Available online at www.sciencedirect.com
Journal of Photochemistry and Photobiology B: Biology 89 (2007) 131–138 www.elsevier.com/locate/jphotobiol
Tumour-localizing and -photosensitising properties of meso-tetra(4-nido-carboranylphenyl)porphyrin (H2TCP) Clara Fabris a,*, M.Grac¸a H. Vicente b, Erhong Hao b, Elisabetta Friso a, Lara Borsetto a, Giulio Jori a, Giovanni Miotto c, Paolo Colautti d, Davide Moro d, Juan Esposito d, Alice Ferretti d, Carlo Riccardo Rossi e, Donato Nitti e, Guido Sotti e, Marina Soncin a a
Department of Biology, University of Padova, via Ugo Bassi, 58/B, 35121 Padova, Italy b Department of Chemistry, Louisian State University, Baton Rouge, USA c Department of Biochemistry, University of Padova, Italy d Laboratori di Legnaro, National Institute of Nuclear Physics, Italy e Istituto Oncologico Veneto, Padova, Italy
Received 19 June 2007; received in revised form 28 September 2007; accepted 28 September 2007 Available online 1 October 2007
Abstract A water-soluble meso-substituted porphyrin (H2TCP) bearing 36 boron atoms, which appeared to be an efficient photodynamic sensitiser (singlet oxygen quantum yield = 0.44), was studied for its accumulation by murine melanotic melanoma cells (B16F1). The amount of H2TCP in the cells increased with the porphyrin dose in the incubation medium up to, and at least, 100 lM concentrations with no significant cytotoxic effect in the dark. Moreover, the H2TCP uptake increased with the incubation time reaching a plateau value corresponding with the recovery of 0.4 nmol of H2TCP per mg of cell proteins after 24 h incubation. Fluorescence microscopy observations showed that the porphyrin was largely localized intracellularly, exhibiting a discrete distribution in the cytoplasm with a pattern which was closely similar to that observed for the endosomal probe Lucifer yellow. The photosensitising efficiency of the H2TCP toward B16F1 cells was studied for different irradiation (1–15 min) and incubation (1–24 h) times. Nearly complete (>95%) cell mortality was obtained upon incubation with 20 lM H2TCP and 10 min irradiation with red light (600–700 nm, 20 mW/cm2). The porphyrin was also accumulated in appreciable amounts by the tumour tissue after intravenous injection to C57BL/6 mice bearing a subcutaneously transplanted melanotic melanoma. Maximum accumulation in the tumour was achieved by administration of H2TCP dissolved in the ternary mixture 20% dimethylsulfoxide (DMSO)–30% polyethyleneglycol (PEG 400)–50% water. Thus, this porphyrin could act as both a photodynamic therapy agent and a radiosensitising agent for boron neutron capture therapy. 2007 Elsevier B.V. All rights reserved. Keywords: Photodynamic therapy; Boron neutron capture therapy; Porphyrin; Carborane; Melanotic melanoma
1. Introduction Among the modalities for tumour treatment which are currently under active investigation in view of palliative or curative applications, boron neutron capture therapy (BNCT) and photodynamic therapy (PDT) represent two emerging therapeutic strategies [1,2]. Both modalities
*
Corresponding author. Tel.: +39 049 8276334; fax: +39 049 8276344. E-mail address:
[email protected] (C. Fabris).
