Enhanced photodynamic activity of meso-tetra(4-hydroxyphenyl)porphyrin by incorporation into sub-200 nm nanoparticles

European Journal of Pharmaceutical Sciences 18 (2003) 241–249 www.elsevier.com / locate / ejps

Enhanced photodynamic activity of meso-tetra(4-hydroxyphenyl)porphyrin by incorporation into sub-200 nm nanoparticles 2 ´ Yvette Niamien Konan, Myriam Berton 1 , Robert Gurny*, Eric Allemann

School of Pharmacy, University of Geneva, 30 quai E. Ansermet, CH-1211 Geneva 4, Switzerland Received 31 July 2002; received in revised form 2 January 2003; accepted 6 January 2003

Abstract A photosensitizer, meso-tetra(hydroxyphenyl)porphyrin (p-THPP) was incorporated into sterile submicronic nanoparticles of poly( D,Llactide-co-glycolide) (50:50 and 75:25 PLGA) and poly( D,L-lactide) (PLA). With all polymers used, sub-130 nm p-THPP-loaded nanoparticles with similar drug loadings and entrapment efficiencies were produced using the emulsification–diffusion technique. The photodynamic activity (photocytotoxicity) of these nanoparticles was evaluated on EMT-6 mammary tumour cells in comparison with the free drug. The influence of drug concentration (3–10 mg / ml), incubation time (5–60 min) and light dose (6–9 J / cm 2 ) on p-THPP photocytotoxic efficiency was investigated. With all p-THPP formulations tested, cell viability decreased with increasing values of these parameters. The beneficial effect of nanoencapsulation compared to free drug was highlighted at drug concentrations up to 6 mg / ml and short incubation times (15–30 min). The most important photocytotoxicity was observed with 50:50 PLGA nanoparticles allowing low drug doses and short drug administration–irradiation intervals for local photodynamic therapy.  2003 Elsevier Science B.V. All rights reserved. Keywords: Photodynamic therapy; Meso-tetra(4-hydroxyphenyl)porphyrin; Nanoparticles; Poly( D,L-lactide-co-glycolide); Poly( D,L-lactide); EMT-6 mammary tumour cells

1. Introduction Photodynamic therapy (PDT) of cancer is based on the combined use of visible light-induced activation of photosensitizers (PSs) resulting in selective photochemical damage of tumour cells with minimal effect on the surrounding normal tissues (Jori, 1996; Renno and Miller, 2001). Although this therapy is becoming an established modality of treatment for a variety of diseases, its widespread clinical application remains limited because of the numerous drawbacks of the commonly used PSs including haemotoporphyrin derivatives (e.g. Photofrin  ) (Dougherty et al., 1975; Razum et al., 1987; Dougherty, 1987; Mironov et al., 1990; Lin, 1991). Hence, in order to improve the phototherapeutical protocols, several strategies including the development of second-generation PSs with

*Corresponding author. Tel.: 141-22-702-6146; fax: 141-22-7026567. E-mail address: [email protected] (R. Gurny). 1 Present address: Novartis Pharma AG, CH-4002 Basle, Switzerland. 2 Present address: Bracco Research S.A., CH-1228 Plan-les-Ouates, Geneva, Switzerland.

more favorable properties, are being investigated (Kreimer-Birnbaum, 1989). One of these compounds, meso-tetra(4-hydroxyphenyl)porphyrin (p-THPP) has been shown to be about 4–6 times more potent than Photofrin  in its ability to photosensitize skin, brain and implanted PC6 plasma cell tumours (Berenbaum et al., 1986; Kreimer-Birnbaum, 1989). Pronounced hydrophobicity characterizes most porphyrins including p-THPP. This property is considered to be one of the parameters responsible for the affinity of the PSs for neoplastic tissues (Lin, 1991; Pandey et al., 1996; Henderson et al., 1997). However, this hydrophobicity leads to poor solubility of these molecules in physiologically acceptable media and often impedes their parenteral administration. To overcome this problem, polar substituents including sulphonic acid, carboxylic acid and hydroxyl groups were added to the peripheral positions of the macrocycle (ten Wolde et al., 1996; Lin, 1991). Alternatively, hydrophobic PSs have been incorporated into colloidal carriers such as liposomes (Ricchelli et al., 1993), emulsions (Garbo et al., 1998), microparticles (Bachor et al., 1991a,b; Li and McCarthy, 1999) or ´ nanoparticles (Brasseur et al., 1991; Allemann et al.,

