Journal of Photochemistry and Photobiology B: Biology 97 (2009) 8–17
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Comparison of photodynamic efficacy of tetraarylporphyrin pegylated or encapsulated in liposomes: In vitro studies Kinga Nawalany a, Aleksandra Rusin b, Mariusz Ke˛pczyn´ski a, Alexei Mikhailov b, Gabriela Kramer-Marek b, Mirosław S´nietura b, Jan Połtowicz c, Zdzisław Krawczyk b, Maria Nowakowska a,* a b c
Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Kraków, Poland Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Wybrzez_ e Armii Krajowej 15, 44-100 Gliwice, Poland Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, 30-239 Kraków, Poland
a r t i c l e
i n f o
Article history: Received 28 December 2008 Received in revised form 10 April 2009 Accepted 2 July 2009 Available online 12 July 2009 Keywords: Photodynamic therapy Porphyrins Sterically stabilized liposomes Cancer cells
a b s t r a c t Two photosensitizing systems: (1) tetrakis(4-hydroxyphenyl)porphyrin (p-THPP) encapsulated in sterically stabilized liposomes (SSL) and (2) p-THPP functionalized by covalent attachment of poly(ethylene glycol) (p-THPP–PEG2000) were studied in vitro. The dark and photo cytotoxicity of these systems were evaluated on two cell lines: HCT 116, a human colorectal carcinoma cell line, and DU 145, a prostate cancer cell line and compared with these determined for free p-THPP. It was demonstrated that both encapsulation in liposomes as well as attachment of PEG chain result in pronounced reduction of the dark cytotoxicity of the parent porphyrin. The liposomal formulation showed higher than p-THPP–PEG2000 photocytotoxicity towards both cell lines used in the studies. Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction Photodynamic therapy (PDT) is a clinically approved method for treatment of cancer and some other diseases. It involves a simultaneous interaction between the photosensitizer, light and oxygen. The photosensitizer is preferentially accumulated in tumor tissue and subsequently activated with light of appropriate energy. The activated (electronically excited) molecules of photosensitizer are quenched by the molecular oxygen. The process results in formation of active oxygen species, especially singlet oxygen, which oxidizes the tumor cell components leading to their damage and consecutive death (necrosis, apoptosis or autophagy) [1–6]. The PDT being the localized treatment has considerably fewer side effects than other procedures, such as chemotherapy or radiotherapy. Many potential photosensitizers for PDT have been developed and studied, including porphyrins, chlorins, and phthalocyanines [7,8]. Tetraarylporphyrins (TArPs) are the most commonly studied derivatives of porphyrin because of their accessibility and good spectral/photochemical properties. It was demonstrated that they have the ability for preferential accumulation and retaining in malignant tissue. However, biomedical application of porphyrins in PDT is considerably hindered by their hydrophobicity leading
* Corresponding author. Tel.: +48 12 663 2250; fax: +48 12 634 0515. E-mail address:
[email protected] (M. Nowakowska). 1011-1344/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2009.07.005
to the limited solubility in aqueous cell environment. Porphyrins are known to aggregate in aqueous solution. That is expected to have an adverse effect on the PDT efficacy, as the aggregates will not function as singlet oxygen generators. Various approaches were undertaken to improve the water solubility and tumor-specificity of porphyrin photosensitizers [9,10]. One of the most popular strategies for increasing the therapeutic activity of porphyrin derivative agents is its ‘‘pegylation”. That is based on the formation of a soluble macromolecular derivative by conjugation of the active agent with poly(ethylene glycol) (PEG) chains. PEG was chosen for that procedure as it is a watersoluble polymer that exhibits protein resistance of low toxicity, and non-immunogenicity. It has been shown that due to the hydrophilic nature of the PEG chains the aggregation of pegylted porphyrin was reduced, while selectivity of accumulation in cancer cells increased. Several PEG–porphyrin conjugates for PDT were developed and studied [11–16]. Hornung et al. [13,14] have used tetrakis(m-methoxypolyethylene glycol) derivative of mTHPC for study of selective targeting in a rat ovarian cancer model. The authors demonstrated that pegylation of drug results in pronounced improvement of selective targeting to tumor cells. Another approach to overcome the problem of porphyrin low solubility is their encapsulation in lipid vesicles [17]. It has been observed that the liposomal encapsulation of anticancer drugs significantly alters their biodistribution and pharmacokinetics, resulting in some reduction in toxicity and in improvement of targeting to desired tissues. Liposomes are spherical vesicles
