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Time-gated fluorescence spectroscopy of porphyrin derivatives incorporated into cells
Journal of Photochemistry and Photobiology, B: Biology, 6 (1990) 39-48
39
T I M E - G A T E D F L U O R E S C E N C E S P E C T R O S C O P Y OF P O R P H Y R I N D E R I V A T I V E S I N C O R P O R A T E D INTO CELLS* R. CUBEDDU, R. RAMPONI and P. TARONI C.E.Q.S.E.-C.N.R., Istituto di Fisica del Politecnico, Piazza L. da Vinci 32, Milano (Italy) G. CANTI and L. RICCI Dipartimento di Farrnacologia dell'Universitd, Via Vanvitelli 10, Milano (Italy) R. SUPINO Istituto Nazionale Tumori, Via Venezian 1, Milano (Italy) (Received October 5, 1989; accepted December 5, 1989)
Keywords. Haem a t opor phyr i n derivative, photodynamic therapy, time-gated fluorescence.
Summary Time-gated fluorescence s p e c t r o s c o p y was p e r f o r m e d on the tumourlocalizing fraction (TLF) of haem a t opor phyri n derivative (HPD) incorporated into cells. Three different cell lines were incubated with 20 and 5 /zg ml-1 of TLF for various time periods; they were t hen washed and resuspended in buffer. Fluorescence decay m e a s ur e m ent s and time-integrated and timegated spectra were t hen obtained from the cell suspensions. Similar experiments were r e pe a t e d using HPD containing 60% of the active material. The experimental results show a modification of the emission spectra for both drugs depending on the incubation time; this modification is more significant for the TLF. In particular, the emission peak observed in aqueous solution at 615 nm is shifted to 630 nm as a c o n s e q u e n c e of incorporation into cells, and the gated spectra indicate that the fluorescence emission is mainly related to m o n o m e r s and unfolded polymeric chains. The ratio between the intensities of the two peaks depends on the relative amount of the TLF; the peak at 615 nm is m or e p r o n o u n c e d for HPD. The results obtained seem to indicate that both the composition c,f the drug and the metabolic properties of the biological environment strongly influence the uptake process and the fluorescence behaviour of the i ncor por at ed sensitizer.
*Paper presented at the Congress on Photodynamic Therapy of Tumours, Sofia, Bulgaria, October, 1989.
1011-1344/90/$3.50
© Elsevier Sequoia/Printed in The Netherlands
40 1. I n t r o d u c t i o n
Haematoporphyrin derivative (HPD) is the most extensively used photosensitizing drug for the local treatment of malignant tumours by photodynamic therapy. It consists of a mixture of porphyrins, monomers and covalently-bound complexes (seven or eight molecules on average), which exhibit a strong tendency to form aggregates of hydrophobic origin in aqueous solution. The fraction formed by the covalently-bound aggregates (hereafter called the tumour-localizing fraction (TLF)) is that responsible for the tumour affinity of the drug. However, it exhibits a low fluorescence quantum yield and a low singlet oxygen formation yield. In contrast, the monomeric fraction does not show any tumour specificity, but its fluorescence and photodynamic efficiency are much higher. HPD, once injected into a living body, is likely to undergo several modifications which lead to higher fluorescence and singlet oxygen formation yields [1, 2 ]. Indeed, the biological substrate (in particular the cell and cytoplasm organelle membranes, which are known to be the main binding sites for HPD [3, 4]) creates a highly hydrophobic environment. As shown in previous studies [1, 5], porphyrins tend to interact with polar hydrophobic structures, probably through delocalized charge interactions, with consequent breaking of the hydrophobic aggregates, followed by monomerization and/or unfolding of the polymeric chains; this results in spectral modifications similar to those observed in the presence of proteins and in cells and biopsy samples [6-8]. Through chromatographic techniques, it is possible to prepare haematoporphyrin derivatives containing various percentages of TLF. Both standard HPD (commercialized under the tradename of Photofrin) and a purified version consisting of TLF alone (Photofrin II) have been used in clinical practice with similar results, the only difference being the dose administered to the patient. In this work, we have investigated the photophysical properties of the TLF and HPD containing 60% TLF incorporated into cells and their dependence on the uptake time. Different cell lines were considered: cells were collected from ascitic tumours implanted in mice and suspended in culture medium immediately before the experiment (P388 and L1210) or stabilized i n vitro (P388); these cells were used to verify whether or not the modifications induced in the drug by the cellular microenvironment depend on the cell line and, for the same cell line, on the cell metabolic and functional characteristics (these can be markedly different in culture cells and in cells collected directly from the animal). Time-integrated and time-gated fluorescence emission spectra and spectrum-integrated decay waveforms were measured for all samples to discriminate between the contributions of the different fluorescent species in the drug and to study the dependence of their relative equilibria on the uptake time. Indeed, time-dependent equilibria were observed between the species which interact with the cellular hydrophobic structures and those which behave as in an aqueous environment; this suggests that phagocytosis contributes to the uptake of aggregates, followed by modification
41 of their structure and/or configuration on interaction with the cellular structures. This was confirmed by the absence of a continuous trend in the observed spectral modifications with uptake time. The pharmacokinetics of the different fluorescent species were dependent on the cell line and the metabolic and functional characteristics. 2. M a t e r i a l s a n d m e t h o d s 2.1. C h e m i c a l s