1011-1344/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2007.09.012
involve the systemic injection of an intrinsically non toxic radio- or photo-sensitising agent to the patient, followed by the specific irradiation of the diseased area with thermal neutrons in the case of BNCT [3,4] or selected visible light wavelengths for PDT [5]. In particular, BNCT represents a binary modality for cancer treatment that involves the irradiation of 10B-enriched tumour lesions with low energy (thermal, 0.025 eV) neutrons, resulting in the release of highly cytotoxic particles, 4He2+ (a-particle of 1.42 MeV of energy) and 7Li3+ ions of 0.84 MeV of energy. These ions are capable of causing severe damage to biological
132
C. Fabris et al. / Journal of Photochemistry and Photobiology B: Biology 89 (2007) 131–138
materials; the toxic effect is most likely confined within the 10 B-containing cell since such ions display limited paths of travel in tissue (9–5 lm, respectively, i.e., about one cell diameter) [1,3,4]. On the other hand, PDT is based on the combined action of visible photons with photosensitising agents and molecular oxygen leading to a sequence of photochemical and photobiological reactions that result in irreversible damage to the tumour. During this process the electronic energy initially absorbed by the photosensitiser is transferred to ground-state oxygen to produce singlet oxygen (1O2) and oxygen radicals, which further react with cellular components to cause cell death [6]. At present, regulatory approval is limited to borono-phenylalanine and sodium borocaptate [7,4] for BNCT, as well as to Photofrin, a complex mixture of haematoporphyrin derivatives, and 5-aminolevulinic acid (ALA), a metabolic precursor of protoporphyrin IX, for PDT [8]. A large number of patients have been treated by either technique worldwide with objectively positive results. However, for both techniques, severe limitations exist owing to the relatively low selectivity of tumour targeting by the above mentioned radio- or photo-sensitising agents coupled with the small efficiency of their interaction with the incident radiation [3,5]. Recently, new perspectives have been opened in this field by the synthesis of chemically pure boron-labelled porphyrins and phthalocyanines [9], where the tetrapyrrolic derivative can act as a vehicle for the transport of significant amounts of boron to the neoplastic lesion; at the same time, many newly developed porphyrins and phthalocyanines have been shown to possess satisfactory tumour-photosensitising properties which are markedly superior to those typical of Photofrin [10]. In principle, such boronloaded porphyrins/phthalocyanines could allow the development of combination therapies based on the sequential application of BNCT and PDT, which have the possibility to act synergistically given the different mechanisms of action of the two therapeutic approaches. In this paper, we describe our findings on the affinity for tumour cells in vitro and for tumour tissues in vivo of a
water-soluble meso-substituted tetra(nido-carboranylphenyl)porphyrin (H2TCP) carrying 36 boron atoms per molecule, which should thus exhibit a large cross section for interaction with neutrons [11]. The effect of the large boron clusters on the photodynamic activity of this porphyrin was also tested. The chemical structure of H2TCP is shown in Fig. 1. 2. Materials and methods 2.1. Porphyrin synthesis H2TCP [meso-tetra(4-nido-carboranylphenyl)porphyrin] was prepared by chemical synthesis in the Department of Chemistry at Louisiana State University in Baton Rouge, USA following a synthetic route similar to that previously published [11]. Our optimized synthesis of H2TCP uses BF3 Æ OEt2 (0.1 mmol) as the acid catalyst in the condensation of 4-carboranylbenzaldehyde (1 mmol) with pyrrole (1 mmol); under these conditions H2TCP was obtained in 53.9% yield after purification by column chromatography on silica gel using dichloromethane/hexane 1:2 for elution, and recrystallization from dichloromethane/methanol. The chemical structure of H2TCP was characterized by standard spectroscopic and chemical analytical techniques. 2.2. Determination of singlet oxygen quantum yield The quantum yield (UD) of singlet oxygen generation by H2TCP was measured by following the decrease in the fluorescence emission of 9,10-dimethyl-anthracene (DMA) upon its photosensitised conversion into the corresponding non-fluorescent 9,10-endoperoxide [12]. In a typical experiment, a 20 lM DMA and 1.4 lM H2TCP solution in methanol was placed in a quartz cuvette of 1 cm optical path and irradiated for different periods of time at 20 ± 2 C under gentle magnetic stirring by 600–700 nm light. This wavelength interval was isolated from the emission of a halogen lamp by the insertion of broadband optical filters (Waldmann, Schwenningen, Germany). The fluence-rate was 100 mW/cm2. The DMA fluorescence emission was recorded in the 380–550 nm wavelength range with excitation at 360 nm. The first-order rate constant of the photoprocess was obtained by plotting ln F0/F as a function of the irradiation time, where F0 and F represent the fluorescence intensity at time 0 and at time t, respectively. The rate constant was then converted into 1O2 quantum yield by comparison with the rate constant for DMA photooxidation sensitized by haematoporphyrin (Hp) for which UD was shown to be 0.65. 2.3. Fluorescence quantum yield
Fig. 1. Chemical structure of H2TCP.
