Journal ofPhotochemistry

and Photobiology,

B: Biology,

5 (1990) 49 - 67

49

SYNTHESIS, CELLULAR UPTAKE OF, AND CELL PHOTOSENSITIZATION BY A PORPHYRIN BEARING A QUINOLINE GROUP P. MORLItiRet, M. MOMENTEAUb, C. CANDIDEC, V. SIMONINa, R. SANTUSd, J.-C. MAZIfiREC, L. DUBERTRETa, S. GOLDSTEINC and G. HePEa aLaboratoire de Dermatologie, INSERM U.312, H8pital Henri Mondor, 94010 Creteil (France) bLabomtoire de Biophysique Molbculaire, INSERM U.219, Znstitut Curie, Section de Biologie, Centre Universitaire, Bat. 112, 91405 Orsay (France) CLaboratoire de Biochimie, Faculte de Mkdecine Saint Antoine, 27 rue Chaligny, 75012 Paris (France) dLaboratoire de Physico-Chimie de I’Adaptation Biologique, INSERM U.312, Museum National d’Histoire Naturelle, 43 rue Cuvier, 75231 Paris Cedex 05 (France) (Received March 29,1989;accepted

Keywords. Porphyrin, photodynamic therapy.

July 11,1989)

lipoproteins,

porphyrin

uptake,

photosensitization,

Summary A tetraphenyl porphine linked to a 7-chloroquinoline (5,10,15,20tetraphenyl-l-3-{ 4( 4-aminobutyl)7-chloroquinoline}propioamidoporphine, TPPQ) was synthesized and examined as a potential photosensitizer for photodynamic therapy (PDT) of proliferative diseases. With respect to haematoporphyrin, TPPQ is a good in vitro photodynamic sensitizer producing singlet oxygen in 1% Triton Xl00 solutions. As with other hydrophobic porphyrins used in PDT, blood lipoproteins strongly bind TPPQ. Thus one low density lipoprotein (LDL) can incorporate 25 TPPQ molecules and 17 TPPQ molecules are taken up by one high density lipoprotein (HDL). Cell delivery of TPPQ using HDL or human serum albumin (HSA) as carrier is rather weak. However, an efficient TPPQ delivery to human skin fibroblasts is observed, partly aided by receptor-mediated endocytosis of LDL. Fluorescence spectroscopy shows that the cellular localization of TPPQ is both carrier and time dependent. During its delivery with LDL, TPPQ does not significantly impair the endocytosis of LDL-receptor complexes. After delivery with LDL, TPPQ is as efficient as other haematoporphyrin derivatives used in the PDT of cancers in photosensitizing human skin fibroblasts.

*Author to whom correspondence should be addressed. loll-1344/90/$3.50

0 Elsevier Sequoia/Printed in The Netherlands

50

1. Introduction The photodynamic therapy (PDT) of cancer relies on the properties of some solid tumours to take up and retain non-toxic porphyrins [l]. Activation of drug by visible light then causes tumour destruction. The deficiencies of the presently used haematoporphyrin derivatives such as Photofrin II (Phot II) are the ill-defined nature of their components and the long-lasting photosensitivity of the skin of patients. In theory, these secondary effects could be overcome by increasing the differential uptake of porphyrins by tumours using drug targeting techniques. Low density lipoproteins (LDLs) used as porphyrin carriers [2, 31 have thus been shown to be an interesting delivery system because the number of LDL receptors is increased by an order of magnitude in many tumour cell lines [4, 51. However, rapid porphyrin exchange with other lipoproteins cannot be avoided. In particular, high density lipoproteins (HDLs) whose lifetime is rather long in human blood (approximately 1 week) [6] are suspected as the main cause of the long-lasting skin sensitivity observed after PDT treatment [ 71. Another approach for limiting secondary effects would be to synthesize new porphyrins which are rapidly excreted from blood. Some compounds possess this property. Quinoline derivatives such as chloroquine form strong complexes with porphyrins [El]. This complex formation is believed to be responsible for enhanced excretion in porphyria cutanea tarda patients where chloroquine has been shown to be an effective drug [9, lo]. Furthermore, quinoline derivatives such as chloroquine are weak lysosomotropic bases which inhibit the lysosomal function [ll]. This inhibition leads to relief of inflammatory processes mediated by lysosomal hydrolases [ 121. In a search for chemically well-defined porphyrins that may be useful in photodynamic therapy of cancers or other diseases, we have synthesized a tetraphenylporphine derivative linked via a diamino linker chain to a quinoline derivative whose chemical structure is close to chloroquine (see Fig. 1).

CH,-CH,-CO-NH-(CH,),-NH

Fig. 1. Molecular

structure

of TPPQ.

51

In this paper we report the physicochemical properties, serum protein affinity and cellular uptake of this model compound. The latter was undertaken in the absence and presence of LDL, HDL or human serum albumin (HSA) as carriers. In situ fluorescence of the incorporated 5,10,15,20tetraphenyl-l-3-~4-(4-aminobutyl)7-chloroquinoline}propioamidoporphine (TPPQ) was monitored to study the effects of these different carriers on its intracellular localization. In addition, the effect of TPPQ on the LDL endocytosis pathway was investigated. We demonstrate that this compound is as efficient as Phot II in inducing skin fibroblast photosensitization.