HPD was prepared following the procedure described by Lipson et al. [9] and was stored as a lyophilized powder, ready to be dissolved in buffer. It was tested by analytical high performance liquid chromatography (HPLC) and was found to contain 60% of the tumour-localizing fraction (TLF). The TLF was obtained as described by Keir et al. [10] and was supplied as a powder. Both drugs were kindly provided by the Department of Chemistry, Paisley College of Technology (Paisley, U.K.). 2.2. Cell c u l t u r e s a n d t r e a t m e n t s 2.2.1. P388 c u l t u r e cells
P388 cells, maintained in DBA/2 mice, were grown i n v i t r o by passaging twice a week in RPMI 1640 (Flow Laboratories, Irvine, Ayrshire, U.K.) containing fl-mercaptoethanol (10 -5 M), 10% foetal calf serum (Flow Laboratories, Irvine, Ayrshire, U.K.), penicillin and streptomycin. Cells in the growing phase at a passage i n v i t r o between the 10th and 30th passage were used for the experiments. 2.2.2. P388 a n d L1210 cells
Hybrid (Balb/cxDBA/2 F1, hereafter called CD2F1) mice of both sexes, 6 - 8 weeks old, from Charled River Breeding Laboratories (Calco, Italy) were used. The chemically induced lymphoid leukaemia L1210 (H-2 d) and the chemically-induced lymphocytic leukaemia P388 (H-2 d) were maintained by weekly intraperitoneal injection into CD2F1 mice. When ascitic tumours developed, the cells were collected and extensively washed. 2.2.3. Cell t r e a t m e n t
Cells of all lines were diluted to 108 cm -3 in Hank's balanced salt solution (HBSS); they were then treated with the drug (TLF or HPD containing 60% TLF) at different concentrations (5 and 20 t~g ml-1), and incubated in the dark. Every 30 min (up to 120 min and, in several cases, up to 180 rain), 1 cm 3 was centrifuged and resuspended in phosphate-buffered saline (PBS) (pH 7.4) for the measurements. Cell viability was checked using the Trypan blue dye exclusion test. 2.3. E x p e r i m e n t a l
apparatus Fluorescence decay waveforms and time-integrated and time-gated fluorescence spectra were obtained using the system described in ref. 11. The
42 excitation source was a mode-locked Ar + ion laser (Coherent CR-18) tuned at 364 nm, with a pulse duration of approximately 150 ps. The repetition rate of approximately 75 MHz was r e duc e d to approximately 750 kHz using an acousto-optic pulse-picker (Coherent 7200). The samples were contained in a quartz cuvette (path length, 1 cm). The emitted fluorescence was collected at 90 ° through a m o n o c h r o m a t o r (Jarrell-Ash 82-410) for the selection of the observation wavelength, and was detected by a microchannel-plate photomultiplier (Hamamatsu R1564U-01). Cut-off filters (Kodak Wratten) were used to eliminate any residual excitation light. The overall time resolution of the system was approximately 150 ps. The experimental data were obtained using the time-correlated single phot on counting technique [12]. The output pulses of the time-to-amplitude converter (TAC) were sent to a multichannel analyser (Silena System BS 27), operated in the pulse height analyser (PHA) mode, to measure the decay curves, and to a home-built acquisition unit. This unit allowed the simultaneous collection of the time-integrated emission spectrum, by accumulating all the TAC output pulses contributing to the decay curve, and two time-gated fluorescence spectra by counting only the signals falling within preselected time windows. Therefore a single measurement provided the three fluorescence spectra and the full-spectrum decay curve, obtained by setting the multichannel analyser in the acquisition mode during the whole time interval of the collection of the spectra. Experimental data were sent to a personal c o m p u t e r for storage and analysis. Fluorescence decay waveforms were analysed using a non-linear least-squares fitting method; weighted residuals and their autocorrelation functions were used to judge the quality of the fitting. Wavelength scanning and setting of the gate parameters were c o m p u t e r controlled.
3. R e s u l t s a n d d i s c u s s i o n Typical fluorescence spectra of P388 culture cells treated with 20 /~g ml-1 of TLF for different incubation times are shown in Fig. 1; the values of the fitting parameters for the fluorescence decay after 30 min incubation are r ep o r ted in Table 1. It should be noted that only these uptake data are re po r ted in the table since no significant changes were detected as a function of the uptake time. The same behaviour was observed for all the cell lines studied, independent of the drug used. The time-integrated spectrum collected after 30 min is characterized by the presence of two peaks at 615 nm and 630 nm and a shoulder at approximately 665 nm. According to the results obtained for the TLF in solution [5], three molecular species are present in the fluorescence decay: the fast species is related to aggregates or folded polymeric chains; the intermediate species is related to porphyrin moieties which interact with cellular c om ponent s and/or porphyrin aggregates and the slow species is related to m o n o m e r s and end rings of unfolded polymeric chains. As shown by the values of the relative amplitudes, the long-lived c o m p o n e n t provides the major contribution to the time-integrated spectrum.
43
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680
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Fig. 1. Fluorescence spectra of P388 culture cells treated with 2 0 / z g ml-* of TLF for different incubation times (left to right: 30 min; 60 min; 90 min; 120 min): top, time-integrated spectrum; middle, time-gated spectrum, zero delay, 500 ps width; bottom, time-gated spectrum, 18 ns delay, 6 ns width.
