Journal of Photochemistry and Photobiology, B: Biology, 6 (1990) 6 1 - 6 8

61

CELLULAR UPTAKE AND PHOTOSENSITIZING P R O P E R T I E S O F A N T I C A N C E R P O R P H Y R I N S IN CELL MEMBRANES AND LOW AND HIGH DENSITY LIPOPROTEINS* J. C. MAZIERE"b't, R. SANTUSb, P. MORLIERE ¢, J.-P. REYFTMANNb, C. CANDIDE a, L. MORAa, S. SALMON"' b, C. MAZIEREa, S. GATr a and L. DUBERTRET¢ "Laboratoire de Biochimie, Facultd de Mddecine Saint-Antoine, 27 rue Chaligny, 75012 Paris (France) bLabovatoire de Physico-Chimie de l'Adaptation Biologique, INSERM U312, Museum National d'Histoire Naturelle de Paris, 43 rue Cuvier, 75231 Paris Cedex 05 (France) CLaboratoire de Recherches en Dermatologie, INSERM UM2, Hdpital Henri Mondor, 04410 Crdteil (France) dUniversity of Jerusalem, Hadassah School of Medicine, Jerusalem (Israel) (Received November 24, 1989; accepted December 17, 1989)

Keywords. Anticancer porphyrins, photosensitization, membrane, low density lipoprotein (LDL), high density lipoprotein (HDL).

Summary The mechanisms of the phototoxic effect of anticancer porphyrins used in the photodynamic therapy (PDT) of tumours are not yet completely understood. Irradiation of porphyrins gives rise to singlet oxygen which reacts with key residues of proteins, polyunsaturated fatty acids and cholesterol in membranes, leading to inactivation of various enzymes and transporters. Lipoproteins, mainly low density lipoproteins (LDL), are efficient carriers of anticancer porphyrins in blood and can deliver these photosensitizers to tissues through the apolipoprotein (apo) B/E specific LDL receptor pathway. In this review, we discuss some aspects of anticancer porphyrin transport, cellular uptake and photosensitizing properties in cell membranes and lipoproteins.

1. Introduction During the last 10 years~ an increasing number of studies have been concerned with the photodynamic therapy (PDT) of tumours using porphyrins. *Paper presented at the Photomedicine Meeting organized by the French Society for Photobiology, Paris, November, 1989. t Author to whom correspondence should be addressed.

1011-1344/90/$3.50

© Elsevier Sequoia/Printed in The Netherlands

62 One of the most interesting properties of porphyrins as anticancer drugs is their selectivity, which, following intravenous injection, sometimes results in a high ratio between porphyrin concentration in tumours and porphyrin concentration in normal tissues. However, the mechanisms by which porphyrins can be accumulated in tumours are not yet clearly understood. Moreover, it is well established that singlet oxygen is responsible for cell killing after irradiation of cells loaded with porphyrins. However, the cellular targets and the molecular mechanisms of this photodynamic effect are still not entirely clear and strongly depend on the photosensitizer. Another aspect of porphyrin-induced photosensitization is the possible alteration of their lipoproteinic plasma carriers, i.e. low density lipoproteins (LDLs) and high density lipoproteins (HDLs), after irradiation in the peritumoral microcirculation. Although LDL clearance by tumour cells is rapid, this question has to be considered, and particularly the possible consequences of LDL photosensitization on its interaction with the apolipoprotein (apo) B/E specific receptor. Moreover, it has been demonstrated that porphyrins are also bound to the HDL fraction, which has a low turn-over rate in plasma. Therefore it is also interesting to consider the HDL photosensitization by anticancer porphyrins. In this review we discuss the role of LDLs in the cellular uptake of anticancer porphyrins, some of the effects of porphyrin-induced photosensitization of cells, with particular emphasis on membrane-bound enzymes, and, finally, the changes in LDL and HDL structure which occur after photosensitization of particles preloaded with porphyrin. 2, D o e s t h e LDL apo B/E specific r e c e p t o r play a m a j o r r o l e in cellular u p t a k e o f a n t i c a n c e r porphyrins? The major role of lipoproteins as carriers of hydrophobic porphyrins in plasma was first described 5 years ago [1-3]. Of the lipoproteins, LDLs are of particular interest because they are recognized by a specific receptor (the apo B/E receptor), which results in rapid internalization and delivery of the LDL particle to the lysosomal compartment [4]. Hydrophobic porphyrins are mainly carried in blood by LDLs and could thus be taken up by cells through the very efficient apo B/E receptor pathway. It has been demonstrated previously that the number of LDL receptors is increased in tumour cells compared with their normal counterparts [5-8]. Consequently, it is thought that porphyrin accumulation by tumours results from its delivery to cells via the LDL receptor pathway. Studies from our laboratories have also shown that, in vitro, the anticancer porphyrin mixture Photofrin II (P2) interacts with LDLs, and its delivery to cultured fibroblasts with LDLs as carriers is much more efficient than with albumin or HDLs [9]. When fibroblasts axe precultured under conditions in which the LDL receptor expression is low, less P2 is recovered in cells using P2-1oaded LDLs as donors [9]. The rapid delivery of LDLs to lysosomes via the apo B/E receptor pathway is probably an important factor in the ability of porphyrins to induce cellular