0928-0987 / 03 / $ – see front matter  2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0928-0987(03)00017-4

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1996). Colloidal carrier-associated PS have shown to exhibit greater photodynamic efficiency and selectivity of tumour targeting as compared with the dye administered in ´ a homogeneous aqueous solution (Allemann et al., 1996; ¨ et al., 1999; Konan et al., 2002a). However, in most Worle investigations, the particle mean size used was often greater than 200 nm. Nanoparticles with a smaller mean size (i.e. 100 nm) are preferable not only for the more effective blood-to-tumour transfer of colloidal delivery systems, but also for their longer retention in tumour tissues (Kong et al., 2000). It has been reported that the optimal size for increasing tumour accumulation and blood circulation of GM1 liposomes was in the range of 90 to 200 nm in diameter (Liu et al., 2002). Moreover, such particle size enables to reduce the recognition of these particles by opsonins in the bloodstream leading to a reduction of their uptake by the mononuclear phagocytic system (Nagayasu et al., 1999). In a previous study (Konan et al., 2003), it has been shown that p-THPP could be encapsulated into sterile sub-150 nm biodegradable nanoparticles based on two different poly( D,L-lactide-co-glycolide) and poly( D,L-lactide) polymers with drug loading up to 7% (m / m). The objective of the present work was to evaluate the photodynamic activity of these p-THPP-loaded particles on EMT-6 mouse mammary tumour cells compared to free p-THPP. This study was focused on the influence of drug concentration, incubation time and light dose on the in vitro photocytotoxicity of p-THPP.

2. Materials and methods

2.1. Materials Poly( D,L-lactide-co-glycolide) (PLGA) of copolymer ratio of 50:50 PLGA (Resomer  RG502) and 75:25 PLGA (Resomer  RG752) were obtained from Boehringer (Ingelheim, Germany) and poly( D,L-lactide) (PLA) ( D,LPLA2M) was received from Alkermes (Cincinnati, OH, USA). The weight-average molecular weight (Mw ) of these polymers, previously determined (Konan et al., 2002) by gel permeation chromatography using polystyrene as reference, were 13 000, 16 000 and 19 000 for 50:50 PLGA, 75:25 PLGA and PLA, respectively. p-THPP provided by Aldrich (Steinheim, Germany) was chosen as photosensitizing agent. The chemical structure and absorption spectrum of p-THPP are depicted in Fig. 1. Poly(vinyl alcohol) (PVAL) 87.7% hydrolyzed with a Mw of 26 000 (Mowiol  4-88) (Hoechst, Frankfurt / Main, Germany) was selected as a stabilizing colloid. Benzyl alcohol (Fluka Biochemika, Buchs, Switzerland) was chosen as the organic partially water-miscible solvent. Dimethylsulfoxide (DMSO) and tetrahydroxyfuran (THF) were provided by Sigma–Aldrich (Steinheim, Germany) and Fluka Bio-

Fig. 1. Structural formula and absorption spectrum of p-THPP dissolved in THF.

chemika, respectively. D(1)-Trehalose dihydrate (Sigma, St. Louis, MO, USA) was used as a lyoprotectant. EMT-6 mouse mammary tumour cells were kindly provided by Dr. N. Brasseur (University of Sherbrooke, Quebec, Canada). All other chemicals were of analytical grade and were used without further purification.

2.2. p-THPP solutions A solution of p-THPP was freshly made by dissolving in DMSO (2 mg / ml). Then, the solution was diluted with Waymouth cell growth medium supplemented with 10% fetal bovine serum (FBS) (Gibco  , Life / Technologies, Basle, Switzerland) to reach final concentrations ranging from 2 to 20 mg / ml.