2. Experimental details 2.1. Solvents, chemicals and routine equipment Water was obtained from a Millipore Milli Q ion exchanger downstream of a Millipore Milli RO 4 filtering unit. Solvents for synthesis were of reagent grade and other solvents used were of the best commercially available (spectroscopic or high performance liquid chromatography (HPLC)) grades. Chemicals were obtained from Sigma (HSA), Aldrich (1,4diaminobutane, 4,7-dichloroquinoline) and Prolabo (triethylamine). Phot II was kindly provided by Photofrin Medical Inc. (Raritan, NJ). Chemicals and media used for tissue culture were obtained from Flow (penicillinstreptomycin, trypsin, trypan blue), Boehringer (Earle’s modified Eagle medium (EMEM)), Squibb (amphotericin) and IBF (Ultroser G). Absorption spectra were recorded with either a Perkin-Elmer Lambda 5 or a Varian DMS 100 spectrophotometer. Corrected excitation and uncorrected emission fluorescence spectra were obtained using a Spex F112 fluorometer. 2.2. TPPQ synthesis Proton nuclear magnetic resonance (NMR) spectra of quinoline derivatives and porphine-quinoline (TPPQ) in deuteriochloroform were recorded on a Varian XL 100. Assignments of the resonances to individual protons were based on integration and selective homonuclear decoupling experiments. The spectra were in accordance with the structures assigned. Elemental analyses were performed by the Service Central de Microanalyse du CNRS. 2.2.1. Synthesis of 4-(4-aminobutyl)amino-7-chloroquinoline A mixture of 1,4-diaminobutane (8.8 g, 0.1 mol) and 4,7-dichloroquinoline (9.9 g, 0.05 mol) was refluxed for 4 h. Excess diamine was removed in vacua and the residue was taken up with methanol. After filtration of the insoluble product, the solvent was evaporated under reduced pressure. The solid compound was dissolved in methylene chloride and the solution was successively washed with 0.2 M aqueous ammonium hydroxide and water. It was then dried over Na2S04. Slow evaporation

52

of the solvent gave the desired compound as a crystalline solid. Yield: 4.56 g (43%). Elemental analysis: C&Hi$JsCl (found: C, 62.2%; H, 6.0%; N, 16.9%; Cl, 12.5%;required: C, 62.5%; H, 6.5%; N, 16.8%; Cl, 14.2%). 2.2.2. Synthesis of 5,10,15,20-tetraphenyl-l-3-{4-(4-adninobutyl)7chloroquinolinejpropioamidoporphine (TPPQ) A solution of 5,10,15,20-tetraphenyl-l-(3-propionic acid)porphine (TPP-(CH&-COOH) (137 mg, 0.2 mmol), prepared following the method of Adler et al. [13], in toluene was treated with excess oxalyl chloride (1 ml) at 50 “C for 1 h. The solvent was evaporated to dryness. The residue was taken up in dry methylene chloride (5 ml) and mixed with a solution of 4-(4-aminobutyl)amino-7chloroquinoline (138 mg, 0.5 mmol) and triethylamine (0.2 ml) in the same solvent. The mixture was stirred for 3 h at room temperature; it was then successively washed with water, diluted aqueous HCl, water, aqueous 0.2 M ammonium hydroxide and then dried over NazS04. The title compound was isolated by preparative thin layer chromatography over silica gel plates (Merck, silica gel 60, 2 mm) developed with methylene chloride-methanol (lO:lO, v/v). Yield: 130 mg (72%). Elemental analysis: C,&14$l,0C1 (found: C, 77.1%; H, 5.4%; N, 11.3%; Cl, 3.5%; required: C, 78.4%; H, 5.4%; N, 10.7%; Cl, 3.9%). NMR (CDCl,) 6 (ppm): 8.72 (m, 7 X Hpyr); 8.15 (m, 8 X PheHo + 1 X QC,H); 7.75 (m, 8XPheHm+4 XPheHp+ 1 XQCsH); 78.59 (s, 1 XQCsH); 7.05 (d, 1X Q&H); 5.96 (d, 1 X Q&H); 5.91 (t, 1 X Q-NH); 5.04 (t, 1 X NH-CO); 3.26 (t, 1 X TPP-C&-CH,); 3.15 (m, 1 X-C&-NH-CO); 2.88 (m, 1 X Q--NH-C&); 2.64 (t, 1 X TPP-CH,-CH,); 1.38 (b, NH-CH,-CH,-CII,CH,-NH-); 2.70 (s, 2 X NHTPP). 2.3. Photodynamic properties Irradiations were performed with an Osram HBO 200 W high pressure mercury lamp. The solution was contained in a 1 cm X 1 cm thermostatically controlled optical cell and was continuously bubbled with air. The 405 nm line was isolated by combination of an anti-l-IV glass filter (Wild) and a liquid filter (12-Ccl4 0.75% w/v; path length, 1 cm). The solution to be irradiated contained 0.5 mM histidine (His) and the porphyrin (TPPQ or haematoporphyrin) concentration was adjusted so that the optical density was 0.4 at 405 nm. The His concentration was followed by HPLC using a Chromatem 800 HPLC (Touzart et Matignon) and a Whatman Partisil lo/25 SCX column (inside diameter, 4.6 mm; length, 25 cm). Spectrophotometric determination of His was carried out at 210 nm at the output of a Shimadzu SPD 2A spectrophotometer. The mobile phase was 50 mM NH4H2P04 adjusted to pH 2.3 with H$‘O,. These experiments were performed with TPPQ or haematoporphyrin as photosensitizer. The quantum yields of photosensitized oxidation of His are proportional to the initial slopes of the plots of His concentration us. irradiation time.