TABLE 1 Fluorescence decay time constants and relative peak amplitudes from cells incubated with 20 ~ g ml-1 of drug for 30 min
Sample
T, (ns)
A] (%)
72 (ns)
A2 (%)
~'3 (ns)
A~ (%)
P388 Cult. + TLF P388+TLF L1210 + TLF
15.17 15.19 14.69
39.31 31.49 33.17
4.02 4.17 4.70
31.51 25.22 29.63
0.60 1.14 0.92
29.18 43.29 37.20
P388 Cult. + HPD P388 + HPD L1210÷HPD
14.18 14.38 14.86
60.25 73.48 69.79
3.56 3.25 3.81
27.02 15.06 17.87
0.69 0.59 0.56
12.73 11.46 12.34
44 The appearance of two main peaks indicates that the porphyrin molecules are in the presence of either an aqueous (615 nm) or hydrophobic (630 nm) microenvironment. The assignment of the long-lived c o m p o n e n t is also confirmed by the shape of the delayed spectrum, where the two peaks are still dominant. As expected, the fast and intermediate com ponent s b e c o m e more apparent in the undelayed spectrum, where an e n h a n c e m e n t in the emission between 630 and 680 nm is observed. On increasing the uptake time, the cells seem to incorporate the porphyrin molecules to a larger extent according to the observed dominance of the emission peak at 630 nm and the similarity of the gated spectra. However, for 120 min incubation the peak at 615 nm is again significant and the shoulder at 6 3 0 - 6 8 0 nm is observed in the undelayed spectrum. It is worth mentioning that, for all the uptake times considered, fitting of the fluorescence decays gives values of the exponential c o m p o n e n t s which are similar to those r e p o r t e d in Table 1 for 30 min incubation; this indicates an equilibrium between the fluorescent molecular species as a function of time. Thus the modifications observed in the spectra can be explained in terms of metabolic effects on the TLF, which after incorporation into cells is reduced, possibly by the breaking of large polymeric chains which leads to a consequent partial drug release or weaker binding at cellular sites (615 nm peak). This seems to be consistent with the hypothesis that the uptake mechanism of TLF aggregates is phagocytosis [13]. The results obtained for incubation with 5 ~g ml-1 of TLF (Fig. 2) are consistent with those r e p o r t e d above. In this case, because of the lower drug concentration, most of the molecules are in a hydrophobic environment; the spectra show peaks at 630 nm, while the 615 nm contribution is observed as a shoulder. The metabolic process is also present, since the shoulder at 615 nm is reduced after 60 min incubation and b e c o m e s more significant at 90 min and 120 min incubation. It is worth noting that the relative amplitudes of the exponential c o m p o n e n t s are slightly different with a reduction (to approximately 28%) in the slow c o m p o n e n t and an increase in the intermediate and fast c om ponent s (to approximately 34% and approximately 38% respectively). Due to the lower drug content, the contribution of the cell autofluorescence becom e s significant, particularly for the fast and intermediate components. This is also evident in the undelayed spectra, where an increase in the emission intensity is observed starting from 580 nm in the spectral interval considered. To evaluate the influence of animal metabolism, the same spectroscopic measurements were carried out on P388 cells taken directly from a mouse immediately after sacrifice. As shown in Fig. 3, the spectra collected after 30 min incubation with 20 ]~g m1-1 TLF again exhibit the peaks at 615 nm and 630 nm; after a slight increase in the 630 nm emission at 60 min, the peak at 615 nm b e c o m e s predominant at 90 rain, while the emission at 630 nm is observed as a shoulder. After 120 rain the two peaks are almost equal. Thus animal metabolism seems to play a significant role in the incorporation of the TLF, apparently favouring weak bonds in a water-like environment
45 30
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Fig. 2. Fluorescence spectra of P388 culture cells treated with 5 ;zg ml- * of TLF for different incubation times (left to right: 30 min; 60 min; 90 min; 120 min): top, time-integrated spectrum; middle, time-gated spectrum, zero delay, 500 ps width; bottom, time-gated spectrum, 18 ns delay, 6 ns width. a n d / o r i n d u c i n g b o n d b r e a k i n g in the p o l y m e r i c material with a c o n s e q u e n t release o f m e t a b o l i z e d chains. This b e h a v i o u r is c o n f i r m e d b y the data o b t a i n e d for i n c u b a t i o n with 5 ;~g ml - ] , a l t h o u g h in this case, as previously, the p e a k at 6 3 0 n m is always p r e d o m i n a n t . The u p t a k e m e c h a n i s m of the TLF also s e e m s to be influenced b y the c h a r a c t e r i s t i c s o f the cell line. M e a s u r e m e n t s p e r f o r m e d o n L 1 2 1 0 cells t a k e n f r o m a m o u s e s h o w a p p r e c i a b l e differences f r o m the results o b t a i n e d with P 3 8 8 cells. As s h o w n in Fig. 4, a l t h o u g h the ratio b e t w e e n the p e a k s at 6 1 5 n m a n d 6 3 0 n m c h a n g e s with the i n c u b a t i o n time, a c c o r d i n g to the influence o f animal m e t a b o l i s m , the p e a k at 6 1 5 n m plays a m o r e significant role, a n d is d o m i n a n t b o t h after 30 min a n d 120 min. This e m i s s i o n is also o b s e r v e d as a s e p a r a t e p e a k for i n c u b a t i o n with 5 Izg m l - 1 in c o n t r a s t with the p r e v i o u s m e a s u r e m e n t s . This s e e m s to indicate the p r e s e n c e o f different b i n d i n g sites w h i c h f a v o u r an a q u e o u s e n v i r o n m e n t r a t h e r t h a n a h y d r o p h o b i c
46
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Fig. 3. Fluorescence spectra of P388 cells treated with 20 /zg ml -~ of TLF for different incubation times (left to right: 30 rain; 60 min; 90 rain; 120 min): top, time-integrated spectrum; middle, time-gated spectrum, zero delay, 500 ps width; bottom, time-gated spectrum, 18 ns delay, 6 ns width. e n v i r o n m e n t for this cell line. It is worth n o t i n g that the relative amplitudes of the e x p o n e n t i a l c o m p o n e n t s in the f l u o r e s c e n c e d e c a y are not appreciably different from the previous m e a s u r e m e n t s (see Table 1) a c c o r d i n g to a redistribution effect o f the s a m e molecular moieties. Since the TLF is derived from h a e m a t o p o r p h y r i n derivative via a purification technique, as described in ref. 10, the entire set o f m e a s u r e m e n t s w a s repeated using HPD containing 60% TLF. The effect o f m o n o m e r i c material is evident for all the spectra in a p r e d o m i n a n c e o f the e m i s s i o n at 6 1 5 n m and in an increase in the relative amplitude o f the long-lived c o m p o n e n t . The p e a k at 6 3 0 n m increases with incubation time and is m o r e evident for cells extracted from the animal. The decrease in HPD c o n c e n t r a t i o n from 2 0 /~g m l - 1 to 5 /~g m l - 1 d o e s not lead to a significant c h a n g e in the s h a p e o f the spectra. F r o m these results it appears that the p r e s e n c e o f the m o n o m e r s modifies the uptake p r o c e s s by saturating the binding sites, p o s s i b l y
47
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620
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)
Fig. 4. Fluorescence spectra of L1210 cells treated with 20 /zg ml -z of TLF for different incubation times (left to right: 30 min; 60 min; 90 min; 120 min): top, time-integrated spectrum; middle, time-gated spectrum, zero delay, 500 ps width; bottom, time-gated spectrum, 18 ns delay, 6 ns width.