63 damage after irradiation. Photosensitization of lysosomal m em branes can result in destabilization of lysosomes and release of highly toxic hydrolytic enzymes in cytosol. This has also been dem onst rat ed in our laboratories by microspectrofluorometry on L cell murine fibroblasts [10]. The use of a peptide coupled to rhodamine as a synthetic substrate for acid proteases allows the release of lysosomal proteases in cytosol to be followed continuously. Using this method, we have dem ons t r a t e d that p r o t o p o r p h y r i n induces a greater lysosomal m e m br a ne permeabilization when LDLs are present in the incubation medium [10]. In addition to these i n v i t r o experiments, studies perform ed on animal models have also demonstrated a prominent role of the LDL fraction in porphyrin delivery to t u m o u r cells. Barel e t al. [11 ] have shown that the amount of h a e m a t o p o r p h y r i n r e c o v e r e d in the t um our (MS-2 fibrosarcoma) is higher after intravenous injection of h a e m a t o p o r p h y r i n bound to isolated LDLs as c o m p a r e d with the free drug or with the drug bound to isolated HDLs. The localization ratio (tumours v s . other tissues) is better when h a emato p o r p h y ri n- bound LDLs are used. Kessel [3] using Lewis-Lung-bearing mice, has shown that the clearance of ha em at oporphyri n is faster in the LDL fraction than in HDLs or albumin. Moreover, he has demonstrated that only porphyrins that are not t u m o u r localizers are bound to albumin. He also found a correlation between the distribution pattern of h a e m a t o p o r p h y r i n and the relative n u m b e r of LDL r e c e p t o r s in various tissues. All these studies suggest an important role of LDLs in porphyrin uptake by cells, and support the hypothesis that LDLs may be responsible for selective accumulation of porphyrins by tumours. However, in a recent paper, Kongshauw et al. [12] disagree with this opinion. These workers studied the affinity of various porphyrins for LDLs and their t um our localizing ability. They concluded that there is no clear correlation between these two parameters. For example, h a e m a t o p o r p h y r i n and protoporphyrin, which bind to LDLs, are poorly efficient t u m o u r localizers. In contrast, TPPS4, one of the best t u m o u r localizers, exhibits a high affinity for LDLs. However, they did not take into account the pharmacoldnetics of individual photosensitizers. Thus p r o t o p o r p h y r i n is probably taken up and released by tumours very rapidly (maximum t u m o u r concentration 1 h after injection [13]). Nevertheless, it could be conceived that many other factors influence the tissue localization of anticancer porphyrins i n v i v o . In addition to the LDL r e c e p t o r pathway, a non-specific exchange between LDLs and the plasma m em brane probably takes place, depending m or e or less on the relative affinity of the considered porphyrin for LDLs and the plasma membrane. This partition factor could be affected by the hydrophobicity [14] and by the negative charge of both the porphyrin and the plasma m e m b r a n e [15], the latter factor possibly differing between tissues and t u m o u r cell types. In our experiments, it has b e en observed that P2 uptake by fibroblasts is greater than e x p e c t e d under conditions in which the LDL r e c e p t o r expression is low. We thus suggest that, in addition to the LDL r e c e p t o r pathway, a non-specific uptake may be involved in P2 delivery to fibroblasts [9].