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formed by some amphiphilic compounds, e.g. phosphatidylcholines and ceramides. The molecules of hydrophobic sparingly water-soluble therapeutics may be incorporated into the hydrophobic lipid bilayer. Additional incorporation of lipids with covalently attached PEG into liposomal bilayer membranes causes significant stabilization of liposome suspensions, prevents their aggregation and inhibits protein and cellular interactions with liposomes thereby considerably prolonging their circulation time in a blood stream [18]. These vesicles are known as Stealth liposomes or sterically stabilized liposomes (SSL) and are applied as effective drug delivery supports. Jiang et al. [19] have treated U87 human glioma in rat brain using Photofrin (PF) as a photosensitizer encapsulated in a liposome or in dextrose carriers. PF uptake by tissue was significantly elevated when liposomes were used as vesicles in comparison with the system in which dextrose was used as a carrier. Also, PDT in which the liposome encapsulated photosensitizer was applied, have been shown to be considerably more efficient. Sadzuka et al. [20] have investigated PF dissolved in phosphate-buffered saline and incorporated into liposomes or poly(ethylene glycol)-modified liposomes. They found that the phototoxicity of PF in PEG-liposomes was significantly higher than that for PF in solution or solubilized in ordinary liposomes. This paper presents the results of our studies on two strategies developed to improve water-solubility, tumor-specificity and photoefficiency of porphyrin photosensitizers. They involved encapsulation in SSL liposomes or pegylation of porphyrin molecule. We have compared the solution properties and evaluated the photosensitizing potential of the investigated systems towards two cancer cell lines: HCT 116, a human colorectal carcinoma cell line, and DU 145, a prostate cancer cell line. It was previously reported that induced by PDT programmed death of these cell lines occurs according to different mechanisms. The HCT 116 cells die in p53-dependent apoptotic manner [21] while DU 145 cells mainly by developing autophagy [5,22]. Commercially available porphyrin, tetrakis(4-hydroxyphenyl)porphyrin (p-THPP) was chosen as the parent compound for novel photosensitizer systems. Promising activity and tissue selectivity in photonecrosis of that dye was previously shown by Bonnett et al. [23]. Banfi et al. [24] have shown photodynamic and toxicity profiles for series of porphyrin and chlorin derivatives including p-THPP and compared them with the data obtained for Photofrin under identical experimental conditions. They have reported high photodynamic efficiency for p-THPP on the HCT 116 cell line. 2. Materials and methods 2.1. Materials L-a-Phosphatidylcholine (PC) type XIII-E from egg yolk (99%, solution of 100 mg/mL in ethanol) was obtained from Sigma Chemical Co. (St. Louis, MO). It was a mixture of lipids with the following fatty acid makeup: 33% C16:0 (palmitic), 13% C18:0 (stearic), 31% C18:1 (oleic), and 15% C18:2 (linoleic) (other fatty acids being minor contributors), which gives an average molecular weight of approximately 768 g/mol. N-[Methoxy(polyethylene glycol) 2000] carbonyl-1,2-dipalmitoyl-sn-glycero-3-phospho-ethanolamine, sodium salt (PEG2000-lipid) was obtained from Northern Lipids Inc. (Vancouver, British Columbia, Canada). Polyethylene glycol monomethyl ether mesylate 2000 (PEG2000 mesylate), N,N-dimethylformamide (DMF, absolute; dried over molecular sieve) and diethyl ether (>99.8%) were obtained from Fluka Chemie (Buchs, Switzerland). 5,10,15,20-Tetrakis(4-hydroxyphenyl)porphyrin (pTHPP) was received from Aldrich Chemical Co. (Milwaukee, WI). For all subsequent studies the DMF stock solution of dyes of approximately 1 mM concentration was prepared and used. All
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experiments were conducted in phosphate-buffered saline (PBS) at pH 7.4. p-THPP solution for biological study was prepared by dissolving the dye in DMSO (2 mg/mL stock solution), the solution was diluted to reach the desirable concentration. 2.1.1. Synthesis of the pegylated porphyrin (p-THPP–PEG2000) PEG supported p-THPP (p-THPP–PEG2000) has been prepared by the method described in the literature [25]. Briefly, the reaction of 1 mol equiv of p-THPP with 1.2 mol equiv of the PEG2000 mesylate in dry DMF in the presence of 5.2 mol equiv of potassium carbonate afforded the supported porphyrin derivatives. The crude product was dissolved in CH2Cl2 (ca. 3 mL) and precipitated with diethyl ether (100 mL). Then p-THPP–PEG2000 was purified by column chromatography using CH2H2–MeOH, 20:1 (v/v)/SiO2. p-THPP–PEG2000 was characterized by UV spectroscopy and MALDITOF. From MALDI-TOF we found that the compound was a mixture of derivatives with one or two PEG chains covalently attached to the porphyrin ring. The content of porphyrin chromophore was determined by UV to be 2.5 wt.