53

2.4. Cell culture Fibroblast cultures were established from punch biopsies (1 or 2 mm diameter) of human skin obtained from breast plastic surgery [14]. The cells were propagated in EMEM containing antibiotics (e.g. 100 IU ml-’ penicillin, 100 ,ug ml-’ streptomycin, 50 ng ml-’ amphotericin) and supplemented with 10% foetal calf serum in culture flasks (75 cm2) kept in an atmosphere of 5% C02. For most experiments, cells were further seeded at 6000 cells cme2 in Petri dishes (35 mm) (2.5 ml of a 24 000 cells ml-l suspension in EMEM containing antibiotics and supplemented with foetal calf serum) and grown for 5 days. When required and as specified below, the regular culture medium (see above) was replaced 24 h before experiments by a lipoprotein-deficient culture medium for maximum LDLreceptor expression. This medium consists of EMEM with antibiotics (see above), supplemented with 2% lipoprotein-deficient serum substitute (Ultroser G). 2.5. Incubation procedure and porphyrin uptake determination LDL and HDL, prepared from normolipidaemic human serum by the method of Have1 et al. [15], were diluted in 0.1 M Tris buffer and 0.15 M NaCl, pH 7.4 to achieve a concentration of 30 Ergml-’ in terms of protein content. Assuming molecular weights of around 600 000 and 200 000 for LDL and HDL [6,16], concentrations were thus about 50 nM and 150 nM respectively. HSA was prepared at 750 pg ml-’ (approximately 11 PM) in Tris buffer. A 15 mg ml-’ TPPQ stock solution (15 mM) was prepared in dimethylsulphoxide and diluted to 2 PM with LDL, HDL or HSA solutions. These solutions, LDL loaded with TPPQ (LDL-TPPQ), HDL loaded with TPPQ (HDL-TPPQ) and HSA loaded with TPPQ (HSATPPQ), were then filtered on 0.45 pm Millipore filters and rapidly used. Solutions of TPPQ (0.45 PM) in Tris buffer (Tris-TPPQ) were similarly prepared and used without further filtration. Aliquots (100 - 200 ~1) of these solutions were diluted with 1% Triton Xl00 in Tris buffer (2.5 ml) to allow a fluorometric determination of the porphyrin concentration (A,,, = 419 nm, X,, = 649 nm) to be made. Cells in Petri dishes were washed twice with Tris buffer and then incubated for various times at 37 “C with 1 ml of TPPQ-containing solutions. In experiments using LDL-TPPQ, cells were pre-incubated 24 h before experiments with LDL-deficient medium (see above). After incubation, cells were washed twice with cold Tris buffer and harvested with a rubber policeman in 2 ml cold Tris buffer. Emission and excitation spectra (both at a bandwidth resolution of 9 nm) of the gently stirred cell suspensions were recorded in a 1 cm X 1 cm optical cell set in a thermostatically controlled holder (25 “C). Then, 1 ml of the cell suspension was devoted to protein determination according to the method of Lowry et al. [17]. The remaining 1 ml was added to 1 ml of 2% Triton Xl00 in Tris buffer. Data are therefore expressed in weight of porphyrin incorporated per weight of cellular protein. Experiments were performed twice and

54

each determination was done in triplicate. Excitation and emission spectra are the average of these triplicates although the reproducibility of the spectra was better than 15%. 2.6. Binding to receptors of LDL loaded with TPPQ A TPPQ-LDL solution was prepared as described above to achieve 100 E.cgml-’ LDL and 6 PM TPPQ before filtration. The TPPQ concentration of the filtered solution was fluorometrically determined as approximately 0.5 PM. It was then diluted in Tris buffer to achieve various LDL concentrations (10, 20,40, 70 and 100 pg ml-‘) which were used for incubations (1 h, 1 ml) of cells pre-incubated or not with LDL-deficient medium 24 h before experiments. Cells were incubated, washed and scraped at 4 “C. The TPPQ uptake was determined as described above. Experiments were performed twice in triplicate. 2.7. Competition for binding, internalization and degradation of LDL Fibroblasts were grown in Petri dishes (35 mm) and the culture medium was replaced by lipoprotein-deficient medium 24 h before experiments to achieve maximum LDL-receptor expression. Cells were washed and incubated for 1 h at 4 “C (binding) or 4 h at 37 “C (internalization and degradation) with 10 Erg ‘251-LDL in the presence or absence of various amounts of unlabelled LDL, LDL-TPPQ or LDL loaded with TPP-(CH2)2COOH (LDL-TPP-( CH2),-COOH). TPPQ and TPP-( CH2)2-COOH solutions (6 FM) were prepared by diluting corresponding stock solutions in culture medium (without serum) containing 100 pg ml-l LDL. After filtration (0.45 I.trn Millipore filters), TPPQ and TPP-(CH2)2-COOH concentrations were fluorometrically determined as 1.3 and 2.5 PM respectively. The ‘251-LDL was prepared according to Bilheimer et al. [18] using 1251-Na (Amersham, 13 - 17 mCi pg-‘), and the specific activity was approximately 250 - 300 d.p.m. ng-’ of LDL. After 4 h incubation at 37 “C, the incubation medium was saved for measurements of LDL degradation (see below). Cells incubated at 4 or 37 “C were washed four times with cold Tris buffer, harvested with rubber policemen and centrifuged. The radioactivity in the pellet was measured using an LKB 1275 7 counter. Protein determination was performed on the same sample and results (c.p.m. mg-i cell protein) are expressed as a percentage of the control ( 1251-LDLalone) [19]. For estimation of LDL degradation, 250 ~1 HSA (1%) and 250 ~1 trichloroacetic acid (50%) were added to 500 1.11 of the collected incubation medium (see above) in order to precipitate LDLs that were not internalized. After centrifugation, the supernatant was collected and treated with 10 ~1 KI (40%) and 50 ~1 H202 (30 vol) and vortexed for approximately 1 min. Then CHCl, (2 ml) was added and the mixture was vortexed for a few minutes. The aqueous upper phase containing labelled peptides arising from LDL degradation was collected and submitted to radioactivity counting. Results (c.p.m. mg-’ cell protein) are expressed as a percentage of the control ( 1251-LDLalone) [ 191. Experiments were performed twice in triplicate.