o n the o u t e r cell m e m b r a n e , a n d r e d u c i n g the a m o u n t of material i n c o r p o r a t e d in the h y d r o p h o b i c e n v i r o n m e n t . The results o b t a i n e d s e e m to indicate t h a t b o t h the d r u g c o m p o s i t i o n a n d the m e t a b o l i c p r o p e r t i e s of the biological e n v i r o n m e n t s t r o n g l y influence the u p t a k e p r o c e s s a n d the f l u o r e s c e n c e b e h a v i o u r of the i n c o r p o r a t e d sensitizer. Thus different t u m o u r t y p e s a n d l o c a t i o n s m a y lead to different therapeutic responses and fluorescence mapping. Time-gated fluorescence s p e c t r o s c o p y c a n p r o v i d e a b e t t e r signal-to-noise c o n t r a s t for the p h o t o sensitizer fluorescence. By t a k i n g into a c c o u n t the fact t h a t the lifetimes of the natural f l u o r e s c e n c e are different f r o m t h o s e o f the p o r p h y r i n s , the c h o i c e of a suitable gate allows a b e t t e r discrimination b e t w e e n the f l u o r e s c e n c e emissions. This w a s verified o n the cell lines r e p o r t e d above, where, for i n c u b a t i o n with 5 / z g m l - 1, an i n c r e a s e in c o n t r a s t b y a f a c t o r o f a p p r o x i m a t e l y
48
four w a s o b t a i n e d in the 18 ns d e l a y e d s p e c t r a c o m p a r e d w i t h the c o n t i n u o u s w a v e (CW) spectra.
References 1 R. Cubeddu, R. Ramponi and G. Bottiroli, Time-resolved fluorescence spectroscopy of hematoporphyrin derivative in micelles, Chem. Phys. Lett., 128 (1986) 4 3 9 - 4 4 2 . 2 R. Redmond, E. J. Land and T. G. Truscott, A comparison of the photophysical properties of porphyrins used in cancer phototherapy, in R. V. Bensasson, G. Jori, E. J. Land and T. G. Truscott (eds.), P r / m a r y Photoprocesses in Biology and Medicine, Plenum, New York, 1985, pp. 3 3 5 - 3 3 9 . 3 F. Docchio, R. Ramponi, C. A. Sacchi, G. Bottixoli and I. Freitas, Time-resolved fluorescence microscopy of hematoporphyrin-derivative in cells, L a s e r s Surg. Med., 2 (1982) 2 1 - 2 8 . 4 R. Supino, G. Della Torre, R. Ramponi and G. Bottiroli, Effects of hematoporphyrin-derivative on mouse erythroleukemia cells in the absence of light irradiation, Chem. Biol. Interact., 57 (1986) 2 8 5 - 2 9 4 . 5 R. Cubeddu, R. Ramponi, W.-Q. Liu and F. Docchio, Time-gated fluorescence spectroscopy of the t u m o r localizing fraction of HpD in the presence of cationic surfactant, Photochem. Photobiol., 50 (1989) 1 5 7 - 1 6 3 . 6 G. Bottiroli and R. Ramponi, Equilibrium a m o n g hematoporphyrin-derivative components: influence of the interaction with cellular structures, Photochem. Photobiol., 47 (1988) 209-214. 7 G. Bottiroli, R. Ramponi and A. C. Croce, Quantitative analysis of intraceUular behaviour of porphyrins, Photochem. Photobiol., 46 (1987) 6 6 3 - 6 6 8 . 8 M. Dal Fante, G. Bottiroli and P. Spinelli, Behaviour of h a e m a t o p o r p h y r i n derivative in a d e n o m a s and adenocarcinomas of the colon: a microfluorimetrie study, L a s e r s Med. Sci., 3 (1988) 1 6 5 - 1 7 1 . 9 R. L. Lipson, E. J. Baldes and A. M. Olsen, The use of a derivative of h e m a t o p o r p h y r i n in t u m o r detection, J. Natl. Cancer Inst., 26 (1961) 1-8. 10 W. F. Keir, E. J. Land, A. H. MacLennan, D. J. McGarvey and T. G. Truscott, Pulsed radiation studies of photodynamic seusitizers: the nature of DHE, Photochem. Photobiol., 46 (1987) 5 8 7 - 5 8 9 . 11 R. Cubeddu, F. Docchio, W.-Q. Liu, R. Ramponi and P. Taroni, A system for time-resolved laser fluorescence spectroscopy with multiple picosecond gating, Rev. Sci. Instrum., 59 (1988) 2 2 5 4 - 2 2 5 9 . 12 S. Cova, A. Longoni, A. Andreoni and R. Cubeddu, A semiconductor detector for measuring ultraweak fluorescence decays with 70 ps FWHM resolution, IEEE J. Quantum Electron., 19 (1983) 6 3 0 - 6 3 4 . 13 T. J. Dougherty, Photoseusitizers: therapy and detection of malignant tumors, Photochem. Photobiol., 45 (1987) 8 7 9 - 8 8 9 .