% using the molar absorption coefficient equal to log e422 = 5.6. 2.2. Apparatus UV–Vis absorption spectra of the samples were measured using a Hewlett–Packard 8452A diode-array spectrophotometer equipped with a HP 89090A Peltier temperature control accessory at 25 °C. Steady-state fluorescence spectra of the samples were recorded on an SLM-AMINCO 8100 Instruments spectrofluorimeter at room temperature. Emission spectra were corrected for the wavelength dependence of the detector response by using an internal correction function provided by the manufacturer. Matrix-assisted laser desorption/ionization mass spectrometry time of flight (MALDI–TOF) measurements were done using a Bruker Reflex IV spectrometer (Saxonia, Leipzig, Germany) equipped with a pulsed nitrogen laser emitting at a wavelength of 337 nm. The instrument was operated in reflecting mode. The matrix used was a-cyano-4-hydroxycinnamic acid (CHCA) at the concentration of 10 mM and solvents were acetonitrile/0.1% water solution of trifluoroacetic acid (1/2 vol.). The solutions of polymer and matrix were mixed together and 0.5 lL of this mixture solution was placed on a metal sample plate and air-dried before analysis. The size of liposomes was determined by dynamic light scattering measurements (DLS) using a Malvern Nano ZS light-scattering apparatus (Malvern Instruments Ltd., Worcestershire, UK). The time-dependence autocorrelation function of the photocurrent was acquired every 10 s, with 15 acquisitions for each run. The sample was illuminated with a 633 nm laser, and the intensity of light scattered at the angle of 173° was measured by an avalanche photodiode. The z-average diameter (dz), the polydispersity index (PDI) and the size distribution profiles of the samples were automatically provided by the instrument using cumulants analysis. Intracellular accumulation of porphyrins was observed under Carl Zeiss LSM 510 confocal microscope. Specimens were illuminated with the 488 nm excitation line. The specific fluorescent emission of the porphyrin was collected by a photomultiplier tube after passing through a 560 nm bandpass emission filter. Detection settings were determined using a negative control by adjusting the gain and offset settings to eliminate background. Images were collected using a 40 oil immersion objective (NA 1.3). The pinhole was set to 128 lm. Living cells in culture dishes were observed under inverted Oympus IX80 microscope equipped with differential interference contrast DIC and photographed.
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2.3. Preparation of liposomes
2.7. PDT treatment
PEG2000-lipid was dissolved in ethanol (12.5 mg in 2 mL) to form a stock solution. Appropriate volumes of the stock solution were mixed with 50 lL of the PC lipid solution in a volumetric flask to provide the desired PEG-lipid content. The solvent was evaporated under flow of nitrogen from the flask leaving a phospholipid film. Then the film was dissolved in diethyl ether which was then reevaporated under nitrogen to complete dryness. 10 mM PBS buffer was added till a desired lipid concentration was attained (usually 2.5 mg/mL), and the sample was sonicated for 5 min at 20 °C in a Bransonic ultrasonic bath. The resulting multilamellar vesicle dispersion was subjected to five freeze–thaw cycles from liquid nitrogen to temperature of 60 °C, extruded ten times through two stacked membrane filters with 200-nm pores (Nucleopore Track-Etch Membrane Whatman filters) using the PPH Marker manual extruder, and then passed ten times through membrane filters with 100 nm pores. dz and PD of the liposomal vesicles were in the range of 104–109 nm and 0.025–0.08, respectively. The size of the vesicles is large enough to carry a substantial drug payload and small enough to allow to enter passively though gaps in the endothelial cells lining the walls of the blood vessels which are highly permeable as a result of tumor angiogenesis. There was no effect of the PEG-lipid content on the vesicle size. Vesicles were stored at 4 °C. For biological study p-THPP was incorporated into vesicles during the liposome preparation. To this end 2 mg of the dye was dissolved in 200 lL ethanol solution of the PC lipid with 7 mol% content of the PEG2000-lipid. The lipid film was hydrated with 1 mL of PBS buffer and the preparation of vesicles was continued as above. The final concentrations of p-THPP and lipid were 2.9 mM and 20 mg/mL, respectively.