64 In conclusion, it must be stressed that tumour localization of anticancer porphyrins is probably a very complex process, involving multiple factors. Although there is no definite evidence as yet for a major role of LDLs in "selective" porphyrin accumulation by tumours, i n v i t r o and i n v i v o observations by different groups point to the LDL fraction as one of the important factors involved in tumour localization of anticancer porphyrins.

3. A l t e r a t i o n s o f m e m b r a n e - b o u n d e n z y m e a c t i v i t i e s in photosensitized cells Singlet oxygen reacts with a few amino acids, cholesterol and polyunsaturated fatty acids (PUFA), although its reactivity with PUFA is low [16, 17]. However, secondary reactions may occur in the presence of PUFA, leading to lipid peroxy radicals [18, 19]. The occurrence of PUFA and cholesterol hydroperoxides in ghost membranes photosensitized by haematoporphyrin derivative has been directly demonstrated [20, 21]. In cell membranes, where protein and lipids are in close interaction, these secondary reactions could play an important role in the late dark effects of the photodynamic reaction. Alterations of membrane proteins could thus take place as a consequence of lipid peroxidation in membranes. Of course, direct alteration of membrane proteins also occurs during photosensitization. For example, Dubbeiman et al. [22] have shown that isolated spectrin can be directly affected at the level of histidine (His), tryptophan (Trp) and cysteine (Cys) [22]. Another factor which has to be considered is the possibility of functional alterations of membrane proteins resulting from modifications of their lipidic microenvironment following membrane lipid photoperoxidation. Regardless of the mechanisms, many alterations of membrane-bound systems have been described after porphyrin-induced photosensitization. It appears that all cell compartments are potential targets. In rat liver mitochondria photosensitized by P2, the anion translocation system is markedly inhibited [23]. Enzymes of the mitochondrial respiratory chain such as succinodehydrogenase and cytochrome c oxidase are also rapidly inactivated in L929 fibroblasts photosensitized by haematoporphyrin derivative [24, 25]. In lysosomes, lysosomal membrane destabilization leads to leakage in the cytosol of highly toxic hydrolytic enzymes [10, 26]. Alterations of the endoplasmic reticulum have also been demonstrated: destruction of cytochrome P-450 takes place after haematoporphyrin photosensitization [27]. In intact transformed human fibroblasts, a rapid inactivation of the typical endoplasmic reticulum enzyme Acyl Coenzyme A:Cholesterol-O-Acyl Transferase (ACAT) is found, which controls cellular cholesterol esteriiication [28]. Plasma-membrane-bound enzymes and transporters are also markedly affected following cell photosensitization by porphyrins: for example, 5'-nucleotidase, Na ÷, K +-ATPase and Mg 2+-ATPase are inactivated in a dose-dependent manner when R3230AC mammary carcinoma plasma membranes are irradiated i n v i t r o in the presence of P2 [29]. An interesting observation reported in this

65 paper is that when using "in v i v o - i n vitro" protocols (injection of P2 into rats bearing R3230AC tumours, followed by irradiation of animals and in vitro measurement of enzyme activities on isolated plasma membranes prepared from tumours), only Na ÷, K+-ATPase is inhibited, suggesting that the actual (in vivo) consequences of cell photosensitization could differ from results obtained on in vitro experimental models. Transport functions of plasma membrane such as cycloleucin [30] or T-aminoisobutyric acid [31, 32] uptake are impaired. Leakage of cations following photosensitization by haematoporphyrin has also been reported in L929 fibroblasts by Dubbelman and Steveninck [32]. Thus all cell compartments are important targets for porphyrin-induced photosensitization, probably as a consequence of the wide partition of these hydrophobic compounds into all cell membranes. In contrast with other workers [30], we feel that it is difficult to indicate a particular mechanism which could be responsible for cell killing.