2.3. Nanoparticle preparation and characterization The nanoparticles were prepared using the emulsification–diffusion technique as previously described (Konan et al., 2002a). Briefly, 6 g of organic phase consisting of the polymer (500 mg) dissolved in benzyl alcohol were

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emulsified with 8 g of aqueous solution of PVAL (17% (m / m)). After 15 min of mechanical stirring at 2000 rev. / min, 500 ml of distilled water were added to the resulting emulsion in order to allow complete diffusion of the benzyl alcohol into the aqueous external phase, leading to the formation of nanoparticles. To produce the p-THPPloaded nanoparticles, 50 mg of the photosensitizer were dissolved into the organic phase before the emulsification. Finally, the nanoparticle suspensions were purified by cross-flow filtration using a Sartocon  Slice device fitted with an ultrafiltration membrane (cut-off of 300 000 Da, Sartorius, Goettingen, Germany). Then, the nanoparticle suspensions containing trehalose (trehalose:nanoparticles mass ratio of 2:1) were sterilized by filtering through a Steriflip  filter unit with 0.22 mm Millipore Express姠 (Millipore  , Volketswill, Switzerland). The sterile suspensions were frozen and freeze-dried at 0.001 mbar in a Lyolab CII (Secfroid, Switzerland) as previously described (Konan et al., 2002b,2003). The particle mean size and the polydispersity index (PI) were assessed by photon correlation spectroscopy using a Zetasizer  5000 (Malvern, Worcesterhire, UK). The PI is an indication of the size distribution with values ranging from 0 to 1. The p-THPP content and entrapment efficiency into the freeze-dried nanoparticles were determined spectrophotometrically at 653 nm (spectrophotometer HP8452, Hewlett-Packard, Waldbronn, Germany) as previously described (Konan et al., 2003).

2.4. Cell line The EMT-6 mouse mammary tumour cells were maintained according to an established protocol as previously described (Brasseur et al., 1999). Typically, a monolayer culture of EMT-6 tumour cells was maintained in Waymouth medium (Gibco  , Life / Technologies) supplemented with 10% FBS, 100 mM L-glutamine and 100 U ml 21 penicillin–streptomycin (Gibco  , Life / Technologies) at 37 8C in 5% CO 2 atmosphere. Aliquots of 15310 3 cells per well were plated into 96-well dishes in 100 ml of culture medium and incubated for 24 h at 37 8C before photocytotoxicity assays.

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mg / ml, incubation time of 40 min and light dose of 6 J / cm 2 . The photocytotoxicity of both p-THPP-loaded nanoparticulate formulations and the free p-THPP was evaluated by varying: (i) drug concentrations (3–10 mg / ml); (ii) incubation time (5–60 min); (iii) light dose (6 and 9 J / cm 2 ). Unloaded nanoparticulate formulations were also tested in the same manner at particle concentrations ranging from 2 to 20 mg / ml for incubation time over 24 h. The cytotoxicity of the light alone (6 and 9 J / cm 2 ) against the EMT-6 cells was also assessed.

2.6. Light source Irradiation was performed with a source composed of eight (43100 W and 43150 W) halogen tubes (Haloline  , OSRAM, Germany) placed in a box underneath an aqueous rhodamine refrigerated circulating filter. The fluence rate was measured over the absorbance of peak of p-THPP at 655 nm using a Radiometer / Photometer (Fieldmaster姠, Coherent姠, Germany) fitted with a light sensor (SmartSensors姠 LM-2 VIS, Coherent姠, Germany). Temperature measurements of the rhodamine bath were checked during irradiation in order to avoid hyperthermic conditions.

2.7. MTT assays The viability of treated cells was determined immediately or 18 h after irradiation by means of the 3-(4,5dimethyl-thiazol-2-yl-2,5-diphenyl tetrazolium bromide) assay (MTT, Sigma, Steinheim, Germany). Briefly, after removing the culture medium, 50 ml of MTT solution (1 mg / ml) were added to each well and incubated for 3 h. Then, the formazan crystals were solubilized by adding 100 ml of 10% (m / v) sodium dodecyl sulphate (Sigma– Aldrich) in 0.01 M HCl to each well. The absorbance was determined at 595 nm by means of a microplate reader (Bio-RAD, Hercules, Model 550). For each concentration, average cell viability was calculated from the data of six wells and expressed as a percentage, compared to untreated cells (100%).