Cells were seeded on 3 cm plates (NUNC) at the density 90,000 cells per plate and left for 24 h for adhesion to the bottom. Cells were incubated with various concentration of porphyrins: 1, 2.5, 5, 10 and 20 lM for 4 h. Then the medium containing porphyrins was replaced with phenol red free medium and plates were exposed to the red light source (a halogen lamp 1000 W with heat isolation filter and 610 nm long-pass filters) for 1 min (6 J/cm2 light dose), 2 min and 30 s (15 J/cm2 light dose), 5 min (30 J/cm2 light dose) or 10 min (60 J/cm2 light dose). Control cells were treated neither with porphyrins nor irradiated with red light. Dark control cells after incubation with porphyrins were not irradiated with red light, and light control cells were exposed to red light without prior incubation with porphyrins. All experiments were performed thrice.
2.4. Determination of liposome-binding constants A spectroscopic titration technique was used to determine the binding constants (Kb) of the porphyrin chromophores to lipid vesicles. Details of this technique were described previously [26,27]. To determine the incubation time required to reach equilibrium, the partitioning kinetics of the porphyrin moieties into liposomes were studied. After each addition of an aliquot of lipid, the system was equilibrated and emission spectra of porphyrin chromophore were recorded. Kb is given in units of (mg/mL)1 throughout this study. 2.5. Cell lines The effect of PDT was studied using a prostate adenocarcinoma cell line (DU 145) and a human colon adenocarcinoma cell line (HCT 116), both obtained from American Type Culture Collection. Cells were routinely grown in RPMI 1640 culture medium (Gibco BRL/Life Technologies Inc) supplemented with 10% (v/v) fetal bovine serum at temperature 37 °C in humidified (95%) atmosphere with 5% CO2. 2.6. Internalization of the photosensitizing systems For confocal microscopy examination of intracellular accumulation of porphyrins cells were seeded on NUNC thin glass 4-well slides at the density of 10,000 cells per well and left for 24 h for adhesion to the bottom. Growth medium was replaced with medium containing various photosensitizing systems of equivalent concentration of p-THPP (1 lg/mL). The cells were incubated for 3 h at 37 °C. Afterwards the cells were washed twice with PBS buffer and examined under the confocal microscope. The experiments were performed thrice.
2.8. Cell viability test Cell viability test with MTS CellTiter 96Ò (Promega) was performed 24 h after PDT following standard protocol. In brief, medium in culture plates was replaced with 1 mL of MTS in DMEM without phenol red, and cells were incubated for 1 h at 37 °C. The solution was transferred to the well in 96-well culture plate (Nunc, Denmark). Optical density was determined at 490 nm using a microplate reader (BioTek Instruments, USA). Viability was expressed as a percentage of control. IC50 was defined as a concentration of a drug that decreases cell viability by 50%. 3. Results and discussion 3.1. Characterization of the photosensitizer systems In that paper we have studied two sensitizing systems: (i) pTHPP entrapped in liposomes, which were sterically stabilized with PEG2000-lipid, and (ii) the pegylated p-THPP (p-THPP–PEG2000) dissolved in 10 mM phosphate buffer. The properties of these two systems were compared to those of the free p-THPP. The chemical structures of the investigated compounds are shown in Fig. 1. pTHPP–PEG2000 was synthesized as described in Section 2.1.1. The attachment of monomethyl ether of PEG to p-THPP was performed using PEG mesylate derivative. The PEG-functionalized porphyrin were structurally characterized using MALDI-TOF, absorption and fluorescence spectroscopy. Fig. 2 shows the MALDI-TOF mass spectrum obtained for p-THPP–PEG2000. The analysis of spectrum revealed that p-THPP–PEG2000 consists of mono- and disubstituted porphyrin. The photophysical properties of porphyrins are strongly dependent on their aggregation state and drastically change upon aggregation. Electronic absorption and fluorescence spectra of the sensitizing systems were measured to estimate the extent of aggregation processes of p-THPP and p-THPP–PEG2000 in various environments. Fig. 3A shows absorption spectra of p-THPP in water, DMF and in liposome suspensions, respectively. Fig. 3B presents steady state fluorescence spectra of p-THPP in water, DMF solution as well as solubilized in liposome suspension. Porphyrin absorption spectra are characterized by a strong absorption band near 422 nm (Soret band). A considerable decrease in the intensity of the Soret band was observed in water in comparison to that in DMF and liposome suspensions. The observed changes are due to the formation of porphyrin dimers or higher aggregates as a result of the p–p interaction between the molecules [28]. The changes in the absorption spectra were accompanied by a strong decrease of the fluorescence intensities of the dye in aqueous medium, that was confirmed by the measurements of the emission spectra of p-THPP. The solubilization of porphyrin in hydrophobic liposome
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son with that for free p-THPP [25]. We found that the value of the dimerization constant characteristic of the free porphyrin (1.2 105 M1) decease upon pegylation with 2000 Da polymer chain to 4.5 104 M1. We concluded that attachment of PEG chain to the porphyrin ring enhanced the solubility of the p-THPP in water and polymeric chains prevent to some extent aggregation phenomena of porphyrin chromophores.