The fluorescence quantum yield for H2TCP in methanol was measured by using a Perkin–Elmer LS 50 spectrophotofluorimeter. meso-Tetrakis(p-sulfonatophenyl) porphyrin (TPPS) in neutral aqueous solution was used as a reference
C. Fabris et al. / Journal of Photochemistry and Photobiology B: Biology 89 (2007) 131–138
standard having an absorbance of 0.1 at 420 nm and a fluorescence yield of 0.1 [13]. The fluorescence emission of both porphyrins was recorded between 600 and 800 nm, exciting at 420 nm. The quantum yield was obtained by the following formula: US ¼ UR
F S A R g2 AS F R g20
where FS and FR are the areas of the emission spectra of the investigated substance and the reference, respectively; AS and AR are the absorbance at 420 nm; g and g0 represent the refractive index of the solvent; UR is fluorescence quantum yield of the reference. 2.4. Photostability studies The stability of the porphyrin to white or red light irradiation was determined by preparing a solution of H2TCP in methanol with an absorbance of 0.4 at the maximum peak. This solution was placed in a quartz cuvette with a 1 cm optical path and irradiated at 25 C for different periods of time under gentle magnetic stirring by using 350– 700 nm light (Teclas light source, Lugano, Switzerland, 100 mW/cm2) or 600–700 nm light (Waldmann light source, 100 mW/cm2). After each irradiation, the absorbance spectrum was recorded by a Cary Eclipse 50 scan spectrophotometer and the value of the absorbance at the maximum peak at time 0 was compared with those recorded at the various irradiation times. 2.5. Cell line and culture conditions The cell line B16F1, used in our studies, is a pigmented variant of murine melanoma B16 [14] which differs because of its highly metastatic potential. The cell line cultured as a monolayer at 37 C in a humidified atmosphere with 5% CO2 was grown in Dulbecco’s modified minimal essential medium (DMEM, Sigma, St. Louis, USA) containing 10% heat-inactivated foetal calf serum (FCS, Gibco, Auckland, NZ) and supplemented with 100 units/ml penicillin, 100 lg/ml streptomycin, 0.25 lg/ml amphotericin, and 2 mM glutamine (Sigma). The cell line was routinely checked for the absence of mycoplasma contamination. 2.6. Cellular uptake of H2TCP For the uptake experiments 2 · 105 B16F1 cells were seeded in 25 cm2 plastic flasks (BD Falcon, Franklin Lakes, NJ, USA) and grown for about 20 h in DMEM containing 10% FCS. Under these conditions, cells remained in the logarithmic phase of growth throughout the experiment. H2TCP was diluted to the desired concentration in DMEM containing 10% FCS and 1% methanol and incubated with the cells after removal of the growth medium. The incubation was usually performed at 37 C in a dark humid atmosphere containing 5% CO2.
133
At the end of the incubation period (1–24 h), the medium containing the porphyrin was discarded and the cell monolayer carefully washed twice with phosphate-buffered saline (PBS) and removed from the flasks by exposure to trypsin. The cells were collected by centrifugation at 1100 rpm for 7 min and the pellet was resuspended in 2 ml of a 2% aqueous dispersion of sodium dodecyl sulphate (SDS). After gentle magnetic stirring for 1 h, each sample was divided into two portions: 0.5 ml was stored to assay the protein content by the bicinchoninic acid test [15], and 1 ml was further diluted with 2% SDS. The fluorescence emitted in the 600–800 nm regions was measured for each sample by means of a Cary Eclipse spectrophotofluorimeter (Varian Australia Pty Ltd.). The porphyrin concentration in the samples was then calculated by interpolation with a calibration plot obtained by measuring the fluorescence emission intensity of solutions containing known photosensitiser concentrations. The uptake of the porphyrin by the cells was expressed as nmol of photosensitiser per mg of protein. During each experiment, two flasks were seeded with the same number of cells and were used for cell counting: a correspondence between the protein content of the flasks and the cell number was calculated with an average value of 400 ± 30 lg of protein per 106 B16F1 cells. 2.7. Dark- and photo-toxicity of H2TCP toward B16F1 cells The cytotoxic effect of the porphyrin with and without light was measured. For these experiments, 2 · 105 cells were seeded in 25 cm2 plastic flasks with 5 ml of DMEM containing 10% FCS. After about 20 h, the medium was replaced with DMEM containing 10% FCS, 1% methanol and the desired H2TCP concentration. The cells were incubated with the porphyrin for 24 h. At the end of the incubation period the cells were washed with PBS, removed from the flasks by exposure to trypsin and stained with trypan blue [16] for determination of cell survival. The survival of cells exposed to the porphyrin in the dark was expressed as percentage of viable cells as compared with control samples which had been processed in parallel but not exposed to the porphyrin. In the phototoxicity experiments, after 24 h incubation with 1–20 lM porphyrin, the cells were rinsed twice with PBS containing Ca2+ and Mg2+ ions and irradiated for 1–15 min at 600–700 nm in the same buffer by means of the Waldmann halogen lamp. The fluence rate at the irradiation site was 20 mW/cm2. Immediately after irradiation the cells were brought back to the incubator after replacement of the PBS with complete medium containing 10% FCS. After 24 h the trypan blue exclusion test was applied and the survival of the irradiated cells was evaluated relative to cell samples that were incubated with porphyrin but unirradiated. Control experiments showed that light alone induced no detectable decrease in cell survival, at least under our experimental conditions.