2.5. Photocytotoxicity assay 3. Results The cells were washed and incubated with the different p-THPP formulations. Following incubation, cells were washed twice with phosphate buffer saline (Gibco  , Life / Technologies) and 100 ml of fresh culture medium were added in each well. Finally, the cells were immediately exposed to red light ( l5655 nm). The dark toxicity (not exposed to light) was also determined. In an attempt to obtain the conditions for the comparative study of the different p-THPP nanoparticles versus free drug, preliminary investigations were carried out with aqueous solutions of p-THPP at drug concentrations ranging from 2 to 20

3.1. Nanoparticle preparation and characterization Irrespective of the polymer composition, the nanoparticles showed very similar characteristics in terms of particle size, surface characteristics and redispersibility (Table 1). After freeze-drying in the presence of trehalose, complete redispersion of all formulations was observed in water (Table 1). The nanoparticulate formulations exhibited drug loading values of around 7% and entrapment efficiencies of up to 77%.

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Table 1 Characterization of p-THPP-loaded nanoparticles freeze-dried in the presence of trehalose (mean6S.D., n53) Polymer

Mw a

Mean particle size (nm)

Mean PI b

Drug loading (%) (w / w)

Entrapment efficiency (%) (w / w)

50:50 PLGA 75:25 PLGA PLA

12 500 15 500 18 400

11767 11862 12561

0.20 0.20 0.16

7.860.3 8.160.7 7.360.7

76.963.4 77.067.0 72.868.7

a b

Values determined with blank nanoparticles. Polydispersity index.

3.2. Influence of time delay on photocytotoxic efficiency Firstly, it has to be noted that light alone did not result in a significant decrease in cell viability under the experimental conditions used for this study (data not shown). Control measurements performed with drug-free nanoparticulate formulations (2 to 20 mg / ml) showed no photocytotoxic effect even after 24 h of incubation and irradiation at 9 J / cm 2 (data not shown). Moreover, no dark cytotoxicity was observed under these conditions (data not shown). In order to assess if the photodynamic activity of p-THPP is delayed, the cells were treated with different concentrations of free p-THPP (2–20 mg / ml) for 40 min and irradiated at a light dose of 6 J / cm 2 . The photocytotoxicity was evaluated immediately or 18 h after irradiation. As shown in Fig. 2, the MTT test performed at 18 h post irradiation exhibited a substantial decrease in cell viability when the drug concentration increased from 2 to 12 mg / ml. The lowest viability fraction (10%) was reached at concentrations up to 12 mg / ml where a plateau was observed. In contrast, only a slightly reduced cell

Fig. 2. Time-delayed p-THPP photocytotoxicity on EMT-6 mammary tumour cells. The cells were incubated with p-THPP aqueous solution at drug concentrations ranging from 2 to 20 mg / ml for 40 min. MTT assay was carried out immediately (h) or 18 h (j) after irradiation at a light dose of 6 J / cm 2 (655 nm). Each data point represents the mean (6S.D.) of six values.

viability was noted at these drug concentrations when the viability test was carried out immediately after irradiation. The drug concentrations yielding 50% cell death (IC 50 ) were 15.8 and 7 mg / ml for MTT assays performed immediately or 18 h after light exposure, respectively. Based on these first experiments, a concentration range from 3 to 10 mg / ml and the cell viability assays performed at 18 h after irradiation were used as the appropriate conditions for the subsequent experiments.

3.3. Influence of p-THPP concentrations on photocytotoxic efficiency The influence of drug concentration on p-THPP photocytotoxic efficiency was investigated with p-THPP-loaded nanoparticles compared to free p-THPP after 1 h of incubation and light exposure of 6 J / cm 2 . As shown in Fig. 3, for all the delivery systems tested, cell viability decreased in a drug concentration-dependent manner. The most important differences in photocytotoxicity were observed at 3 mg / ml. At this concentration, all the p-THPP-loaded nanoparticles exhibited a photocytotoxic activity at least twofold greater than free drug. With respect

Fig. 3. Influence of drug concentration on photocytotoxicity of p-THPPloaded nanoparticulate formulations (m, 50:50 PLGA; d, 75:25 PLGA; ♦, PLA) or free p-THPP (j). The EMT-6 tumour cells were incubated for 1 h, at equivalent drug concentrations ranging from 3 to 10 mg / ml for 1 h and irradiated at a light dose of 6 J / cm 2 (655 nm). MTT assay was performed 18 h after light exposure. Each data point is the mean (6S.D.) of six values.

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to the different nanoparticulate formulations, p-THPPloaded 50:50 PLGA nanoparticles exhibited the fastest and highest photocytotoxic activity (Fig. 3). Moreover, no significant difference in photosensitizing activity was found between 75:25 PLGA and PLA nanoparticles. At a drug concentration up to 6 mg / ml, the decrease in cell viability reached a plateau for the four formulations tested.

