Spectroscopic studies of hematoporphyrin-derivative in culture medium

Chem.-Biol. Interactions, 50 (1984) 153--157 Elsevier Scientific Publishers Ireland Ltd.

SPECTROSCOPIC STUDIES IN CULTURE MEDIUM*

OF

153

HEMATOPORPHYRIN-DERIVATIVE

G. BOTTIROLI a, F. DOCCHIO b, I. FREITAS a, R. RAMPONI b and C.A. SACCHI b aCentro di Studio per l'Istochimica del C.N.R., Dipartimento di Biologia Animale, Universitd di Pavia and bCentro di Studio per l'Elettronica Quantistica e la Strumentazione Elettronica del C.N.R., lstituto di Fisica, Politecnico di Milano (Italy)

(Received December 14th, 1983) (Accepted February 5th, 1984)

SUMMARY

This work reports on studies of hematoporphyrin-derivative (HpD) behaviour in culture medium. Absorption, excitation and emission spectra, together with time-resolved fluorescence measurements, were performed. In previous works, similar studies had been carried out on HpD in saline and in lymphocytes: a new porphyrin species (NPS) and the environmental conditions for its formation in saline were studied. A fluorescent emission similar to that presented by the NPS is reported to be more likely in t u m o r rather than in normal HpD-treated cells, it was also found in greater amounts in lymphocytes in the pre-replicative phase, as compared with quiescent ones. The higher NPS content in stimulated rather than in quiescent lymphocytes may be due either to a differential uptake, as compared with other HpD components, or to a differential formation rate in cells, because of different microenvironmental conditions. To distinguish between these two main assumptions, the formation of NPS in culture medium was studied. The process was very slow: no NPS appeared within the first 40 h. The incubation time of lymphocytes in culture medium added with HpD in the experiments performed was only 1 h and therefore a differential formation rate of NPS may explain the higher content found in stimulated lymphocytes.

Key words: Hematoporphyrin-derivative - - P o r p h y r i n s p e c i e s - Culture medium -- Spectroscopic studies -- Time-resolved fluorimetry

*Work supported by Consiglio Nazionale delle Ricerche (CNR) through the Special Program 'Tecnologie Biomediche'. Abbreviations: HpD hematoporphyrin-derivative; NPS, new porphyrin species; PHA, phytohemagglutinin. 0009-2797/84]$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

154 INTRODUCTION

Several authors [1,2; G. Jori, pers. comm.] reported the presence of a HpD emission band in the 570--590 nm range. This band does not naturally occur in fresh HpD solution, and seems likelier in t u m o r than in normal HpD-treated cells. In previous works [3,4], we studied the spectral characteristics and the time-dependent behaviour of HpD in saline and the dependence of the time-resolved fluorescence microscopy of this drug on the cellular functional state in human lymphocytes. The aim of these works was to analyze the characteristics of this drug in solution and to study its interaction with cells. Among the results obtained, the spectroscopic studies of HpD in saline [3] showed that, with aging of the solution, NPS appears with an absorption peak around 405 nm and a main emission band around 575 nm. Timeresolved fluorescence analysis of this species was performed, and a decay time of about 3.5 ns was found. The environmental factors influencing NPS formation were studied, and it was found that one of the most favouring factors is a low oxygen content. HpD behaviour was studied in human lymphocytes, both in the Go (quiescent) phase and in the GI (pre-replicative) phase after stimulation with phytohemagglutinin (PHA) [4]. Fluorescence decay time measurements were performed on single cells in two spectral regions: a red one corresponding to the usual HpD emission band at about 610 nm and a yellow one centered at about 575 nm corresponding to the NPS emission. A decay time of ~ 1 6 ns was f o u n d for the red emission and ~ 3 . 5 ns for the yellow one. The same decay times had been found in HpD in saline and attributed to the monomeric species present in fresh solution and to the NPS, respectively. Analysis of the fluorescence peak amplitude permitted a quantitative evaluation of the drug content. A greater overall HpD content was found in stimulated lymphocytes as compared with the unstimulated ones. This is probably due to different membrane conditions, which determine different transport mechanisms. Examination of the relative percentages of the two fluorescent components (monomer and NPS) showed that the differences in content are much greater for the NPS. Three assumptions may be advanced to explain the greater differences in the 575 nm emission band between stimulated and unstimulated lymphocytes: (i) a differential uptake of NPS as compared with the monomer; (ii) a differential release of NPS as compared with the monomer; (iii) a differential formation rate of NPS in stimulated as opposed to unstimulated lymphocytes, due to different microenvironmental conditions. At this stage, the second assumption may be excluded. In fact, in the experiments on human lymphocytes, these were incubated in culture medium added with HpD for 1 h and then smeared on slides. In this time interval, the uptake process is very important, whereas there is practically no release as yet [1]