39
T I M E - G A T E D F L U O R E S C E N C E S P E C T R O S C O P Y OF P O R P H Y R I N D E R I V A T I V E S I N C O R P O R A T E D INTO CELLS* R. CUBEDDU, R. RAMPONI and P. TARONI C.E.Q.S.E.-C.N.R., Istituto di Fisica del Politecnico, Piazza L. da Vinci 32, Milano (Italy) G. CANTI and L. RICCI Dipartimento di Farrnacologia dell'Universitd, Via Vanvitelli 10, Milano (Italy) R. SUPINO Istituto Nazionale Tumori, Via Venezian 1, Milano (Italy) (Received October 5, 1989; accepted December 5, 1989)
Keywords. Haem a t opor phyr i n derivative, photodynamic therapy, time-gated fluorescence.
Summary Time-gated fluorescence s p e c t r o s c o p y was p e r f o r m e d on the tumourlocalizing fraction (TLF) of haem a t opor phyri n derivative (HPD) incorporated into cells. Three different cell lines were incubated with 20 and 5 /zg ml-1 of TLF for various time periods; they were t hen washed and resuspended in buffer. Fluorescence decay m e a s ur e m ent s and time-integrated and timegated spectra were t hen obtained from the cell suspensions. Similar experiments were r e pe a t e d using HPD containing 60% of the active material. The experimental results show a modification of the emission spectra for both drugs depending on the incubation time; this modification is more significant for the TLF. In particular, the emission peak observed in aqueous solution at 615 nm is shifted to 630 nm as a c o n s e q u e n c e of incorporation into cells, and the gated spectra indicate that the fluorescence emission is mainly related to m o n o m e r s and unfolded polymeric chains. The ratio between the intensities of the two peaks depends on the relative amount of the TLF; the peak at 615 nm is m or e p r o n o u n c e d for HPD. The results obtained seem to indicate that both the composition c,f the drug and the metabolic properties of the biological environment strongly influence the uptake process and the fluorescence behaviour of the i ncor por at ed sensitizer.
*Paper presented at the Congress on Photodynamic Therapy of Tumours, Sofia, Bulgaria, October, 1989.
1011-1344/90/$3.50
© Elsevier Sequoia/Printed in The Netherlands
40 1. I n t r o d u c t i o n
Haematoporphyrin derivative (HPD) is the most extensively used photosensitizing drug for the local treatment of malignant tumours by photodynamic therapy. It consists of a mixture of porphyrins, monomers and covalently-bound complexes (seven or eight molecules on average), which exhibit a strong tendency to form aggregates of hydrophobic origin in aqueous solution. The fraction formed by the covalently-bound aggregates (hereafter called the tumour-localizing fraction (TLF)) is that responsible for the tumour affinity of the drug. However, it exhibits a low fluorescence quantum yield and a low singlet oxygen formation yield. In contrast, the monomeric fraction does not show any tumour specificity, but its fluorescence and photodynamic efficiency are much higher. HPD, once injected into a living body, is likely to undergo several modifications which lead to higher fluorescence and singlet oxygen formation yields [1, 2 ]. Indeed, the biological substrate (in particular the cell and cytoplasm organelle membranes, which are known to be the main binding sites for HPD [3, 4]) creates a highly hydrophobic environment. As shown in previous studies [1, 5], porphyrins tend to interact with polar hydrophobic structures, probably through delocalized charge interactions, with consequent breaking of the hydrophobic aggregates, followed by monomerization and/or unfolding of the polymeric chains; this results in spectral modifications similar to those observed in the presence of proteins and in cells and biopsy samples [6-8]. Through chromatographic techniques, it is possible to prepare haematoporphyrin derivatives containing various percentages of TLF. Both standard HPD (commercialized under the tradename of Photofrin) and a purified version consisting of TLF alone (Photofrin II) have been used in clinical practice with similar results, the only difference being the dose administered to the patient. In this work, we have investigated the photophysical properties of the TLF and HPD containing 60% TLF incorporated into cells and their dependence on the uptake time. Different cell lines were considered: cells were collected from ascitic tumours implanted in mice and suspended in culture medium immediately before the experiment (P388 and L1210) or stabilized i n vitro (P388); these cells were used to verify whether or not the modifications induced in the drug by the cellular microenvironment depend on the cell line and, for the same cell line, on the cell metabolic and functional characteristics (these can be markedly different in culture cells and in cells collected directly from the animal). Time-integrated and time-gated fluorescence emission spectra and spectrum-integrated decay waveforms were measured for all samples to discriminate between the contributions of the different fluorescent species in the drug and to study the dependence of their relative equilibria on the uptake time. Indeed, time-dependent equilibria were observed between the species which interact with the cellular hydrophobic structures and those which behave as in an aqueous environment; this suggests that phagocytosis contributes to the uptake of aggregates, followed by modification
41 of their structure and/or configuration on interaction with the cellular structures. This was confirmed by the absence of a continuous trend in the observed spectral modifications with uptake time. The pharmacokinetics of the different fluorescent species were dependent on the cell line and the metabolic and functional characteristics. 2. M a t e r i a l s a n d m e t h o d s 2.1. C h e m i c a l s