3.4. Influence of incubation time on p-THPP photocytotoxic efficiency The influence of incubation time on p-THPP photodynamic activity was evaluated at three different p-THPP concentrations (3, 6 and 8 mg / ml) for variable incubation times ranging from 5 to 60 min. After irradiation at a dose of 6 J / cm 2 , cell viability generally decreased when the

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incubation time increased. At a drug concentration ofThe photosensitizer was dissolved into culture medium containing DMSO. 3 mg / ml, photocytotoxicity was observed only after 60 min incubation with each delivery system (Fig. 4a). At high drug concentrations (6 and 8 mg / ml), significant reduction of cell viability was observed as early as 15 min of incubation (Fig. 4b and c). At these concentrations, the favourable effect of the nanoparticle formulations in terms of enhancement of PDT efficiency was highlighted at short incubation times (i.e. 15–30 min). The highest photocytotoxic effect was obtained with 50:50 PLGA nanoparticles.

3.5. Influence of light dose on p-THPP photocytotoxicity The effect of light dose on p-THPP photocytotoxicity

Fig. 4. Influence of incubation time on photocytotoxicity of p-THPP-loaded nanoparticulate formulations (m, 50:50 PLGA; d, 75:25 PLGA; ♦, PLA) or free p-THPP (j). The EMT-6 tumour cells were incubated at different equivalent drug concentrations (from 3 to 8 mg / ml) for different times (from 5 to 60 min) and irradiated at a light dose of 6 J / cm 2 (655 nm). MTT assay was performed 18 h after light exposure. Each data point is the mean (6S.D.) of six values.

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was assessed at two incubation times (5 and 15 min) and a drug concentration of 6 mg / ml. The results are reported in Fig. 5. After 5 min of incubation, an increase in light dose from 6 to 9 J / cm 2 yielded no detectable photocytotoxic effect on cell viability after treatment with all p-THPP formulations except with the 50:50 PLGA nanoparticles (Fig. 5a). However, the effect of light dose on photocytotoxicity of p-THPP-loaded nanoparticles was conspicuous after an incubation time of 15 min (Fig. 5b). Indeed, an increase in light dose from 6 to 9 J / cm 2 dramatically affected cell viability when cells were treated with the different nanoparticulate formulations. For instance, 6, 12 and 18% of survival fractions were obtained

Fig. 5. Influence of light dose on photocytotoxicity of p-THPP-loaded nanoparticulate formulations or free p-THPP. The EMT-6 tumour cells were incubated at equivalent drug concentration of 6 mg / ml for 5 or 15 min and irradiated at light doses of 6 (h) or 9 (j) J / cm 2 (655 nm). MTT assay was carried out 18 h after light exposure. Each data point is the mean (6S.D.) of six values.

with 50:50 PLGA, 75:25 PLGA and PLA nanoparticles, respectively, while free p-THPP yielded only 73% of cell viability (Fig. 5b).

4. Discussion The efficacy of novel cancer therapeutics such as PDT has been hindered by the inability to deliver the drug to the tumour at efficient concentrations. Thanks to the appropriate polymer composition and particle size for tumour accumulation properties, nanoparticles have been used as a possible method to overcome delivery issues and to prevent the side effects associated with undesired biodistribution of the free form of the drug (Barratt, 2000). Moreover, there are many options for modifying the biodistribution of nanoparticles by appropriately selecting polymer composition, polymer molecular weight, surface hydrophilicity / hydrophobicity and particle size (Dunne et al., 2000; Kawashima, 2001). Particle size can determine circulation half-life, polymer degradation, drug release profile and accumulation site in the tissue (Dunne et al., 2000; Kawashima, 2001). A particle size of around 100 nm is known to be appropriate for selective uptake by neoplastic cells and thus, might improve the phototherapeutic response during cancer treatment (Nagayasu et al., 1999; Dunne et al., 2000). The carriers with this typical size may extravasate into solid tumour tissue, inflamed and infected sites, where the capillary endothelium is defective (Barratt, 2000). In the present work, we have prepared p-THPP-loaded nanoparticles having the following characteristics: (i) size of around 100 nm, (ii) a high drug loading, and (iii) preserved bioactivity. These particles can be sterilized by filtration and reconstituted rapidly after freeze-drying. In addition, all final freeze-dried nanoparticulate formulations can be refiltered on a 0.22 mm membrane and are stable in terms of mean particle size and drug loading over a period up to 6 months (Konan et al., 2003). The in vitro photodynamic activity was evaluated at the cellular level using EMT-6 mouse mammary tumour cell lines. The results obtained from this study demonstrated that the photocytotoxicity of p-THPP against EMT-6 cells is mainly due to time-delayed effects appearing at 18 h post-irradiation. These findings are consistent with those reported by other authors (Merlin et al., 1992; Rezzoug et al., 1998; Zund et al., 1999). These results may be explained by the fact that the cell damage resulting to photochemical reactions are not immediately lethal. This phenomenon could be due to apoptosis since it has been reported that PDT can lead to an apoptotic response in malignant cells (Agarwal et al., 1991). The time required for initiation of apoptosis varies widely (He et al., 1994). Most cells, in response to inducing PS, go through a latency period, variable in duration, which usually results