55

2.8. Extract&n and HPLC of incorporated porphyrins HPLC analyses were run on a Varian Vista 5600 chromatograph using a Whatman Partisil 5 ODS 3 column (inside diameter, 4.6 mm; length, 25 cm). The spectrophotometric detection of porphyrins was carried out at 415 nm and chromatographs were recorded and analysed with a Varian 4290 integrator. The mobile phase (1 ml min-‘) was a mixture of tetrahydrofuran (0.35 vol) and methanol containing 5 mM phosphate buffer (4:1, v/v) (0.65 vol) adjusted to pH 6.7. Cells were seeded and grown to almost confluency in 75 cm2 culture flasks (approximately 1.5 X lo5 cells per Petri dish, approximately SO% confluency). TPPQ solutions (7 ml), prepared as described above, were used for incubations. After incubation and washing, cells were harvested in 2 ml Tris buffer. The cell suspension was extracted twice with 2 ml of methanol-chloroform (l:l, v/v). After centrifugation the lower phase was collected, evaporated to dryness under argon and then taken up with 100 ~1 of methanol-tetrahydrofuran (1 :l, v/v). These conditions allowed successive elution of the porphyrin derivative resulting from the amide bond splitting (TPP-(CH,),COOH) of the parent compound TPPQ and the unsubstituted porphyrin 5,10,15,20-tetraphenylporphine (TPP). 2.9. Cell photosensitization Nearly confluent cells (approximately 1.5 X 10’ cells per Petri dish, approximately 80% confluency) were pre-incubated the day before the experiments in LDL-deficient medium. Incubation was performed 24 h later in EMEM (without phenol red) in the presence of 30 pg ml-’ LDL loaded with the desired amount of TPPQ or Phot II (see below). Prior to incubation, these solutions were filtered through 0.45 pm Millipore filters. After filtration, porphyrin concentrations were determined as above using fluorometric techniques (X,,, = 405 nm, h,, = 630 nm for Phot II). Incubations were performed for 17 h with 1 ml of solution containing 0.2 Erg ml-’ TPPQ or 0.5 or 1.5 Erg ml-’ Phot II. Before irradiation, cells were washed twice with Tris buffer and left with 1 ml of this buffer. Irradiations were carried out with an Osram 2.5 kW xenon lamp in a Kratos lamphouse equipped with a water filter (length, 10 cm). The 405 nm light was obtained at the output of a GM 252 Kratos monochromator whose input and output slits were set at 6.0 mm (bandwidth, 20 nm). The beam was reflected at a right angle onto a thermostatically controlled horizontal holder (4 cm X 4 cm) containing the Petri dishes. A fluence rate of 1.5 X 1016 quanta s-l cmp2 (72.5 W me2) was determined using a ferrioxalate actinometer [20]. Cell damage was estimated using the trypan blue staining test on irradiated and unirradiated Petri dishes, with or without porphyrin. Some Petri dishes were saved for fluorometric estimation of the TPPQ content of the cells, as described above. As far as Phot II is concerned, it is generally accepted that cationic detergent such as cetyltrimethyl ammonium bromide (CTAB) promotes its unstacking or dissociation [21]. However, in this investigation, Triton Xl00 was used since we have determined that this detergent is as

56

good as CTAB in monomerizing Phot II [22,23]. For the try@n blue test, the medium was removed from the Petri dishes and replaced by 0.5 ml of 0.05% trypsin. After 10 min at 37 “C, 0.1 ml of foetal calf serum was added; 10 min before counting the living and dead cells, 0.1 ml of 0.5% trypan blue was added. Each Petri dish was counted three times for averaging purposes and each experiment was carried out in triplicate.

3. Results and Discussion 3.1. Synthesis, physicochemical and in vitro photodynamic properties of TPPQ 3.1.1. Synthesis of the porphyrin The synthesis of TPP bearing the quinoline chain was performed following a similar procedure to that described previously [24]. The first step involved the preparation of aminoquinoline derivative which was easily obtained by condensation of 4,7-dichloroquinoline with l,Cdiaminobutane as reported by Bolte et al. [25]. In a second step, 4-(4aminobutyl)amino-‘lchloroquinoline was condensed with the acid chloride derivative of propionic-TPP obtained by treatment with oxalyl chloride. The expected TPPQ (Fig. 1) was then purified by thin layer chromatography on silica gel plates and isolated as purple crystals. This molecule is capable of adopting a folded conformation in which the two aromatic moieties slightly interact. Indeed, the NMR spectrum shows an upfield shift (approximately 0.5 ppm) of quinoline protons in comparison with the same protons in free quinoline (taken as the reference product) due to the ring current effect of the porphyrin macrocycle. 3.1.2. Physicochemical properties Absorption maxima and extinction coefficients are reported in Table 1. In ethanol or 1% Triton X100, a sharp Soret band is recorded, whereas in aqueous medium, in which TPPQ is poorly soluble (see below), this band is much broader and red shifted (approximately 16 nm). This could be due (as known for other porphyrins [26]) to aggregation which disappears in organic solvents or micelles; however, it may also involve (as stated above) the interaction between the porphyrin ring and the quinoline group in a folded configuration. The fluorescence spectra are characteristic of porphyrins, two bands are observed whose wavelength maxima are similar in all solvents (see Table 1). However, the fluorescence yield is 40 times lower in aqueous medium than in Triton X100. These fluorescence properties are reminiscent of those of other hydrophobic porphyrins such as protoporphyrin [27] or Phot II, the porphyrin mixture used in the PDT of cancers [ 211. In these cases, the fluorescence quenching in water has also been attributed to interactions between porphyrin rings in aggregates.

57 TABLE 1 Absorption maxima, molar extinction coefficients and fluorescence emission maxima of TPPQ in ethanol, 1% Triton Xl00 in phosphate-buffered saline and phosphate-buffered saline Solvent

Absorption (Extinction

maxima (nm) coefficients (M-l cm-‘))

Ethanol

414 (400000)

513 (18900)

546 (6000)

589 (5600)

644 (2800)

649

715

1% Triton Xl00

419 (400000)

514 (18500)

547 (6000)

589 (5100)

645 (3000)

649

716

Phosphate buffer

430 (95000)

518 (16500)

552 (7000)

592 (5500)

644 (4000)

655

718

Fluorescence maxima (nm) (X,x, = 517 nm)