134
C. Fabris et al. / Journal of Photochemistry and Photobiology B: Biology 89 (2007) 131–138
2.8. Intracellular localization The B16F1 cells were plated on BDH cover glass and incubated for 24 h prior to incubation with the porphyrin. The cells were then exposed to 10 lM H2TCP and incubated for 24 h in the dark. Fluorescence microscopy analysis was performed by using an Olympus IMT-2 fluorescence microscope equipped with a refrigerated CCD camera (Micromax, Princeton Instruments). A 75W Xenon lamp was used as the excitation source. Fluorescence images obtained with a 60 · 1.4 NA oil immersion objective (Olympus) were acquired and analysed with the imaging software Metamorph (Universal Imaging). A blind deconvolution algorithm was applied to the images for non-destructive reduction of the haze from out of focus planes (Autodeblur 2D; Autoquant Imaging Inc.). The cellular distribution of the porphyrin fluorescence was compared with that observed for Lucifer Yellow (Molecular Probes, Leiden, The Netherlands), used as marker for the endosomes. Lucifer Yellow (10 nM) was added to the cell monolayers and after 60 min at 37 C the slides were washed twice with PBS containing Ca2+ and Mg2+ ions and analysed. For the porphyrin fluorescence detection a set of filters with 400 nm excitation and 620 nm emission was used, while for LY 425 nm excitation and 520 nm emission were used. 2.9. Nuclear fragmentation At 1–6 h after the end of the treatment (3–5 min irradiation at 20 mW/cm2), photosensitised and control cells were rinsed twice with PBS and incubated in the dark for 10 min with PBS containing Ho¨echst dye HO33342 (HO342) at a concentration of 5 lg/ml. Nuclear fragmentation was assessed by fluorescence microscopy (Zeiss, Germany). The excitation wavelength was 353–377 nm with emission monitored at 420–450 nm. 2.10. Pharmacokinetic studies with tumour-bearing mice Female C57BL/6 mice (20–22 g body weight) were supplied from Charles River Laboratories (Como, Italy) and kept in standard cages with free access to tap water and standard dietary chow. Animal care was performed according to the guidelines established by the Italian Committee for humane treatment of experimental animals. The B16F1 pigmented melanoma was subcutaneously transplanted into the upper flank of the mice by injecting 20 ll (106 cells) of a sterile cell suspension in PBS. On the seventh day after transplantation, when the tumour diameter was about 0.6 cm, the mice were intravenously injected with 5 mg/kg of the H2TCP dissolved in aqueous solution (20% DMSO, 30% PEG 400 and 50% water) or incorporated into Cremophor EL emulsion prepared by a modification of the procedure described by Morgan et al. [17]. Typically, 150 ll H2TCP in methanol were added to 500 ll of Cremophor EL (Sigma) and sonicated until the
porphyrin was completely dispersed. The suspension was brought to a final volume of 10 ml by stepwise addition of Dulbecco buffer, filtered through 0.45 lm filters and the H2TCP incorporation yield was measured by spectrophotometry. At predetermined times after porphyrin injection in either formulation, the mice (five animals at each time point) were sacrificed. The blood (ca.1 ml) was also collected and the plasma was isolated by centrifugation at 3000 rpm for 15 min. The H2TCP levels in plasma were determined by spectrophotofluorimetric analysis (excitation 410 nm, fluorescence emission collected between 600 and 800 nm) after dilution with 2% SDS. Tumour, liver, and other selected tissues were homogenized in 2% SDS, suitably diluted in the same solvent and the resulting solutions were analysed at the spectrophotofluorimeter. The fluorescence intensity was converted into porphyrin concentration by interpolation with a calibration plot. This procedure was shown to extract at least 90% of the porphyrin from the tissue specimens. 