4. P h o t o s e n s i t i z a t i o n o f LDLs a n d H D L s

We have recently examined the possible photosensitization of LDLs and HDLs in protoporphyrin- and P2-1oaded particles. Protoporphyrin- and P2induced photosensitization of human LDL gives rise to lipid peroxidation products such as malonaldehyde and oxysterols specific for singiet oxygen attack, e.g. 5-a-hydroperoxycholesterol [33]. A decrease in free lysine amino groups and tryptophan residues of apolipoprotein B (apo B) is also observed [33]. Moreover, cross-links between apo B and products of arachidonic acid peroxidation occur. Lipofuscin-like pigments are formed and apo B is strongly aggregated [33, 34]. All of these alterations result in a loss of photosensitized LDL recognition by the apo B/E receptor of human fibroblasts [33]. However, although not recognized by fibroblasts, these LDLs have abnormal metabolic effects such as a decrease in oleic acid incorporation into triacylglycerols [34], suggesting a non-specific uptake by cells of toxic lipid peroxidation products from photosensitized LDLs. HDL photosensitization by anticancer porphyrins has also been studied. Using human HDL3 loaded with protoporphyrin or P2, lipid peroxidation products and a decrease in the number of tryptophan residues of apolipoproteins are observed [35]. However, it must be stressed that, in contrast with LDLs, lysine residues are not modified by the subsequent dark reactions (Table 1). This suggests that interactions between the lipidic core and the apolipoproteins axe quite different in LDLs and HDLs [35]. Although LDLs play a special role in porphyrin delivery to cells, it must be kept in mind that a non-negligible fraction of blood porphyrin is recovered in the HDL fraction, which is cleared at a slower rate. Thus the occurrence of altered HDLs in the peritumoral circulation after irradiation of patients must be considered. Experiments concerning the metabolic effects of these photosensitized HDLs are in progress in our laboratories. In addition to its interest

66 TABLE 1 Photosensitization of human LDL and HDL3 by the anticancer porphyrin mixture Photofrin II Irradiation t i m e (min)

12 24 36

LDL

HDL 3

L y s (%)

Trp(%)

L y s (%)

Trp(%)

92 87 81

54 42 34

98 98 97

48 40 32

LDL and HDL3 were prepared by stepwise ultracentrffugation according to Havel et al. [36], and were taken as the 1.024-1.050 and 1.125-1.21 fractions respectively. Lipoprotein samples (0.2 mg ml-~) in phosphate-buffered saline were loaded with Photofrin II (final concentration, 18 ~g m1-1) and then submitted to irradiation at 20 °C using the 405 nm emission ray of an OSRAM HBO 200 W mercury lamp. Intact lysine and tryptophan residues were measured according to Habeeb [37] and Candide et al. [33]. Data from Candide et al. [33] and Mazi~re et al. [35]. i n t h e field o f p h o t o d y n a m i c t h e r a p y o f c a n c e r , e s p e c i a l l y i n t h e p h a r m a cokinetics of a n t i c a n c e r p o r p h y r i n s a n d in the p a t h o p h y s i o l o g y of porphyrias, t h i s e x p e r i m e n t a l m o d e l ( t h e p h o t o s e n s i t i z a t i o n o f i s o l a t e d l i p o p r o t e i n s ) is also very useful in the study of lipid-protein interactions in lipoproteins.

Acknowledgments J. C. M a z i ~ r e g r a t e f u l l y a c k n o w l e d g e s L a L i g u e N a t i o n a l e F r a n q a i s e C o n t r e le C a n c e r , C o m i t 6 d e P a r i s , f o r f i n a n c i a l s u p p o r t . S. G a t t w i s h e s t o thank the Museum National d'Histoire Naturelle for financial support during his stay in Paris.

References 1 G. Jori, E. Beltramini, B. Reddi, A. Salvato, L. Pagnan, L. Ziron, L. Tomio and T. Tsanov, Evidence for a major role of plasma lipoproteins as hematoporphyrin carriers in vivo, Cancer Lett., 24 (1984) 291 -297. 2 J. P. Reyftmann, P. Morli~re, S. Goldstein, R. Santus, L. Dubertret and D. Lagrange, Interaction of human serum low density lipoproteins with porphyrins: a spectroscopic and photochemical study, Photochem. Photobiol., 40 (1984) 721-729. 3 D. Kessel, Porphyrin lipoprotein association as a factor in porphyrin localisation, Cancer Left., 33 (1986) 183-188. 4 M. S. Brown and J. L. Goldstein, Receptor-mediated control of cholesterol metabolism, Science, 101 (1976) 150-154. 5 Y. K. Ho, G. R. Smith, M. S. Brown and J. L. Goldstein, Low density lipoprotein (LDL) receptor activity in human acute myelogenous leukemia cells, Blood, 56 (1979) 1099-1114. 6 D. Gal, P. C. MacDonald, J. C. Porter and E. R. Simpson, Cholesterol metabolism in cancer cells in monolayer culture, III, Int. J. Cancer, 28 (1981) 315--319. 7 J. C. Mazibre, C. Mazi&re and J. Polonovski, Cholesterol metabolism in normal and SV40transformed hamster fibroblasts; effect of LDL, Biochimie, 53 (1981) 221-226.