HPD was prepared following the procedure described by Lipson et al. [9] and was stored as a lyophilized powder, ready to be dissolved in buffer. It was tested by analytical high performance liquid chromatography (HPLC) and was found to contain 60% of the tumour-localizing fraction (TLF). The TLF was obtained as described by Keir et al. [10] and was supplied as a powder. Both drugs were kindly provided by the Department of Chemistry, Paisley College of Technology (Paisley, U.K.). 2.2. Cell c u l t u r e s a n d t r e a t m e n t s 2.2.1. P388 c u l t u r e cells
P388 cells, maintained in DBA/2 mice, were grown i n v i t r o by passaging twice a week in RPMI 1640 (Flow Laboratories, Irvine, Ayrshire, U.K.) containing fl-mercaptoethanol (10 -5 M), 10% foetal calf serum (Flow Laboratories, Irvine, Ayrshire, U.K.), penicillin and streptomycin. Cells in the growing phase at a passage i n v i t r o between the 10th and 30th passage were used for the experiments. 2.2.2. P388 a n d L1210 cells
Hybrid (Balb/cxDBA/2 F1, hereafter called CD2F1) mice of both sexes, 6 - 8 weeks old, from Charled River Breeding Laboratories (Calco, Italy) were used. The chemically induced lymphoid leukaemia L1210 (H-2 d) and the chemically-induced lymphocytic leukaemia P388 (H-2 d) were maintained by weekly intraperitoneal injection into CD2F1 mice. When ascitic tumours developed, the cells were collected and extensively washed. 2.2.3. Cell t r e a t m e n t
Cells of all lines were diluted to 108 cm -3 in Hank's balanced salt solution (HBSS); they were then treated with the drug (TLF or HPD containing 60% TLF) at different concentrations (5 and 20 t~g ml-1), and incubated in the dark. Every 30 min (up to 120 min and, in several cases, up to 180 rain), 1 cm 3 was centrifuged and resuspended in phosphate-buffered saline (PBS) (pH 7.4) for the measurements. Cell viability was checked using the Trypan blue dye exclusion test. 2.3. E x p e r i m e n t a l
apparatus Fluorescence decay waveforms and time-integrated and time-gated fluorescence spectra were obtained using the system described in ref. 11. The
42 excitation source was a mode-locked Ar + ion laser (Coherent CR-18) tuned at 364 nm, with a pulse duration of approximately 150 ps. The repetition rate of approximately 75 MHz was r e duc e d to approximately 750 kHz using an acousto-optic pulse-picker (Coherent 7200). The samples were contained in a quartz cuvette (path length, 1 cm). The emitted fluorescence was collected at 90 ° through a m o n o c h r o m a t o r (Jarrell-Ash 82-410) for the selection of the observation wavelength, and was detected by a microchannel-plate photomultiplier (Hamamatsu R1564U-01). Cut-off filters (Kodak Wratten) were used to eliminate any residual excitation light. The overall time resolution of the system was approximately 150 ps. The experimental data were obtained using the time-correlated single phot on counting technique [12]. The output pulses of the time-to-amplitude converter (TAC) were sent to a multichannel analyser (Silena System BS 27), operated in the pulse height analyser (PHA) mode, to measure the decay curves, and to a home-built acquisition unit. This unit allowed the simultaneous collection of the time-integrated emission spectrum, by accumulating all the TAC output pulses contributing to the decay curve, and two time-gated fluorescence spectra by counting only the signals falling within preselected time windows. Therefore a single measurement provided the three fluorescence spectra and the full-spectrum decay curve, obtained by setting the multichannel analyser in the acquisition mode during the whole time interval of the collection of the spectra. Experimental data were sent to a personal c o m p u t e r for storage and analysis. Fluorescence decay waveforms were analysed using a non-linear least-squares fitting method; weighted residuals and their autocorrelation functions were used to judge the quality of the fitting. Wavelength scanning and setting of the gate parameters were c o m p u t e r controlled.
3. R e s u l t s a n d d i s c u s s i o n Typical fluorescence spectra of P388 culture cells treated with 20 /~g ml-1 of TLF for different incubation times are shown in Fig. 1; the values of the fitting parameters for the fluorescence decay after 30 min incubation are r ep o r ted in Table 1. It should be noted that only these uptake data are re po r ted in the table since no significant changes were detected as a function of the uptake time. The same behaviour was observed for all the cell lines studied, independent of the drug used. The time-integrated spectrum collected after 30 min is characterized by the presence of two peaks at 615 nm and 630 nm and a shoulder at approximately 665 nm. According to the results obtained for the TLF in solution [5], three molecular species are present in the fluorescence decay: the fast species is related to aggregates or folded polymeric chains; the intermediate species is related to porphyrin moieties which interact with cellular c om ponent s and/or porphyrin aggregates and the slow species is related to m o n o m e r s and end rings of unfolded polymeric chains. As shown by the values of the relative amplitudes, the long-lived c o m p o n e n t provides the major contribution to the time-integrated spectrum.
43
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Fig. 1. Fluorescence spectra of P388 culture cells treated with 2 0 / z g ml-* of TLF for different incubation times (left to right: 30 min; 60 min; 90 min; 120 min): top, time-integrated spectrum; middle, time-gated spectrum, zero delay, 500 ps width; bottom, time-gated spectrum, 18 ns delay, 6 ns width.