3. Results 3.1. Photophysical and photochemical studies The fluorescence emission quantum yield of H2TCP was found to be 0.015, i.e. substantially lower than the 0.1 quantum yield found for TPPS: on this basis, one can safely expect that the boronated derivative is characterized by a high triplet quantum yield, since porphyrins generally exhibit an inefficient non-radioactive decay from the first excited singlet state owing to the low vibrational component of their 0–0 transition [18]. In actual fact, the DMA photooxidation studies indicate that UD for this porphyrin is 0.44, that is close to the 0.5–0.6 quantum yield value typical of several photodynamically active porphyrins [19]. Exposure of the H2TCP porphyrin to either full spectrum visible light or the clinically useful red light for up to 20 min using the experimental conditions specified in Section 2 caused a negligible decrease in the intensity of the visible absorption bands. This process, generally defined as photobleaching, is usually related to irreversible destruction of the tetrapyrrolic macrocycle. The observed high photostability of H2TCP guarantees that the photosensitiser concentration in the irradiated lesion does not significantly decrease throughout the treatment time with no consequent reduction in the efficiency of the photoprocess. 3.2. Cellular uptake of H2TCP The effect of the porphyrin concentration on the uptake of H2TCP by B16F1melanotic melanoma cells after 24 h incubation is shown in Fig. 2a. The porphyrin was accumulated by melanocytes in amounts which steadily increased with increasing photosensitiser concentration in the incubation medium. No apparent saturation of H2TCP binding
0.25
100
0.20
80
Cell survival (%)
nnmoles H2TCP/mg of protein
C. Fabris et al. / Journal of Photochemistry and Photobiology B: Biology 89 (2007) 131–138
0.15
0.10
60
40
20
0.05
0
0.00 10
5
1
20
50
controls
100
Fig. 2a. Effect of the porphyrin concentration on the uptake of H2TCP by B16F1 cells. The incubation time was 24 h. Average of three experiments ± standard deviation.
5
10
20
50
100
H2TCP concentration (µM)
H2TCP concentration (µM)
Fig. 3a. Effect of the H2TCP concentration on the survival of B16F1 cells incubated for 24 h with different concentrations of porphyrin. Values represent means ± standard deviation of at least three separate experiments.
0.6
100
0.5
80
Cell survival (%)
nmoles H2TCP/mg of protein
135
0.4
0.3
0.2
60
40
20
0.1 0
0.0
1
3
6
18
24
Incubation time (h) Fig. 2b. Effect of the incubation time on the uptake of 20 lM H2TCP by B16F1 melanotic melanoma cells. Average of three experiments ± standard deviation.
by the cells was observed up to a 100 lM porphyrin concentration. It is worth underlining that the recoveries reported in Fig. 2a have been measured after two cell-washing steps, hence they represent tightly bound porphyrin. The amount of H2TCP accumulated by B16F1 cells also increased with increasing the incubation time and a plateau value was not reached up until at least 24 h as shown in Fig. 2b. 3.3. Dark- and photo-toxicity of H2TCP toward B16F1 cells The dark cytotoxicity of H2TCP was investigated in B16F1 cells. In all cases, no detectable decrease in the survival of melanocytes was caused by incubation of the cells in the dark with the porphyrin concentrations as large as 50 lM (Fig. 3a). Only upon addition of a 100 lM H2TCP dose in the incubation medium was a modest
controls
1
5
10
20
Irradiation time (min) Fig. 3b. Effect of the irradiation time on the survival of B16F1 cells incubated for 24 h with 20 lM H2TCP. Irradiations were performed by using 600–700 nm light at a fluence-rate of 20 mW/cm2. Values represent mean ± standard deviation of three experiments.
(