67 8 G. Norata, G. Canti, L. Ricci, A. Nicolln, E. Trezzi and A. L. Catapano, In vivo assimilation of low density lipoproteins by a fibrosarcoma tumour line in mice, Cancer Lett., 25 (1984) 203-208. 9 C. Candide, P. Morli~re, J. C. Mazi~re, S. Goldstein, R. Santus, L. Dubertret, J. P. Reyftmann and J. Polonovski, In vitro interaction of the photoactive anticancer porphyrin derivative photofrin II with low density lipoprotein and its delivery to cultured human fibroblasts, FEBS Lett., 207 (1986) 133-138. 10 P. Morli~re, E. Kohen, J. P. Reyftmann, R. Santus, C. Kohen, J. C. Mazi~re, S. Goldstein, W. F. Mangcl and L. Dubertret, Photosensitization by porphyrins delivered to L cell fibroblasts by human low density lipoproteins. A microspectrofluorometric study, Photochem. Photobiol., 45 (1987) 183-191. 11 A. Barel, G. Jori, P. Perin, A. Pagnan and S. Biffanti, Role of high-, low- and very low density lipoproteins in the transport and tumor-delivery of hematoporphyrin in vivo, Cancer Lett., 32 (1986) 145-150. 12 M. Kongshauw, J. Moan and S. B. Brown, The distribution of porphyrins with different tumour localising ability among human plasma proteins, Br. J. Cancer, 59 (1989) 184-188. 13 D. Kessel, T. J. Dougherty and T. G. Truscott, Photosensitization by dipophyrins joined via methylene bridges, Photochem. Photobiol., 48 (1988) 741-744. 14 D. K. Gaffney and F. Sieber, Binding of merocyanine 540 by cells labelled with anthroyloxy fatty acids, Photochem. PhotobioL, 49 (1989) WPM-Cll, 685. 15 O. M. Smith and F. Sieber, Partitioning of tumour cells differing in their susceptibility to merocyanine-sensitized photoinactivation, Photochem. PhotobioL, 4P (1989) WPM-C10 685. 16 C. S. Foote, Photooxidation of biological model compounds, in M. A. J. Rodgers and E. L. Powers (eds.), Oxygen and Oxy-Radicals in Chemistry and Biology, Academic Press, New York, 1981, pp. 425--431. 17 C. Vervet-Bizet, M. Dellinger, D. Brault, M. Rougee and V. Bensasson, Singlet molecular oxygen quenching by saturated and unsaturated fatty-acids and by cholesterol, Photochem. Photobiol., 4P (1989) 321-326. 18 E. N. Frankel, Chemistry of free radicals and singlet oxidation of lipids, Prog. Lipid Res., 23 (1985) 197-221. 19 J. P. Reyftmann, R. Santus, P. Morli~re and E. Kohen, Effect of microenvironment and iron salts on lipid peroxidation photosensitized by porphyrins in ionic micelles, Photobiochem. Photobiophys., 9 (1985) 183-192. 20 A. F. P. M. De Goeij and J. Van Steveninck, Photodynamic effects of protoporphyrin on cholesterol and unsaturated fatty acids in erythrocyte membranes in protoporphyria and in normal red blood cell, Clin. Chim. Acta, d8 (1976) 115-122. 21 J. P. Thomas and A. W. Girotti, Photooxidation of cell membranes in the presence of hematoporphyrin derivative: reactivity of phospholipid and cholesterol hydroperoxides with glutathione peroxidase, Biochim. Biophys. Acta, 952 (1988) 297-307. 22 T. M. A. R. Dubbelman, A. F. P. M. De Goeij and J. Van Steveninck, Photodynamic effects of protoporphyrin on human erythrocytes. Nature of the cross-linking of membrane proteins, Biochim. Biophys. Acta, 511 (1978) 141-151. 23 A. Atlante, G. Moreno, S. Pasarella and C. Salet, Hematoporphyrin derivative (Photofrin II) photosensitization of isolated mitochondria: impairment of anion translocation, Biochem. Biophys. Res. Commun., 141 (1986) 584-590. 24 S. L. Gibson and R. Hill, Photosensitization of mitochondrial cytochrome c oxidase by hematoporphyrin derivative and related porphyrins in vitro and in vivo, Cancer. Ides., 43 (1983) 4191-4197. 25 R. Hilf, D. B. Smail, R. S. Murant, P. B. Leakey and S. L. Gibson, Hematoporphyrin derivative-induced photosensitivity of mitochondrial succinate dehydrogenase and selected cytosolic enzymes, Cancer Res., 44 (1984) 1483-1488. 26 R. Santus, C. Kohen, E. Kohen, J. P. Reyftmann, P. Morli~re, L. Dubertret and P. M. Tocci, Permeation of lysosomal membranes in the course of photosensitization with methylene blue and hematoporphyrin: study by cellular microspectrofluorometry, Photochem. Photobiol., 38 (1983) 71-77.