TABLE 1 Fluorescence decay time constants and relative peak amplitudes from cells incubated with 20 ~ g ml-1 of drug for 30 min
Sample
T, (ns)
A] (%)
72 (ns)
A2 (%)
~'3 (ns)
A~ (%)
P388 Cult. + TLF P388+TLF L1210 + TLF
15.17 15.19 14.69
39.31 31.49 33.17
4.02 4.17 4.70
31.51 25.22 29.63
0.60 1.14 0.92
29.18 43.29 37.20
P388 Cult. + HPD P388 + HPD L1210÷HPD
14.18 14.38 14.86
60.25 73.48 69.79
3.56 3.25 3.81
27.02 15.06 17.87
0.69 0.59 0.56
12.73 11.46 12.34
44 The appearance of two main peaks indicates that the porphyrin molecules are in the presence of either an aqueous (615 nm) or hydrophobic (630 nm) microenvironment. The assignment of the long-lived c o m p o n e n t is also confirmed by the shape of the delayed spectrum, where the two peaks are still dominant. As expected, the fast and intermediate com ponent s b e c o m e more apparent in the undelayed spectrum, where an e n h a n c e m e n t in the emission between 630 and 680 nm is observed. On increasing the uptake time, the cells seem to incorporate the porphyrin molecules to a larger extent according to the observed dominance of the emission peak at 630 nm and the similarity of the gated spectra. However, for 120 min incubation the peak at 615 nm is again significant and the shoulder at 6 3 0 - 6 8 0 nm is observed in the undelayed spectrum. It is worth mentioning that, for all the uptake times considered, fitting of the fluorescence decays gives values of the exponential c o m p o n e n t s which are similar to those r e p o r t e d in Table 1 for 30 min incubation; this indicates an equilibrium between the fluorescent molecular species as a function of time. Thus the modifications observed in the spectra can be explained in terms of metabolic effects on the TLF, which after incorporation into cells is reduced, possibly by the breaking of large polymeric chains which leads to a consequent partial drug release or weaker binding at cellular sites (615 nm peak). This seems to be consistent with the hypothesis that the uptake mechanism of TLF aggregates is phagocytosis [13]. The results obtained for incubation with 5 ~g ml-1 of TLF (Fig. 2) are consistent with those r e p o r t e d above. In this case, because of the lower drug concentration, most of the molecules are in a hydrophobic environment; the spectra show peaks at 630 nm, while the 615 nm contribution is observed as a shoulder. The metabolic process is also present, since the shoulder at 615 nm is reduced after 60 min incubation and b e c o m e s more significant at 90 min and 120 min incubation. It is worth noting that the relative amplitudes of the exponential c o m p o n e n t s are slightly different with a reduction (to approximately 28%) in the slow c o m p o n e n t and an increase in the intermediate and fast c om ponent s (to approximately 34% and approximately 38% respectively). Due to the lower drug content, the contribution of the cell autofluorescence becom e s significant, particularly for the fast and intermediate components. This is also evident in the undelayed spectra, where an increase in the emission intensity is observed starting from 580 nm in the spectral interval considered. To evaluate the influence of animal metabolism, the same spectroscopic measurements were carried out on P388 cells taken directly from a mouse immediately after sacrifice. As shown in Fig. 3, the spectra collected after 30 min incubation with 20 ]~g m1-1 TLF again exhibit the peaks at 615 nm and 630 nm; after a slight increase in the 630 nm emission at 60 min, the peak at 615 nm b e c o m e s predominant at 90 rain, while the emission at 630 nm is observed as a shoulder. After 120 rain the two peaks are almost equal. Thus animal metabolism seems to play a significant role in the incorporation of the TLF, apparently favouring weak bonds in a water-like environment
45 30
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Fig. 2. Fluorescence spectra of P388 culture cells treated with 5 ;zg ml- * of TLF for different incubation times (left to right: 30 min; 60 min; 90 min; 120 min): top, time-integrated spectrum; middle, time-gated spectrum, zero delay, 500 ps width; bottom, time-gated spectrum, 18 ns delay, 6 ns width. a n d / o r i n d u c i n g b o n d b r e a k i n g in the p o l y m e r i c material with a c o n s e q u e n t release o f m e t a b o l i z e d chains. This b e h a v i o u r is c o n f i r m e d b y the data o b t a i n e d for i n c u b a t i o n with 5 ;~g ml - ] , a l t h o u g h in this case, as previously, the p e a k at 6 3 0 n m is always p r e d o m i n a n t . The u p t a k e m e c h a n i s m of the TLF also s e e m s to be influenced b y the c h a r a c t e r i s t i c s o f the cell line. M e a s u r e m e n t s p e r f o r m e d o n L 1 2 1 0 cells t a k e n f r o m a m o u s e s h o w a p p r e c i a b l e differences f r o m the results o b t a i n e d with P 3 8 8 cells. As s h o w n in Fig. 4, a l t h o u g h the ratio b e t w e e n the p e a k s at 6 1 5 n m a n d 6 3 0 n m c h a n g e s with the i n c u b a t i o n time, a c c o r d i n g to the influence o f animal m e t a b o l i s m , the p e a k at 6 1 5 n m plays a m o r e significant role, a n d is d o m i n a n t b o t h after 30 min a n d 120 min. This e m i s s i o n is also o b s e r v e d as a s e p a r a t e p e a k for i n c u b a t i o n with 5 Izg m l - 1 in c o n t r a s t with the p r e v i o u s m e a s u r e m e n t s . This s e e m s to indicate the p r e s e n c e o f different b i n d i n g sites w h i c h f a v o u r an a q u e o u s e n v i r o n m e n t r a t h e r t h a n a h y d r o p h o b i c
46
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Fig. 3. Fluorescence spectra of P388 cells treated with 20 /zg ml -~ of TLF for different incubation times (left to right: 30 rain; 60 min; 90 rain; 120 min): top, time-integrated spectrum; middle, time-gated spectrum, zero delay, 500 ps width; bottom, time-gated spectrum, 18 ns delay, 6 ns width. e n v i r o n m e n t for this cell line. It is worth n o t i n g that the relative amplitudes of the e x p o n e n t i a l c o m p o n e n t s in the f l u o r e s c e n c e d e c a y are not appreciably different from the previous m e a s u r e m e n t s (see Table 1) a c c o r d i n g to a redistribution effect o f the s a m e molecular moieties. Since the TLF is derived from h a e m a t o p o r p h y r i n derivative via a purification technique, as described in ref. 10, the entire set o f m e a s u r e m e n t s w a s repeated using HPD containing 60% TLF. The effect o f m o n o m e r i c material is evident for all the spectra in a p r e d o m i n a n c e o f the e m i s s i o n at 6 1 5 n m and in an increase in the relative amplitude o f the long-lived c o m p o n e n t . The p e a k at 6 3 0 n m increases with incubation time and is m o r e evident for cells extracted from the animal. The decrease in HPD c o n c e n t r a t i o n from 2 0 /~g m l - 1 to 5 /~g m l - 1 d o e s not lead to a significant c h a n g e in the s h a p e o f the spectra. F r o m these results it appears that the p r e s e n c e o f the m o n o m e r s modifies the uptake p r o c e s s by saturating the binding sites, p o s s i b l y
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Fig. 4. Fluorescence spectra of L1210 cells treated with 20 /zg ml -z of TLF for different incubation times (left to right: 30 min; 60 min; 90 min; 120 min): top, time-integrated spectrum; middle, time-gated spectrum, zero delay, 500 ps width; bottom, time-gated spectrum, 18 ns delay, 6 ns width.