3.1.3. Photodynamic activity of TPPQ There is ample evidence that porphyrin photosensitization involves singlet oxygen as the major oxidizing species (for a recent review see ref. 1). Light absorption by porphyrins leads to population of their first excited triplet state from which singlet oxygen is produced by energy transfer to molecular oxygen (the so-called type II photodynamic process) [ 281. Type II photodynamic properties induced by TPPQ as photosensitizer were investigated using His as the photodynamic substrate in air-saturated Tris buffer containing 1% Triton Xl00 (which helps to solubilize TPPQ). His is a well-known water-soluble singlet oxygen trap [28]. The concentration of His after various irradiation times was determined using HPLC as described in Section 2. Experiments were performed with TPPQ and haematoporphyrin (which was used as the reference photosensitizer). TPPQ is a good type II photodynamic photosensitizer; the quantum yield of photosensitized oxidation of His obtained using this photosensitizer is equal to 0.65 times that determined for haematoporphyrin (data not shown). 3.2. Interaction of TPPQ with serum proteins and uptake by fibroblasts 3.2.1. Interaction of TPPQ with lipoproteins and albumin As expected, TPPQ exhibits a poor solubility in aqueous solutions. Solutions of TPPQ (0.45 PM) in Tris buffer can be left at room temperature for a few hours without any precipitation as shown by fluorometry performed on the supernatant after addition of Triton Xl00 to monomerize the porphyrin. However, 24 h later the loss of material due to precipitate formation is about 50%. Addition of LDL or HDL to an aqueous TPPQ solution enhances the fluorescence intensity by an order of magnitude and induces a sharpening of the Soret absorption band peaking at 420 nm (data not shown). Based on previous observations of protoporphyrin [2] and Phot II [29],

58

this can be interpreted as resulting from the incorporation and monomerization of TPPQ in lipoproteins. The number of TPPQ molecules incorporated per molecule of lipoprotein can be determined, as previously carried out with protoporphyrin and Phot II [2, 291, by recording the TPPQ fluorescence intensity as a function of increasing lipoprotein concentration. Curves with a plateau are obtained. The intercept of the initial slope with the plateau level leads to the estimate that one LDL molecule can incorporate about 25 TPPQ molecules. The same determination leads to 17 molecules of TPPQ incorporated per HDL molecule. In contrast with solutions prepared in Tris buffer, no loss of TPPQ is observed 24 h after addition of 2 PM TPPQ to solutions containing 30 ~.cg ml-’ LDL, 30 pg ml-’ HDL or 750 l.(g ml-’ HSA. However, immediate ultrafiltration (0.45 pm) causes a decrease in TPPQ concentration to 0.45 PM in HDL or HSA solutions and 0.25 PM in LDL solutions. It may be estimated that, after filtration, 5, 3 and 0.04 TPPQ molecules still interact with one LDL, HDL and HSA molecule respectively. The last value demonstrates the very weak affinity of HSA for TPPQ. These results suggest some relationships between the solubilization of protoporphyrin, Phot II and TPPQ by lipoproteins. However, the mode of interaction of these porphyrins with lipoproteins must be different, since protoporphyrin and Phot II are not removed from lipoproteins by ultrafiltration on 0.45 pm Millipore filters [22]. Ultrafiltration experiments suggest the existence of two types of site. One type concerns the weakly bound TPPQ molecules which may remain on the filter. The second deals with porphyrin molecules which cannot be retained by filters because they are tightly bound and somewhat buried in the protein matrix or lipidic core as observed with protoporphyrin [2] and Phot II [22]. Furthermore, the results illustrate the importance of lipoproteins, particularly LDL, as porphyrin carriers as previously reported by ourselves [2] and other workers [313.2.2. Porphyrin uptake by fibroblasts: kinetics and quantitative determination As described in Section 2, all protein-containing solutions were filtered before incubation with fibroblasts. LDL was loaded with 0.25 PM TPPQ; Tris buffer and the HDL or HSA solutions contained 0.45 PM TPPQ. The LDL, HDL and HSA concentrations were 30, 30 and 750 ~.cg ml-’ respectively which correspond to their physiological ratios. Moreover, the chosen LDL concentration ensures specific binding of LDLs to their cell surface receptors [19,30]. The amount of porphyrin incorporated after incubation of fibroblasts with Tris-TPPQ, LDL-TPPQ, HDL-TPPQ and HSA-TPPQ was determined by fluorometry after washing of cells and addition of Triton Xl00 (final concentration, 1%). This leads to rapid solubilization of the cells [23] and monomerization of the porphyrin which allows accurate concentration determinations to be made.

59

-0

5

10

1s

20

2s

Incubation Time (hours)

Fig. 2. TPPQ uptake as a function of the incubation time: 0, Tris-TPPQ, 0.45 PM TPPQ; m, LDL-TPPQ, 0.25 PM TPPQ, 30 pg ml-’ LDL. Incubation with 1 ml in Petri dishes (35 mm) at 37 “C. TPPQ was assessed by fluorescence measurement (see Section 2 for details).

As can be seen in Fig. 2, the incorporation of TPPQ is different in the presence and absence of LDL. After short incubation times, incorporation of TPPQ in cells is much more extensive in the absence of LDL than in the presence of LDL. This result is not unexpected. The availability of drugs for cells is reduced in the presence of proteins. As discussed above, the decrease in TPPQ incorporation observed after approximately 7 h of incubation in Tris buffer (see Fig. 2) may be due to porphyrin precipitation in the incubation medium. In contrast, TPPQ uptake reaches a plateau after approximately 5 h of incubation with TPPQ-loaded LDL. For the sake of clarity, data obtained with HDL and HSA are not reported since, after incubation for 4 h, the incorporation of TPPQ does not exceed approximately 0.09 and approximately 0.18 ng TPPQ pg-’ cell protein respectively (compared with approximately 3.5 for incubation in Tris buffer and approximately 0.8 for incubation in the presence of LDL). After incubation for 24 h, the incorporated TPPQ in the presence of HDL and HSA remains low, e.g. approximately 0.10 and approximately 0.32 ng TPPQ cell protein respectively, compared with 2.5 in Tris buffer and apccg-’ proximately 1.1 in the presence of LDL. Interestingly, after 15 min of incubation in the presence of HSA and HDL, 0.07 and 0.12 ng TPPQ pgM1 cell protein are already incorporated. Thus longer incubations do not strongly enhance TPPQ incorporation. This phenomenon is not observed during incubation in Tris buffer or with LDL. Therefore it seems that for HDL and HSA, there is a very fast but limited uptake. Consequently, the data suggest that LDLs are good vectors for carrying TPPQ into the cells. In order to examine the effect of incubation conditions on the cellular localization of TPPQ, the fluorescent properties of TPPQ were studied in situ since it is well known that porphyrin fluorescence (emission and excitation spectra and quantum yields) is sensitive to the microenvironment.