155 The aim of this work is to evaluate the role of the first and third assumptions. To this end we studied the time-dependent behaviour of HpD in culture medium and its spectral and time-resolved fluorescence properties. MATERIALS AND METHODS

Chemicals Chromosome Medium B with PHA and Chromosome Medium A without PHA from Seromed (F.R.G.) were used. HpD was purchased from Oncology Research and Development Inc. (Cheektowaga, NY, U.S.A.) under the trade name of Photofrin. Pure NPS was obtained from a solution of fresh HpD in saline, bubbling it with nitrogen (chromatographic grade) for 1 h and keeping it for 1 week at 37°C, in accordance with the results reported recently [3]. Fresh HpD and NPS were added to the culture media in order to obtain a final drug concentration of 3 × 10 -6 M. To study the time-dependent behaviour, all solutions were kept in the dark at 37°C. Spectroscopy A Beckman DU ® 8 UV-Visible computing spectrophotometer, equipped with a ~,-scanning compuset TM and sample holder under electronic temperature control (accuracy: T + 0.1°C) was used to record absorption spectra. Excitation and emission spectra were measured by means of an Applied Photophysics spectrofluorometer, model SP-Z, equipped with an EMI 9558/Q photomultiplier tube and an Ortec Photon Counter System. Excitation spectra were automatically corrected for the o u t p u t of the lamp (Thorn, 250 W D.C. XE/D) by means of a Rhodamine quantum counter. Fluorescence-decay-time measurements Fluorescence-decay-time measurements were performed by means of a pulsed-laser microfluorometer developed in our laboratories and extensively described in Docchio et al. [5]. A nitrogen-laser-pumped dye-laser (10 -a M solution of ~-NPO in ethanol) was used as the excitation source. This gives pulses of ~ 2 0 0 ps duration, 50 Hz repetition rate and was tuned at ~ 4 1 0 nm. Suitable optics and a microscope were used to focus the excitation light on the sample. The purpose of the microscope was to reproduce the experimental conditions used in measurements on HpD-treated cells, thus making immediate comparisons significant [4]. RESULTS Absorption and fluorescence characteristics of fresh HpD and of NPS were evaluated in culture medium with and without PHA. No differences were found due to the presence of PHA. The fresh HpD absorption spectrum in the Soret region consists of a broad band with two peaks centered at 370 nm and 405 nm. The spectrum can be interpreted in terms of a general equilibrium between unspecified dimeric