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in the death of greater than 80% of a cell population in 1–3 days (Dougherty et al., 1998). Prior to studying the photocytotoxicity of the p-THPPloaded nanoparticulate formulations against EMT-6 tumour cells versus free p-THPP, the biocompatibility of the drugfree PLGA or PLA nanoparticles was assessed. As expected, EMT-6 cell viability was not affected after treatment with these nanoparticles. This is consistent with the known biocompatibility and safety of PLA / PLGA polymers. The biocompatibility of 50:50 PLGA nanoparticles against a human umbilical vein endothelial cell line treated with particle concentrations ranging from 62.5 to 500 mg / ml for 48 h has also been demonstrated (Davda and Labhasetwar, 2002). PDT is a complex therapy, the efficiency of which is dependent on several parameters including photochemical properties of PS, delivery systems, biological state of the tumour, physical localization and the amount of PS in treated tissue, drug injection-light activation interval time and the light dose (Konan et al., 2002a). The present study focused on some of these parameters including drug concentration, incubation time and light dose. Regardless of the nature of the delivery system, cell viability was drug concentration-dependent. The beneficial effect of the nanoparticle concept over the free drug was evidenced mainly at a drug concentration of 3 mg / ml. This suggests that the therapeutic index of p-THPP can be improved by nanoencapsulation since low drug concentrations could be used for satisfactory photocytotoxicity. The use of small drug concentrations could also be a means of shortening or minimizing the undesirable effects. At concentrations up to 6 mg / ml, the decrease in cell viability reached a plateau. This might suggest that the intracellular drug concentration yielding complete cell death was certainly reached with all the p-THPP formulations. The p-THPP photocytotoxicity, seen as early as 5 min, increased gradually with the incubation time. These results are consistent with those obtained by other groups (Brasseur et al., 1999). On the other hand, recent work (Rezzoug et al., 1998) has shown that the meso-tetra(hydroxyphenyl)chlorin (m-THPC) photodynamic activity against HT29 cells was not affected by the cell incubation time (3 or 24 h) and that the dye cellular uptake was the same at the two incubation times. This could be explained by the fact that the maximum accumulation of m-THPC in cells was achieved after incubation times up to 3 h and the increase in cell death reached a plateau at this incubation time. In our case, p-THPP-loaded 50:50 PLGA nanoparticles produced the fastest and most significant in vitro photocytotoxicity against EMT-6 tumour cells. Since incubation time reflects the kinetics of cellular uptake of the dye or delivery systems, short incubation times may suggest a rapid uptake of these nanoparticles by EMT-6 tumour cells. This could be a great advantage for the treatment of conditions such as age-related macular degeneration where irradiation has to be carried out rapidly