o n the o u t e r cell m e m b r a n e , a n d r e d u c i n g the a m o u n t of material i n c o r p o r a t e d in the h y d r o p h o b i c e n v i r o n m e n t . The results o b t a i n e d s e e m to indicate t h a t b o t h the d r u g c o m p o s i t i o n a n d the m e t a b o l i c p r o p e r t i e s of the biological e n v i r o n m e n t s t r o n g l y influence the u p t a k e p r o c e s s a n d the f l u o r e s c e n c e b e h a v i o u r of the i n c o r p o r a t e d sensitizer. Thus different t u m o u r t y p e s a n d l o c a t i o n s m a y lead to different therapeutic responses and fluorescence mapping. Time-gated fluorescence s p e c t r o s c o p y c a n p r o v i d e a b e t t e r signal-to-noise c o n t r a s t for the p h o t o sensitizer fluorescence. By t a k i n g into a c c o u n t the fact t h a t the lifetimes of the natural f l u o r e s c e n c e are different f r o m t h o s e o f the p o r p h y r i n s , the c h o i c e of a suitable gate allows a b e t t e r discrimination b e t w e e n the f l u o r e s c e n c e emissions. This w a s verified o n the cell lines r e p o r t e d above, where, for i n c u b a t i o n with 5 / z g m l - 1, an i n c r e a s e in c o n t r a s t b y a f a c t o r o f a p p r o x i m a t e l y
48
four w a s o b t a i n e d in the 18 ns d e l a y e d s p e c t r a c o m p a r e d w i t h the c o n t i n u o u s w a v e (CW) spectra.
References 1 R. Cubeddu, R. Ramponi and G. Bottiroli, Time-resolved fluorescence spectroscopy of hematoporphyrin derivative in micelles, Chem. Phys. Lett., 128 (1986) 4 3 9 - 4 4 2 . 2 R. Redmond, E. J. Land and T. G. Truscott, A comparison of the photophysical properties of porphyrins used in cancer phototherapy, in R. V. Bensasson, G. Jori, E. J. Land and T. G. Truscott (eds.), P r / m a r y Photoprocesses in Biology and Medicine, Plenum, New York, 1985, pp. 3 3 5 - 3 3 9 . 3 F. Docchio, R. Ramponi, C. A. Sacchi, G. Bottixoli and I. Freitas, Time-resolved fluorescence microscopy of hematoporphyrin-derivative in cells, L a s e r s Surg. Med., 2 (1982) 2 1 - 2 8 . 4 R. Supino, G. Della Torre, R. Ramponi and G. Bottiroli, Effects of hematoporphyrin-derivative on mouse erythroleukemia cells in the absence of light irradiation, Chem. Biol. Interact., 57 (1986) 2 8 5 - 2 9 4 . 5 R. Cubeddu, R. Ramponi, W.-Q. Liu and F. Docchio, Time-gated fluorescence spectroscopy of the t u m o r localizing fraction of HpD in the presence of cationic surfactant, Photochem. Photobiol., 50 (1989) 1 5 7 - 1 6 3 . 6 G. Bottiroli and R. Ramponi, Equilibrium a m o n g hematoporphyrin-derivative components: influence of the interaction with cellular structures, Photochem. Photobiol., 47 (1988) 209-214. 7 G. Bottiroli, R. Ramponi and A. C. Croce, Quantitative analysis of intraceUular behaviour of porphyrins, Photochem. Photobiol., 46 (1987) 6 6 3 - 6 6 8 . 8 M. Dal Fante, G. Bottiroli and P. Spinelli, Behaviour of h a e m a t o p o r p h y r i n derivative in a d e n o m a s and adenocarcinomas of the colon: a microfluorimetrie study, L a s e r s Med. Sci., 3 (1988) 1 6 5 - 1 7 1 . 9 R. L. Lipson, E. J. Baldes and A. M. Olsen, The use of a derivative of h e m a t o p o r p h y r i n in t u m o r detection, J. Natl. Cancer Inst., 26 (1961) 1-8. 10 W. F. Keir, E. J. Land, A. H. MacLennan, D. J. McGarvey and T. G. Truscott, Pulsed radiation studies of photodynamic seusitizers: the nature of DHE, Photochem. Photobiol., 46 (1987) 5 8 7 - 5 8 9 . 11 R. Cubeddu, F. Docchio, W.-Q. Liu, R. Ramponi and P. Taroni, A system for time-resolved laser fluorescence spectroscopy with multiple picosecond gating, Rev. Sci. Instrum., 59 (1988) 2 2 5 4 - 2 2 5 9 . 12 S. Cova, A. Longoni, A. Andreoni and R. Cubeddu, A semiconductor detector for measuring ultraweak fluorescence decays with 70 ps FWHM resolution, IEEE J. Quantum Electron., 19 (1983) 6 3 0 - 6 3 4 . 13 T. J. Dougherty, Photoseusitizers: therapy and detection of malignant tumors, Photochem. Photobiol., 45 (1987) 8 7 9 - 8 8 9 .