60

3.2.3. Porphyrin uptake by fibroblasts: differences in localization as revealed by fluorescence measurements Emission spectra were recorded after various incubation and washing times. Two characteristic fluorescence emission bands were observed with maxima at 650 - 653 nm and 714 - 715 nm, depending on the incubation media and conditions. Similar emission spectra were obtained for incubation in Tris buffer, LDL, HDL and HSA. Furthermore, similar spectra were obtained for incubations of 1, 4 and 24 h, 1 h followed by washing for 3 h and 4 h followed by washing for 20 h (data not shown). These results are not surprising since similar emission properties were observed in all solvents used (see Section 3.1.2 and Table 1). Excitation spectra were recorded after incubation in Tris buffer or LDL and are shown in Fig. 3 after normalization at the excitation maxima. It can be seen that the excitation spectra exhibits significant differences depending on the incubation mode and on the presence or absence of LDL. For example, incubation with Tris-TPPQ leads to broader excitation spectra than incubation with LDL-TPPQ. Incubation with HDL-TPPQ or HSATPPQ leads to slightly broader spectra than incubation with LDL-TPPQ, but sharper spectra than incubation with Tris-TPPQ (data not shown). For Tris-TPPQ, the broad excitation spectra obtained after 1 h or 4 h incubation or 1 h incubation plus washing for 3 h are superimposed (Figs.

0 350

\ 400

450

400

350

Wavelength

450

(nm)

Fig. 3. Normalized excitation fluorescence spectra of TPPQ in cell suspensions (see Section 2). For an easy comparison of spectral shapes, all the fluorescence intensities have been assumed to be unity at the excitation maximum. Incubation with LDLTPPQ (a, b); incubation with Tris-TPPQ (c, d); a, 1 h incubation; A, 1 h incubation plus 3 h washing; 0, 4 h incubation; +, 4 h incubation plus 20 h washing. Concentrations and incubation conditions are given in Fig. 2. Washing was performed with whole culture medium.

61

3(c) and 3(d)). Sharper spectra are recorded after 24 h incubation (spectrum not shown) or after 4 h incubation plus washing for 20 h. In contrast, the excitation spectrum observed after 1 h incubation with LDL-TPPQ is already rather sharp and becomes sharper after washing for 3 h (see Fig. 3(a)). Figure 3(b) illustrates the same behaviour after 4 h incubation followed by washing for 20 h. These results not only suggest that the intracellular localization of TPPQ depends on the presence of LDL during incubation, but also that timedependent localization changes occur rapidly in the presence of LDL. Figure 4 shows the ratio of the fluorescence intensities measured before and after addition of Triton Xl00 to the cell suspension. After cell disruption and solubilization in Triton X100, porphyrin fluorescence originates solely from molecules in Triton Xl00 micelles where it is no longer dependent on the cellular microenvironment. Therefore this ratio is a measure of the relative cellular TPPQ fluorescence efficiency which depends on the fluorescence quantum yield and the absorption properties, both controlled by the TPPQ environment. As can be seen this ratio depends on the incubation conditions and washing times and, for given incubation times, it is always higher in the presence than in the absence of protein. This closely parallels the emission spectra results and suggests that, in the absence of proteins in the incubation medium, incorporated TPPQ is poorly fluorescent (as observed in aqueous solutions). Washing for 3 h after 1 h incubation in the presence of LDL gives rise to a large increase in this ratio (factor of 4), whereas there is practically no change after similar incubations in Tris buffer or in the presence of HSA. However, 4 h incubation in Tris buffer or HSA followed by washing for 20 h increases this ratio. All these

Tris

60

10

40

0

I

I+3

4

4+20

0

Incubation + washing conditions (hours)

Fig. 4. Ratio of fluorescence intensities measured before and after addition of Triton Xl00 to cehs incubated with TPPQ in Tris buffer LDL or HSA as a function of incubation and washing conditions: 1, 1 h incubation; 1 + 3, 1 h incubation plus 3 h washing; 4, 4 h incubation; 4 + 20, 4 h incubation plus 20 h washing (see Section 2 for details). Incubations with HSA-TPPQ: 0.45 /JM TPPQ, 750 pg ml-i HSA; incubation conditions and other concentrations are given in Fig. 2.