1
3.2. Interaction of p-THPP with SSL liposomes Loading efficiency of the liposomes is very important when they are considered as delivery vehicles. This efficiency is related to the partitioning coefficient of porphyrin between the lipid vesicles and aqueous phase and can be quantitatively described by so-called binding constant, which is defined as follows [29]:
2
p
1
p
2000
2
K b ¼ cL =ðcw ½LÞ
1
where cL and cw are porphyrin concentrations in the lipid vesicles and aqueous phase, respectively. [L] is concentration of lipid. The binding constant of p-THPP to liposome membrane as a function of PEG-lipid concentration was previously examined in our laboratory using the spectroscopic titration technique [30]. The affinity of the porphyrin to SSL liposomes was investigated in the range from 0 to 10 mol% of PEG2000-lipid content. It was found that the incorporation of as much as 3 mol% PEG-lipid to bilayer caused an increase of Kb value from 105 ± 35 (mg/mL)1 to 237 ± 58 (mg/mL)1. The further increase of the partitioning coefficient on addition of PEG2000-lipid was not so pronounced. For biological study the system containing 7 mol% was chosen. At this PEG2000-lipid content the value of Kb is equal to 270 ± 58 (mg/mL)1 [30]. Since the concentration of lipid which we used in vitro studies was equal to 20 mg/mL, one can easily calculate that the concentration ratio of the porphyrin bound to liposomes and dissolved in aqueous phase was as high as 5400. This result confirms that SSL liposomes possess good loading efficiency of porphyrins.
2
Fig. 1. Chemical structures of the investigated porphyrins.
2806,91
800 4658,88
Intensity (a.u.)
1200
ð1Þ
400
3.3. Cellular uptake and the dark cytotoxicity 0 2000
3000
4000
5000
6000
m/z Fig. 2. MALDI-TOF mass spectrum of p-THPP–PEG2000.