68 27 D. R. Bickers, R. Dixit and H. Mukhtar, Hematoporphyrin photosensitization of epidermal microsomes results in destruction of cytochrome P-450 and in decreased monooxygenase activities and h e m e content, Biochem. Biophys. Res. Commun., 108 (1982) 1 0 3 2 - 1 0 3 9 . 28 C. Candide, J. C. Mazidre, R. Santus, C. Mazi~re, P. Morli~re, J. P. Reyftmann, S. Goldstein and L. Dubertret, Photosensitization of WI26-VA4 transformed h u m a n fibroblasts by low density lipoprotein loaded with the anticancer porphyrin mixture Photofrin II: evidence for endoplasmic reticulum alteration, Cancer Lett., 44 (1989) 1 5 7 - 1 6 1 . 29 S. L. Gibson, R. S. Murant and R. Hilf, Photosensitizing effects of hematoporphyrin derivative and Photofrin II on the plasma m e m b r a n e enzymes 5'-nucleotidase, Na+K+-ATPase, and Mg2÷-ATPase in R3230AC m a m m a r y adenocarcinomas, CancerRes., 48 (1988) 3 3 6 0 - 3 3 6 6 . 30 D. Kcssel, Site of photosensitization by derivatives of hematoporphyrin, Photochem. Photobiol., 44 (1986) 4 8 9 - 4 9 3 . 31 J. Moan, J. McGhie and P. B. Jacobsen, Photodynamic effects on cells in vitro exposed to hematoporphyrin-derivative and light, Photochem. Photobiol., 37 (1983) 5 9 9 - 6 0 4 . 32 T. M. A. R. Dubbelman and J. Van Stevenick, Photodynamic effects of hematoporphyrinderivative on t r a n s m e m b r a n e transport systems of murine L929 fibroblasts, Biochim. Biophys. Acta, 771 (1984) 2 0 1 - 2 0 7 . 33 C. Candide, J. P. Reyftmann, R. Santus, J. C. Mazi~re, P. Morli~re and S. Goldstein, Modification of epsilon-amino groups of lysines, cholesterol oxidation and oxidized lipid-apoprotein cross-link formation by porphyrin-photosensitized oxidation of h u m a n low density lipoproteins, Photochem. Photobiol., 48 (1988) 1 3 7 - 2 4 6 . 34 C. Candide, Thes/s, University of Paris VII, 1989. 35 J. C. Mazidre, S. Salmon, R. Santus, J. P. Reyftmann, C. Candide, P. Morli~re, C. Mazidre and L. Dubertret, Lipid peroxidation and cellular functions: in vitro models and relation to in vivo observations, in Free Radicals, Lipoproteins and M e m b r a n e Lipids, Plenum, New York, in the press. 36 R. J. Havel, H. A. Eder and J. H. Bragdon, The distribution and chemical composition of ultracentrifugally separated lipoproteins in h u m a n serum, J. Clin. Invest., 34 (1955) 1343-1353. 37 A. F. Habeeb, Determination of free amino groups in proteins by trinitrobenzenesulfonic acid, A~ml. Biochem., 14 (1966) 328--336.

Cellular uptake and photosensitizing properties of anticancer porphyrins in cell membranes and low and high density lipoproteins.

The mechanisms of the phototoxic effect of anticancer porphyrins used in the photodynamic therapy (PDT) of tumours are not yet completely understood. ...
428KB Sizes 0 Downloads 0 Views