Light-Harvesting Ionic Dendrimer Porphyrins as New Photosensitizers

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Bioconjugate Chem. 2003, 14, 58−66

Light-Harvesting Ionic Dendrimer Porphyrins as New Photosensitizers for Photodynamic Therapy Nobuhiro Nishiyama,† Hendrik R. Stapert,†,‡ Guo-Dong Zhang,† Daisuke Takasu,§ Dong-Lin Jiang,§ Tetsuo Nagano,| Takuzo Aida,§ and Kazunori Kataoka*,† Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8656, Japan, Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Japan, and Department of Bioorganic Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo. Received August 23, 2002

Photodynamic therapy (PDT) is a promising therapeutic modality for treatment of solid tumors. In this study, third-generation aryl ether dendrimer porphyrins (DPs) with either 32 quaternary ammonium groups (32(+)DPZn) or 32 carboxylic groups (32(-)DPZn) were evaluated as a novel, supramolecular class of photosensitizers for PDT. DPs showed a different cell-association profile depending on the positive or negative charge on the periphery, and both DPs eventually localized in membrane-limited organelles. In contrast, protoporphyrin IX (PIX), which is a hydrophobic and relatively low molecular weight photosensitizer used as a control in this study, diffused through the cytoplasm except the nucleus. Confocal fluorescent imaging using organelle-specific dyes indicated that PIX induced severe photodamage to disrupt membranes and intracellular organelles, including the plasma membrane, mitochondrion, and lysosome. On the other hand, cells treated with DPs kept the characteristic fluorescent pattern of such organelles even after photoirradiation. However, notably 32(+)DPZn achieved remarkably higher 1O2-induced cytotoxicity against LLC cells than PIX. Furthermore, both dendrimer porphyrins had far lower dark toxicity as compared with PIX, demonstrating their highly selective photosensitizing effect in combination with a reduced systemic toxicity.

INTRODUCTION

Molecular nanomeric-scaled devices prepared through chemical process have recently been recognized to be useful for high-throughput screening, molecular diagnosis, and targeting therapy (1). In particular, dendrimers, the three-dimensional tree-like branched macromolecules, have received intense attention especially in the field of drug and gene delivery (1-5). On the basis of the delivery of modern organic synthetic chemistry, dendrimers having intriguing structures can be tailored in size, functional peripheral groups, and inner cavity for incorporation of a variety of molecules. Such custom-made dendrimers offer the design of novel types of delivery systems used for nanomedicines, with controlled interaction with cells and biological compounds. We have recently reported extensively on dendrimer porphyrins in which a porphyrin chromophore is spatially isolated by the aryl ether dendrimer framework (Figure 1) (69). The dendrimer porphyrins can transport absorbed energy to the porphyrin center over relatively large distance via the dendritic architecture, thereby mimicking the antenna complex and bacteriochlorophyll photo* Corresponding author. Phone: +81-3-5841-7138. Fax: +813-5841-7139. E-mail: [email protected]. † Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo. ‡ Present address: Philips Research, prof. Holstlaan 4, 5656 AA, Eindhoven, The Netherlands. E-mail:henk.stapert@ philips.com. § Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo. | Department of Bioorganic Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo.

Figure 1. Chemical structure of third-generation ionic dendrimer porphyrins. (32(+)DPZn, X ) CONH(CH2)2N+Me3Cl-; 32(-)DPZn, X ) COO-H+)

system (8, 9). On the other hand, a series of porphyrin compounds is known to effectively produce highly toxic singlet oxygen (1O2) through excitation by light of a characteristic wavelength, and some are being used as photosensitizers for photodynamic therapy (PDT). Conseqently, dendrimer porphyrins may have a potential as a novel type of photosensitizer used for PDT.

10.1021/bc025597h CCC: $25.00 © 2003 American Chemical Society Published on Web 12/19/2002