bilayer results in increase of dye absorbance and its fluorescence intensity. This indicates that solubilization of dye in liposomes protects it against aggregation. Thus, p-THPP enclosed in SSL is present in the monomeric form. Fig. 3C shows the absorbance spectrum of the p-THPP–PEG2000, in water and in DMF. One can notice that the profile of the absorption spectra is strongly solvent-dependent. The shape of the spectrum of pegylated porphyrin in aqueous solution is typical of porphyrin aggregates (wide Soret band of reduced intensity) while that recorded in DMF solution features the absorbance at 422 nm which is typical of monomeric porphyrin. In order to determine the effect of the pegylation on aggregation processes, the absorption spectra of p-THPP and p-THPP–PEG2000 were compared at the same concentration of the chromophores (Fig. 3E). As can be seen, the spectrum of the free porphyrin is broader and red shifted in comparison to that characteristic of the pegylated one. These results suggest that the attachment of PEG chain to porphyrin reduce to some extent aggregation processes of the dye in aqueous solution. In our previous paper we have presented results of the detailed studies on dimerization in the series of pegylated porphyrin systems with various polymer chain length in compari-
The efficiency of PDT treatment is highly dependent on the photosensitizer cellular uptake and accumulation in malignant tissues [31,32]. Also intracellular aggregation state of the photodynamic agent is very important, since aggregates are not active in PDT. In a current studies, the cellular uptake of the p-THPP entrapped in SSL liposomes and p-THPP–PEG2000 was evaluated using a prostate cancer cell line DU 145 and a colon cancer cell line HCT 116. These were compared with results obtained for free p-THPP. To assess the cellular uptake the confocal laser scanning microscopy was used. The obtained micrographs after 3 h incubation with the photosensitizer systems are shown in Fig. 4. Increases in the intensity of fluorescence do not necessarily correlate with increased cellular uptake of the porphyrin, as the intracellular aggregation state of the dye has been reported to dramatically affect the fluorescence intensity [33]. However, several conclusions regarding cellular uptake of the studied photosensitizing systems and their intercellular aggregation can be drawn from the confocal microscopy experiments: (i) the growing intensity of intracellular fluorescence of porphyrins indicating their accumulation in cells was observed in that experiment. These results have demonstrated that the active agent from both sensitizing systems can enter the cancer cells; (ii) the cellular uptake is dependent both on the type of the cell line and the photosensitizer system; (iii) the photosensitizer uptake of DU145 cells is much lower than that observed in HCT 116 cells for all photosensitizer systems;
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1.2
Absorbance
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Absorbance
0.15
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0.00 350
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Wavelength (nm) Fig. 3. Absorption and emission spectra of p-THPP and p-THPP–PEG2000 in different environments; (A) p-THPP (c = 2.48 106 M) in water, DMF and liposomal dispersion; (B) p-THPP (c = 2.47 108 M, kex = 422 nm) in water, DMF and liposomal dispersion; (C) p-THPP–PEG2000 in water and in DMF (c = 1.02 106 M); (D) p-THPP–PEG2000 (c = 2 108 M, kex = 422 nm) in water and in DMF; (E) comparison of the absorption spectra in aqueous solution of p-THPP and p-THPP–PEG2000 at the same concentration of chromophore (c = 1.02 106 M).
(iv) SSL liposomes loaded with p-THPP are rapidly internalized within cells to a considerably greater extent than the pegylated porphyrin dissolved in culture medium; (v) content of the monomeric form of porphyrin chromophore in the cellular plasma is the highest after internalization of the liposomal formulation. In the previous studies we presented data on the properties of pegylated p-THPP in aqueous solution and their interaction with li-
pid membranes [30]. The DLS and atomic force microscopy experiments have shown that the pegylated porphyrins form large polymer clusters (aggregates) in water solution. Using the fluorescence quenching measurements it was shown that the porphyrin chromophores are situated deeply inside these clusters and thus they are less accessible. Formation of such structures in aqueous solution results in reduction of the affinity of dyes for partitioning into liposomal membrane. The value of Kb characteristic of the free p-THPP decrease ninefold after attachment of 2000 Da PEG chain
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Fig. 4. Intracellular fluorescence of porphyrin in confocal laser scanning microscope. (A–D) DU 145 cells, (E–H) HCT 116 cells; (A, E) control – untreated cells; (B, F) p-THPP in medium; (C, G) p-THPP–PEG2000 in buffer (1 lg/mL, after 3 h); (D, H) p-THPP in liposomes (1 lg/mL, after 3 h).
