the value for the monomeric dication reported by Harriman and Richoux [ 181. The evaluation of decay data at higher pH, however, shows two components, a short component increasing from 3.5 ns to an average value of 5.0 ns as the pH value exceeds 5, and a long decay time fluctuating between 9.6 ns and 12 ns [ 191. The increase in the decay time of the short component between pH 5 and pH 6 indicates a change of the protonation state, the 5 ns value might therefore be assigned to the monocation. However, we should mention that Abraham et al. [20] argue against the existence of a monoprotonated species. The long component shows an average decay time of 11.4 ns in the range between pH 6 and pH 9. This decay component is assigned to the neutral molecule. As shown in Fig. 3 both the neutral form and the dication are present at pH 5. Since the absorption band of the diacid overlaps with the emission band of the unprotonated species, the fluorescence emission of the neutral species is partially quenched which results in a somewhat shorter decay time of 9.6 ns. 3.3. Tim-resolved measurem,ents of single cells Time-resolved measurements of epithelial cells from rats (RR 1022) incubated with 10e5 M TPPSl and kept in the dark show two decay components: 2.6 + 0.9 ns and 10.6 + 0.9 ns. After irradiation (350-450 run) with approximately 7 J cm-2, the decay times change to 4.6 L-0.3 ns and 13.4 + 1 .O ns [ 191. Within error margins these fluorescence decay tunes are in agreement with those obtained from a buffer solution of pH 5 and those with a pH value greater than or equal to 6 respectively. As pH 5 only occurs in cells within lysosomes [21], we conclude that TPPS, is mainly localized in the lysosomes before irradiation which is in agreement with recent findings of Berg and coworkers [ 7,22 ] and Riick et al. [ 23 ]. During irradiation, lysosomes are damaged by the photodynamic action of the photosensitizer, and TPPS, is released into the surrounding cytoplasm (approximately pH 7.4) which is supported by the increase in the decay time of the short component. 3.4. MicrospectroJluorcmRtric m.emrem,ents in single cells Figure 5 shows the fluorescence emission spectra obtained from epithelial cells from rats (RR 1022) incubated with lop5 M TPPS( kept in the dark (Fig. 5(a)) and after irradiation with 6 J cme2 at 420f 12 run (Pig. 5(b)). Corresponding to our measurements in solution, the unprotonated species is excited around 400 run (upper curve), whereas the dication is preferentially excited around 440 run (lower curve). Compared with the solution emission spectra (Fig. 2(a)) the maxima of the unprotonated species are red shifted by about 10 nm, whereas the spectrum of the dication is slightly blue shifted, showing a maximum at about 660 run. Since coexistence of both species is connected with a pH value around 5 we conclude using the same argument














Fig. 5. Fluorescence spectra of TPPS, in a single RR 1022 cell: (a) before irradiation, (b) after irradiation with 6 J cm-‘; upper curves, excitation at 400 * 5 nm; lower curves, excitation at 440+5 nm.

as above that TPPS, is localized in the lysosomes. During irradiation we observed a temporary decrease in the fluorescence intensities for both species without any changes in the spectra. This decrease was relatively weak for the unprotonated and strong for the dicationic species. This is in agreement with photodynamic damage of the lysosomes. The corresponding fluorescence excitation spectra which were observed at 615 nm for the dicationic and 715 nm for the neutral species showed maxima at 420-440 nm and 400-420 nm respectively. These maxima did not change after irradiation with 3 or 6 J cmW2. Thus there is no evidence for different cellular binding sites within the lysosomes. At higher irradiation doses (greater than or equal to 12 J cmp2) a drastic fluorescence increase of both spectra was observed, which was concomitant with the onset of fluorescence in the nucleus as described by Ri.ick et al. [231. Our recent videomicroscopic measurements confirm the redistribution of TPPSl fluorescence from individual granules to the cytoplasm (at about 6 J cm-‘) and to the cell nucleus (at about 12 J cm-2) using an excitation wavelength at about 420 MI. Previous studies [24, 251 have shown that the combination of timeresolved and microscopic methods can be applied to localize porphyrin sensitizers and their individual components (e.g. monomers, aggregates) withii single cells. In addition, as demonstrated in the present article, light-


induced photodynamic reactions can be probed on a subcellular level using the same methods.


The authors wish to thank Dr. E. Unsold and Professor R. Steiner for their stimulating contributions. We are also grateful to K. Hauf for her perfect technical assistance. This work was supported by the Bundesministerium fur Forschung und Technologie grant 070 600 35 (Photodynamic Laser Therapy).

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Intracellular localization of meso-tetraphenylporphine tetrasulphonate probed by time-resolved and microscopic fluorescence spectroscopy.

The effects of solvent pH on spectral properties and fluorescence decay kinetics were investigated in order to characterize the microenvironment of me...
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