62

results confirm that localization depends on the presence and nature of the carriers. It is clear that timedependent changes in the localization also depend on these factors. 3.2.4. Porphyrin uptake by fibroblasts: HPLC of incorporated porphyrins The changes described above in the shape and intensity of the fluorescence spectrum could also be due to modification of the fluorescent molecule. In order to investigate this possibility, incubations were performed for 1 h with Tris-TPPQ or LDL-TPPQ followed or not by washing for 3 h since marked differences in fluorescence have been observed under these conditions. HPLC analysis carried out on extracts of cellular porphyrins revealed only TPPQ. The unsubstituted porphyrin TPP and the acidic porphyrin derivative TPP-(CH&-COOH were not detected (as concluded from the elution times obtained with authentic samples (data not shown)). This suggests that TPPQ is not degraded by lysosomal peptidases if carried to the lysosomes by LDLs. 3.3. Binding, internalization and degradation of LDL loaded with TPPQ 3.3.1. Binding of LDL-TPPQ to receptors: effect of LDL-recep tor expression Using LDLs as carriers, the TPPQ uptake may be mediated by the LDL internalization pathway, i.e. binding to specific LDL receptors followed by endocytosis [ 19,301. In order to investigate this possibility, cells were incubated for 1 h at 4 “C! with various amounts of LDL (10 100 pg) loaded with TPPQ at a constant LDL to TPPQ ratio. These conditions (temperature and LDL concentration) were chosen because they allow the specific binding of native LDLs to their cell surface receptors. As a result, the amount of cell-bound LDL us. the amount of LDL in the incubation medium (in the range 10 - 100 c(g ml-‘) exhibits a plateau as illustrated in ref. 29. Thus if TPPQ uptake at 4 “C in the presence of LDL is mostly associated with specific binding of LDLs to their receptors, it should exhibit a similar plateau. As shown in Fig. 5, TPPQ uptake does not exhibit a plateau, but rather increases linearly with the LDL content in the incubation medium. Furthermore, when cells are preincubated with LDL-deficient medium 24 h before the experiments, an increase in TPPQ uptake of only about 50% is observed with respect to experiments performed on cells cultured in regular medium (EMEM + serum). Pre-incubation with LDL-deficient medium favours LDL-receptor expression [ 19, 301. Under our experimental conditions, this pre-incubation enhances the number of LDL receptors on human skin fibroblasts by a factor of approximately 2 as measured by i2%labelled LDL binding (data not shown). Therefore our results suggest that, in the presence of LDL, the LDL internalization pathway via coated pits is not the only process involved in TPPQ uptake; other pathways may also contribute, e.g. the exchange of TPPQ molecules

63

20

40 LDL/TPPQ

60

SO

100

@g/ml)

Fig. 5. TPPQ uptake as a function of TPPQ-loaded LDL concentration and effect of the receptor expression on TPPQ uptake: n, cells grown in regular culture medium (see Section 2); 0, -cells pre-incubated 24 h before experiments with lipoprotein-deficient culture medium (2% Ultroser G, see Section 2). Incubations took place at 4 “C for 1 h. TPPQ uptake was assessed by fluorescence measurement. Results are expressed in nanograms of TPPQ per microgram of cell protein. TPPQ:LDL = 0.5 pM:lOO pg. For the sake of clarity, standard deviations are not shown and are between 7% and 10%.

between LDL and the plasma membrane transfer after unspecific binding of LDL.

may take place favoured

by lipid

3.3.2. Effect of TPPQ on the LDL endocytosis pathway As the LDL-receptor pathway is partly involved in TPPQ uptake, we investigated whether binding of LDLs to their receptors, internalization of LDL-receptor complexes and intra-lysosomal degradation of LDL were inhibited by TPPQ which contains the possibly lysosomotropic quinoline group. For this purpose, cells were incubated under different conditions (time and temperature, see below) with a constant amount of ‘251-labelled LDL and increasing amounts of native LDL or LDL loaded with TPPQ. Native LDL competes with the binding, internalization and degradation of 1251-labelled LDL. If the presence of TPPQ in LDL inhibits its binding, internalization and degradation, LDL containing TPPQ will not be able to compete with the binding, internalization and degradation of 12SI-labelled LDL as efficiently as native LDL. As shown in Fig. 6(a), there is no significant difference between the abilities of native LDL and LDL-TPPQ to compete with native ‘251-labelled LDL after 1 h incubation at 4 “C!. Experiments were also carried out with TPP-(CHz)2-COOH-loaded LDL. No difference was observed. Since at 4 “C, LDL binding to the receptors is not followed by internalization, it may be concluded that TPP-(CH2)2-COOH and TPPQ do not alter the ability of LDLs to recognize their specific receptors at the cell surface. These experiments were also carried out at 37 “C for an incubation time of 4 h. At this temperature, internalization of the LDL-receptors rapidly occurs (approximately l/2 h). LDL apoprotein degradation into small peptides in the lysosomes is much slower (approximately 2 - 4 h). It is shown in Fig. 6(b) that LDL loaded with TPP-(CH&COOH is internalized with the same efficiency as native LDL. Therefore the presence

64

0

25

50

75

0

25

50

75

0

25

50

75

100

[LDL], [LDLITPP-(CH J ,-COOH] or [LDL/TPPQl @g/ml) Fig. 6. Competition between lzSI-LDL and native LDL (0), LDL-TPPQ (0) or LDLTPP-(CH2)2-COOH (m). (a) LDL binding to the receptors, 1 h incubation at 4 “C, washed and harvested cells were y counted. (b) LDL internalization, 4 h incubation at 37 “C, washed and harvested cells were y counted. (c) LDL degradation, 4 h incubation at 37 “C, ‘2SI-labelled peptide release in the medium was estimated by y counting. (lzSI-LDL = 10 pg; incubation volume, 0.5 ml; results (c.p.m. mg-’ cell protein) are expressed as a percentage of the control ( lzs I-LDL alone); average standard deviation was approximately lO’%of the mean values; see Section 2 for details.)

of the porphyrin ring does not impair the internalization process. Figure 6(b) also shows that very little, if any, inhibition of internalization is observed for LDL loaded with TPPQ (taking into account standard deviations). Thus it can be suggested that the presence of the quinoline chain does not significantly modify the expression of the endocytosis of receptor-LDL complexes. The degradation process is monitored by measuring the release of ‘251-peptides in the incubation medium as described in Section 2. As shown in Fig. 6(c), degradation is not inhibited by LDL-TPP-(CH,),-COOH (compared with native LDL) and only a slight, if any, inhibition is observed for LDL-TPPQ. For LDL-TPPQ, the extent of inhibition is practically the same for internalization and degradation. Therefore the slight inhibition of degradation may be due to the slight inhibition of internalization. These experiments were designed to evaluate the effect of TPPQ incorporated in LDL on the endocytosis pathway. No TPPQ was present in the cells before the experiments, since TPPQ uptake and LDL endocytosis occurred concomitantly. However, the presence of intracellular TPPQ before LDL incubation could alter the endocytosis pathway of native LDL. In order to verify this hypothesis, cells were incubated for 15 h with 0.45 PM TPPQ in EMEM supplemented with 2% Ultroser G; they were assayed, after washing, for LDL degradation by measuring the ‘2SI-peptide release after 4 h incubation with ‘251-labelled native LDL. Under these -I of cell protein. No difference conditions, TPPQ uptake was about 4 ng 1.18 in LDL degradation was observed whether the cells were pre-incubated with TPPQ for 15 h or not (data not shown). Therefore it can be concluded that the presence of intracellular TPPQ does not modify the degradation, binding and internalization of native LDL. There is no evidence for transport of TPPQ in the lysosomes under these incubation conditions.