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light for 1 min (6 J/cm2), 2.5 min (15 J/cm2), 5 min (30 J/cm2) or 10 min (60 J/cm2). The observed reduction of cell viability for three tested photosensitizing systems occurred at the dose of 15 J/cm2. That dose was chosen for further experiments, aiming to indicate the concentration of a photosensitizer leading to 50% reduction of cell viability (IC50). The values of concentrations of the tested porphyrin forms, leading to 50% inhibition of cell proliferation (IC50) are collected in Table 1. The sensitivity of PDT with p-THPP photosensitizers varies substantially between two cell lines (see Fig. 5). However, there is no doubt that there is a profound cytotoxic effect after PDT carried out with the use of porphyrin encapsulated in liposomes and that effect is much higher than that when PEG functionalized porphyrin was applied. IC50 value for p-THPP encapsulated in liposomes calculated for HCT 116 cells equals to 1.43 lg/mL, whereas this value for pegylated p-THPP was 9.1 lg/mL. In more resistant DU 145 IC50 exceeds 20 lg/mL for both photosensitizing systems. The efficiency of free p-THPP was found to be considerably greater than of other p-THPP formulations tested under the experimental conditions (see Fig. 6) and we obtained the IC50 values for free p-THPP dissolved in DMSO the values of 0.8 lg/mL and 2.4 lg/mL for HCT 116 and DU 145 cell lines, respectively (Table 1). The efficiencies of each photosensitizing systems in PDT are highly dependent on their intercellular accumulation level and their subcellular localization, resulting from particular uptake pathways. Thus, there is no simple correlation between the level of cellular uptake and the photocytotoxicity. That indicates that the subcellular localization may be crucial for the photodynamic efficiency. Considering the differences in physicochemical properties of the photosensitizers used one can expect that the PDT effect induced by various investigated systems (p-THPP-loaded liposomes, free p-THPP and p-THPP–PEG2000) is different. The preferential sites of intracellular localization of the similar systems have been previously reported by many authors. Due to its lipophilicity, free p-THPP tends to be localized in the cytoplasmic membrane [35]. In contrast, some TArPs were reported to be localized in the mitochondria or in the peri-nuclear region [32]. p-THPP-loaded
[30]. The lowest cellular uptake of the p-THPP–PEG2000 can be explained based on the described properties. Since the partitioning of the photosensitizers bearing one or two PEG chains into lipid bilayer is decreased, the tendency for crossing plasma membranes should be reduced and consequently lower cellular uptake will be observed compared to the free porphyrin, as was found indeed in these studies. The low uptake of the pegylated porphyrin during the short incubation (3 h) was previously reported by Sibrian-Vazquez [16]. Low dark toxicity is one of the most important criteria of photosensitizer usefulness in clinical PDT. The dark toxicities of free p-THPP, the PEG-functionalized porphyrin and that entrapped in liposomes were investigated in DU 145 and HCT 116 cells exposed to increasing concentrations of each porphyrin for 24 h. The results were presented in Table 1. Both photosensitizing systems developed in that studies were found (using a MTS assay) to be nontoxic in the dark at the concentration up to 50 lg/mL while free p-THPP have shown high dark toxicity. The value of IC50 for free p-THPP was estimated to 5.4 lg/mL and 14 lg/mL for HCT 116 cells and for DU 145 cell line, respectively. The very low toxicity of pegylated porphyrin derivatives might result from the good biocompatibility and low toxicity of PEG. Our results showing the lack of dark cytotoxicity of the p-THPP– PEG2000 are in line with these reported by Sibrian-Vazquez et al. [16]. They investigated TArPs functionalized with 1–4 PEG chains. All compounds were found to be non-toxic in the dark up to 250 lM after uptake into human HEp2 cells. Also, the observed lack of dark cytotoxicity of the p-THPP enclosed in the SSL supports the common opinion that liposomalization reduces systemic toxicity of the drugs [35]. 3.4. PDT effects The reduction of HCT 116 and DU 145 cells viability 24 h after irradiation (PDT) was assessed with the MTS assay. Initially, the phototoxic effect was tested in cells preincubated with 1 lg/mL concentration of porphyrin chromophore and irradiated with red Table 1 The values of IC50 at 15 J/cm2 dose of the red light. IC50 dark cytotoxicity (lg/mL)
Photosensitizing system
p-THPP in DMSO p-THPP in SSL liposomes p-THPP–PEG2000
IC50 photo cytotoxicity (lg/mL)
HCT 116
DU 145
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5.4 >50 >50
14 >50 >50
0.8 1.43 9.1
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Fig. 5. Cells viability measured by MTS assay 24 h after PDT with p-THPP–PEG2000 or p-THPP encapsulated in liposomes in HCT 116 cells (A) and DU 145 (B).