156 and monomeric forms. In this case moreover, the absorption spectrum depicts the interaction of HpD with proteins. Compared with the spectrum of HpD in saline, this interaction produces two main effects: a red shift by about 10 nm of the m o n o m e r band, and an increase in this band's height, due to a shift of the dimer-monomer equilibrium towards the latter form. This agrees with the data reported in the literature concerning the interactions of porphyrins with serum proteins [6]. As to the slight red shift of the dimer band, it is likely to be only an apparent shift, due to overlapping of the m o n o m e r and dimer bands. The fluorescence excitation spectrum of fresh HpD in culture medium shows a peak corresponding to the monomer-protein peak in the absorption spectrum. According to data in the literature [7], the dimer species is not fluorescent. The emission spectrum is the one typical of the monomer, with a redshift of about 10 nm. The fluorescence decay time was measured, and found to be about 16 ns: that is, the same as t h a t found in saline. The absorption/excitation spectra of NPS in culture medium exhibits, in the Soret region, a single peak at ~ 4 1 5 nm, i.e. red-shifted by about 10 nm, compared with the NPS in saline. The corresponding emission bands present a comparable red shift. This red shift shows an interaction with proteins, thus supporting the assumption advanced recently [3] that the NPS is a monomeric species. The fluorescence decay time is about 3.3 ns. Once the fluorescence characteristics of m o n o m e r and NPS in culture medium had been determined, a study was made of the formation process of NPS in this environment. Fresh HpD was added to culture medium, and absorption, excitation and emission spectra were recorded at time intervals of 4 h. Fluorescence decay times were measured in the two spectral regions corresponding to the m o n o m e r (around 620 nm) and to the NPS (around 585 nm) emission. Up to about 40 h of aging, only the m o n o m e r excitation and emission bands, and its typical decay time of ~ 1 6 ns were found. For longer aging, a small emission band at about 585 nm and a corresponding excitation peak at ~ 4 1 5 nm appear. Simultaneously, with the 16 ns decay time, another one of ~3.3 ns was found, in the fluorescence decay waveform. Therefore, in normal incubation conditions for cells, we may exclude the NPS formation in culture medium within the first 40 h. DISCUSSION The measurements performed show that the formation of NPS in culture medium added with fresh HpD is much slower than the incubation time of lymphocytes. Therefore no NPS is present during the uptake process. The results found in our previous experiments on human lymphocytes [4] and recalled in the introduction, can be explained in the light of the measurements in culture medium reported in this work. The greater overall uptake of HpD by stimulated, as opposed to unstimulated, lymphocytes

157 may be assumed to be due to different membrane conditions. Since the only fluorescent species present during the uptake process is the monomeric one, there should be a differential NPS formation rate in the cells in the two different functional states, due to dissimilar microenvironmental conditions. It is reasonable to assume that the lower oxygen content in stimulated lymphocytes (their oxygen consumption is obviously greater, due to their higher metabolic activity) favours more rapid formation of NPS. The interpretation given to the results obtained in human lymphocytes may be extended to tumOr cells, where an 'NPS-like' emission band seems to be more likely as compared with normal cells. The overall greater HpD uptake may again be explained by the different membrane conditions as between normal and t u m o r cells. And again, the anoxic environment typical of most t u m o r tissues may account for the higher NPS formation rate in t u m o r cells. REFERENCES 1 M.W. Berns, A. Dahlam, F.M. Johnson, R. Burns, D. Sperling, M. Guiltinan, A. Siemens, R. Walter, W. Wright, M. Hammer-Wilson and A. Wile, In vitro cellular effects of Hematoporphyrin Derivative, Cancer Res., 42 (1982) 2325. 2 W.J.M. Van der Putten and M.J.C Van Gemert, Hematoporphyrin derivative fluorescence spectra in vitro and in an animal tumor, Proc. Laser '81 Opto-Elektronik. (1981) 256. 3 G. Bottiroli, I. Freitas, F. Docchio, R. Ramponi, C.A. Sacchi, The time-dependent behaviour of Hematoporphyrin-Derivative in saline: a study of spectral modifications, Chem.-Biol. Interact., 49 (1984) 1. 4 F. Docchio, R. Ramponi, C.A. Sacchi, G. Bottiroli and I. Freitas, Time-resolved fluorescence spectroscopy of Hematoporphyrin-derivative (HpD) in human lymphocytes, Chem.-Biol. Interact., 50 (1984) 135. 5 F. Docchio, R. Ramponi, C.A. Sacchi, G. Bottiroli and I.Freitas, An automatic pulsed-laser microfluorometer with high spatial and temporal resolution, J. Microsc. 134 (1984) 151. 6 J. Moan and S. Sommer, Fluorescence and absorption properties of the components of hematoporphyrin derivative, Photobiochem. Photobiophys. 3 (1981) 93. 7 S.B. Brown, H Hatzikonstantinov and D.G. Herries, The structure of porphyrins and haems in aqueous solution, Int. J. Biochem., 12 (1981) 701.