65

3.4. Cell photosensitization As described in Section 2, prior to irradiation, cells were incubated for 17 h in EMEM, followed by addition of LDL (30 pg ml-‘) loaded with porphyrin (TPPQ or Phot II). Fluorometric measurements show that 75 ng TPPQ (10’ cells)-‘, 50 ng Phot II (10’ cells))’ and 200 ng Phot II (10’ cells)-’ are taken up when cells are incubated with LDL solutions containing 0.2 Erg ml-’ TPPQ, 0.5 E.cgml-’ Phot II and 1.5 pg ml-’ Phot II respectively. Taking the lower TPPQ incubation concentration into consideration, it can be seen that TPPQ is taken up at least as efficiently as Phot II. According to the trypan blue exclusion test, these photosensitizer concentrations do not induce cytotoxicity in the absence of irradiation. The results of cell photosensitization are shown in Fig. 7. As can be seen, TPPQ induces cell photosensitization with an efficiency similar to that of Phot II. No cell death was observed in the controls incubated with unloaded LDL and irradiated under the same conditions.

0.21 0

, 1

, 2

, 3

,I 4

Irradiation time (min)

Fig. 7. Survival fraction of cells incubated with TPPQ- or Phot-II-loaded LDL as a function of irradiation time. Cells were incubated for 17 h with 1 ml of solution at 37 “C. 0, 0.2 pg ml-’ TPPQ, 30 l.(g ml-’ LDL; A, 0.5 pg ml-’ Phot II, 30 pg ml-* LDL; w, 1.5 pg ml-’ Phot II, 30 /.fg ml-’ LDL. irradiations at 405 nm.

4. Concluding

remarks

This newly synthesized porphyrin containing a quinoline group presents some interesting features with respect to other photosensitizers such as Phot II presently used in the PDT of proliferative diseases. TPPQ uptake partly occurs via receptor-mediated LDL endocytosis. This is a major advantage in view of the increased number of LDL receptors in all proliferative cells. The presence of the quinoline group, which inhibits the photodynamic properties of the TPP moiety in aqueous media, is not a limiting factor with respect to cell photosensitization via LDL delivery as judged by the comparable photocytotoxicity of Phot II and TPPQ towards fibroblasts. The expected fast elimination of TPPQ from the cell, which would reduce long-lasting skin photosensitivity, remains to be investigated as does the toxicity of TPPQ and the preferential uptake by tumour cells.

66

Acknowledgments Grants from “1’Association pour la Recherche contre le Cancer” and “INSERM” (C.R.E. 852021) are gratefully acknowledged. J.-C. Maziere and R. Santus wish to thank “la Ligue Nationale contre le Cancer” and “la Fondation pour la Recherche Medicale” respectively for financial support.

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67 18 D. W. Bilheimer, S. Eisenberg and R. I. Levy, The metabolism of very low density lipoprotein. I. Preliminary in uiuo and in vitro observations, Biochem. Biophys. Acta, 260 (1972) 212 - 221. 19 J. L. Goldstein and M. S. Brown, Binding and degradation of LDL by cultured human fibroblasts: comparison of cells from a normal subject and from patient with homozygous familial hypercholesterolemia, J. Biol. Chem., 249 (1974) 5153 - 5162. 20 C. A. Parker, Photoluminescence of Solutions, Elsevier, Amsterdam, 1968, pp. 208 216. 21 J. P. Keene, D. Kessel, E. J. Land, R. W. Redmond and T. G. Truscott, Direct determination of singlet oxygen sensitized by haematoporphyrin and related compounds, Photochem. Photobiol., 43 (1986) 11’7 - 121. 22 P. Morliere and R. Santus, unpublished observations, 1986. 23 G. Moreno, A. Atlante, C. Sale& R. Santus and F. Vinzens, Photosensitivity of DNA replication and respiration to haematoporphyrin derivative (photofrin II) in mammalian CV-1 cells, Znt. J. Radiat. Biol, 52 (1987) 213 - 222. 24 M. Momenteau, B. Look, E. Bisagni and M. Rougee, Five-coordinate iron(I1) porphyrins derived from meso (Y, 0, y, 6 mesotetraphenylporphyrin: synthesis, characterization and coordinating properties, Can. J. Chem., 57 (1979) 1804 - 1813. 25 J. Bolte, C. Demuinck and J. Lhomme, Synthetic models of DNA complexes with antimalarial compound. 2. The problem of guanine specificity in chloroquine binding, J. Med. Chem., 20 (1977) 106 - 113. 26 R. F. Pasternack, P. R. Huber, P. Boyd, G. Engasser, L. Franesconi, E. Gibbs, P. Fasella, G. Cerio Ventura and L. de C. Hinds, On the aggregation of meso-substituted water-soluble porphyrins, J. Am. Chem. Sot., 94 (1972) 4511 - 4517. 27 A. Lamola, Fluorescence study of protoporphyrin. Transport and clearance, Acto Dermatovener. (Stockholm) Suppl., 100 (1982) 57 - 66. 28 C. S. Foote, Quenching of singlet oxygen, in H. H. Wasserman and R. W. Murray (eds.), Singlet Oxygen, Academic Press, New York, 1976, pp. 139 - 171. 29 C. Candide, P. Morliere, J.-C. Mazidre, 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. 30 J. L. Goldstein, R. G. W. Anderson and M. S. Brown, Coated pits, coated vesicles and receptor-mediated endocytosis, Nature (London), 279 (1979) 679 - 685.