K. Nawalany et al. / Journal of Photochemistry and Photobiology B: Biology 97 (2009) 8–17
100
Hct 116 DU 145
Cell viability (%)
80 60 40 20 0 0
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p-THPP concentration (µg/ml) Fig. 6. Cells viability measured by MTS assay 24 h after PDT with p-THPP in HCT 116 and DU 145 cells.
nanoparticles are sequestered in intracytoplasmic compartments such as lysosomal compartments [35] or both early and late endo-
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somes [34]. TArPs bearing one or two PEG chains are preferentially localized in sensitive organelles like mitochondria and endoplasmic reticulum (ER), while those containing three or four PEG chains are localized preferentially in the lysosomes [16]. SSL liposomes enter the cells by endocytosis and are accumulated in the lysosomes [35]. The observed in this work higher PDT activity of the free p-THPP compared to that embedded in SSL can be explained assuming different localization of the photosensitizer inside the cell. Based on literature we suggest that the liposomal formulation of p-THPP is localized in lysosomes, while free porphyrin may be present in more sensitive organelles such as mitochondria. The systematic studies are currently performed in our laboratories to confirm that assumption. The effect of PDT on cellular morphology (Fig. 7) was assessed under the inverted microscope for the system of porphyrin entrapped in SSL. It was dependent on the dose of porphyrin used for photosensitization. The proliferation of cells after PDT was limited; cells were rounded and detached from the bottom of the plate. The membrane blebbing of many cells after irradiation was also noticed. Porphyrin in SSL liposomes without irradiation (dark control in Fig. 7) was not toxic for the cells even at high concentration (50 lg/mL), but the density of cells was slightly lowered after treatment with that dose of porphyrin.
Fig. 7. Changes of HCT 116 cells morphology after PDT. A, C, E – dark control; B, D, F – cells treated with PDT after 4 h treatment with growing concentration of p-THPP encapsulated in liposomes. A, B – 1 lg/mL, C, D – 10 lg/mL, E, F – 50 lg/mL.
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Our results can be compared to those for the systems in which p-THPP was trapped in nanoparticles. Konan et al. [34,36] have investigated the influence of drug concentration on photocytotoxicity of free p-THPP or p-THPP loaded in poly(D,L-lactide-coglycolide) (PLGA) and poly(D,L-lactide) (PLA) nanoparticles. The photocytotoxicity was evaluated on EMT-6 mammary tumor cells. They have shown that p-THPP trapped in PLGA nanoparticles was more efficient than that in PLA nanoparticles. Additionally, p-THPP in nanoparticles formulation has exhibited higher photodynamic activity in comparison with free p-THPP. The value of IC50 for free p-THPP was estimated to 5 lg/mL, whereas for porphyrin in PLGA nanoparticles to 2 or 3.7 lg/mL, depending on the composition of the copolymer.
4. Conclusions In the present work we have reported the preparation and photodynamic efficacy of two formulations towards human colon adenocarcinoma and prostate cancer cell lines. They contain hydrophobic p-THPP as photochemically active agent. That compound was solubilized in SSL liposomes or modified by covalent attachment of hydrophilic PEG chains. Peripheral functionalization of the porphyrin core by anchoring polymeric chains have changed the physical properties of the tetrapyrolic macrocycle. Pegylation reduced to some extent the aggregation processes of the dye in aqueous solution. The results of the present study show that both pegylation and embedding in SSL liposomes considerably reduce the dark cytotoxicities of the parent porphyrin. The porphyrin uptake of DU 145 cells was much lower than that observed in HCT 116 cells for both photosensitizer systems. The use of liposomes as vehicles transporting hydrophobic photosensitizer molecules have been shown to be advantageous for their use in PDT – it enables their efficient transfer and accumulation in the cells in the non-aggregated form. Moreover, the pegylated porphyrin dissolved in culture medium was less readily taken-up by cells than porphyrin encapsulated in liposomes, probably due to formation of large polymeric clusters. In conclusion, it seems that the system in which porphyrin is embedded into SSL liposomes may provide a method of increasing the selectivity and efficiency of PDT by reducing the photosensitizer aggregation and toxicity, by improving the tumor penetration and uptake and by enhancing phototoxicity in vitro and in vivo. However, the overall phototoxicity efficiency is strongly dependent on the type of the cancer cell line. 5. Abbreviations mTHPC 5,10,15,20-tetrakis-(m-hydroxyphenyl)chlorin L-a-phosphatidylcholine PC PDT photodynamic therapy PEG poly(ethylene glycol) PEG2000-lipid N-methoxy(polyethylene glycol) 2000]carbonyl-1,2dipalmitoyl-sn-glycero-3-phospho-ethanolamine, sodium salt PF Photofrin p-THPP tetrakis(4-hydroxyphenyl)porphyrin SSL sterically stabilized liposomes TArPs tetraarylporphyrins Acknowledgement Project operated within the Foundation for Polish Science Team Programme co-financed by the EU European Regional Development Fund.
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