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after injection of the dye (Miller et al., 1999; Soubrane and Bressler, 2001). The incubation time might be even shortened by the light dose from 6 to 9 J / cm 2 since the photocytotoxicity of p-THPP-loaded nanoparticles was dramatically improved at 15 min of incubation compared to the free dye. A rise in light dose of 1.5 times is enough to induce almost complete cell death. Furthermore, compared to the results obtained with m-THPC (Temoporfin or Foscan  ) (Rezzoug et al., 1998), at equivalent light dose and drug concentration, 3 h of incubation time were required to yield the same in vitro PDT response. The results obtained here are consistent with those obtained by other authors who studied the incorporation of photosensitizers into microparticles (Bachor et al., 1991a– c). In light of data collected in the literature (Bachor et al., 1991a–c; Sharma and Sharma, 1997; Davda and Labhasetwar, 2002), the higher photodamage efficiency of p-THPPloaded nanoparticles compared to free drug could be explained by different hypothesis. Firstly, depending on the delivery systems, photosensitizer could accumulate in EMT-6 tumour cells via different mechanisms and / or rate. Indeed, nanoparticles, which normally enter the cells by endocytosis, can deliver greater drug concentrations compared to free p-THPP (Bachor et al., 1991b; Sharma and Sharma, 1997). In contrast, free p-THPP because of its lipophilic characteristic could be taken into the cell membrane by diffusion, leading to a low intracellular drug concentration (Bachor et al., 1991b; Sharma and Sharma, 1997). The intracellular localization, an important factor in PDT, might also differ between p-THPP-loaded nanoparticles and free p-THPP. Different intracellular localization might induce different photochemical lesions in treated cells. Due to its lipophilicity, free p-THPP tends to localize in the cytoplasmic membrane (Peng et al., 1995). In contrast, p-THPP-loaded nanoparticles are sequestered in intracytoplasmic compartments such as lysosomal compartments; membrane disruption following irradiation leads to pH imbalance and release of enzymes, and rapid cell death (Dougherty et al., 1998). Different photodynamic mechanisms of cytotoxicity could also explain the higher efficiency of p-THPP nanoparticles (Bachor et al., 1991b). Generation of singlet oxygen and additional free radicals may both act as potent cytotoxic agents. Bachor et al. have shown that the quantum yield of these species differed between free and encapsulated PS (Bachor et al., 1991c). The photocytotoxic efficiency of p-THPP incorporated into nanoparticles is dependent on the polymer nature. Indeed, although possessing the same characteristics in terms of polymer molecular weight, crystallinity, particle size and drug loading, these nanoparticles exhibited different in vitro photocytotoxicity in the order: 50:50 PLGA. 75:25 PLGA.PLA. This suggests that the photodynamic activity of the dye was strongly affected by the copolymer molar ratios, which determine the polymer hydrophilicity. As mentioned above, this polymer characteristic may induce different photodynamic mechanisms resulting in

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different levels of phototherapeutic response. It has also been reported that both polymer degradation rate and drug release depend on several parameters including copolymer molar ratios, which control water accessibility to ester linkages (Park, 1995). Hence, the findings observed in the present study could be expected since the hydrolysis-labile ester linkages in PLGA are more accessible to water than those in the homopolymer PLA. Glycolide-rich PLGA such as 50:50 PLGA are more accessible to water than 75:25 PLGA (Park, 1995). Consequently, fast release of p-THPP from the 50:50 PLGA matrix may occur into the intracellular compartments compared to other polymers. Different nanoparticle–cell interactions according to the copolymer molar ratio have also been reported (Prior et al., 2002). Indeed, copolymer molar ratios could influence the rate of carrier uptake and consequently, the intracellular drug concentration.

5. Conclusions This study has demonstrated that the encapsulation of p-THPP into sterile and freeze-dried sub-130 nm nanoparticles should be considered as an effective strategy for delivering p-THPP to tumour cells. The relatively low drug concentrations and short incubation times required to induce satisfactory photodynamic damages after cell treatment, especially with 50:50 PLGA nanoparticles, indicate that these formulations offer superior photoactivity. Hence, the nanoencapsulation of the dye may offer the possibility to inject smaller drug doses resulting in an enhancement of the p-THPP phototherapeutic index and short drug–light interval time. The photoinduced cytotoxicity of the dye is dependent on the PDT parameters such as drug concentration, light dose, drug–light time interval and delivery system nature (e.g. copolymer molar ratio). Considering the potential clinical applications of nanoparticulate formulations for photodynamic therapy, a greater understanding of the biological interactions and the implicated PDT mechanism is of utmost importance. Further research is underway in order to elucidate the phenomena.

Acknowledgements Dr. N. Brasseur is gratefully acknowledged for providing the EMT-6 mouse mammary tumour cells. We thank Dr. O. Jordan for helpful discussion and suggestions.

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