Light-Harvesting Ionic Dendrimer Porphyrins

PDT is a topical and promising method for the localized treatment of solid tumors, and an increasing number of the photosensitizers are presently being explored in the preclinical and clinical study (10-15). However, most of them cause toxic side effects such as prolonged skin photosensitivity due to lack of the specificity to tumors (16). The efficiency of photosensitizers in situ is most likely to be dependent on their local accumulation and specific cellular uptake in the tumor site, stimulating research toward the development of water-soluble and efficient in vivo sensitizer-delivery system with a high potential to target specific organs (17-20). Notably, the higher molecular weight of the dendrimer porphyrins are promising in this regard, since macromolecular compounds can preferentially accumulate in solid tumor due to the enhanced permeability of tumor vascular endothelium and the lack of functional lymphatic drainage in the tumor tissue, known as the so-called enhanced permeability and retention (EPR) effect (21, 22). In addition, being different from low molecular weight photosentizers currently in clinical use which exhibit nonspecific intracellular distribution, the dendrimer porphyrins with the relatively large size and three-dimensional structure are expected to be internalized in membrane-limited organelles, thereby achieving controlled localization in the intracellular compartment. Further, modification of the periphery of dendrimers with various ligands, including charged groups, sugars, and peptides, may open the way to design molecular-targeted PDT to accomplish highly selective death of tumorous cells with minimal side effects to the normal tissue. Here, we report for the first time the feasibility of dendrimer porphyrins with positive or negative charged groups on their periphery as an effective photosensitizer with little nonspecific, dark toxicity for PDT. MATERIALS AND METHODS

Photosensitizers. The synthesis and characterization of the ionic dendrimer porphyrins as given in Figure 1 have been reported previously (6-8). The third-generation aryl ether dendrimer with a Zn-porphyrin center and 32 positively charged (C(O)N(H)(CH2)2N+Me3) groups on its periphery and third-generation aryl ether dendrimer with a Zn-porphyrin center and 32 negatively charged (COO-) groups on its periphery are abbreviated to 32(+)DPZn and 32(-)DPZn, respectively (Figure 1). The protoporphyrin IX (PIX, 8,13-divinyl-3,7,12,17-tetramethyl-21H,23H-porphine-2,18-dipropionic acid) (Aldrich Chemical Co., Inc., U.S.A.) was used as a control photosensitizer in this study. Evaluation of Singlet Oxygen Quantum Yield. The singlet oxygen quantum yield was measured via direct observation of the near-infrared emission at 1268 nm, corresponding to the O2 (1∆g) f O2(3Σg-) transition (23). The experimental setup consists of Ar laser equipment (Innova 70-4; Coherent Inc., U.S.A.) and a near-infrared Ge detector (model 403HS; Applied Detector Co., U.S.A.) cooled by liquid nitrogen, connected to the exit slit of the monochromator (model CT10; JASCO, Japan) with a blaze wavelength at 1250 nm to minimize the grating loss. An IR-80 cutoff filter with 0% transmittance at less than 750 nm and 35% transmittance at 800 nm was placed at the entrance slit of the monochromator. A collecting lens focused the monochromator output onto the detector crystal. The Ar laser output at 514.5 nm was chopped with 800 Hz by an acousto-optic modulator (A160; HOYA, Japan) operated by a driver (110-DS; HOYA, Japan). The signal output from the Ge detector was fed into a model 124A lock-in amplifier via a model 116

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preamplifier (both from E.G. & G. Princeton Applied Research, U.S.A.) and synchronized with the internal reference signal of the lock-in amplifier. The signal output from the lock-in amplifier was fed to an XY recorder, and the emission spectrum was recorded by scanning the grating with a monitor. To minimize photobleaching, the solution was circulated at 4 mL/min using a peristaltic pump through a quartz flow cell (3 × 3 mm). MeOD was used as the solvent. Solutions were bubbled with pure O2 for 1 min before measurement. Molar absorption coefficients at 514.5 nm were determined in MeOD using a JASCO UV/VIS apparatus (model:Ubest-series V-550). Quantitative Analysis of Photosensitizers Associated with the Cells. Quantification of the amount of dendrimers or PIX associated with Lewis Lung Carcinoma (LLC) cells at 4 or 37 °C was performed by utilizing the fluorescence of PIX at 630 nm (excitation at 400 nm) and of 32(+)DPZn and 32(-)DPZn at 600 nm (excitation at 430 nm). Following exposure to dendrimers or PIX for 30 min, 3 h, and 8 h, the cells were washed three times with sterile PBS, harvested after treated with trypsinEDTA solution, and then dissolved in 20% SDS solution prior to fluorescence measurement (n ) 3). Fluorescent measurement was performed by JASCO spectrofluorometer FP-777 (Tokyo, Japan). Confocal Microscopy. For confocal microscopy, LLC cells were cultured onto fibronectin (from bovine plasma, Itoham Foods, Inc., Japan)-coated sterile 35 mm glassbase dishes (Iwaki Glass, Japan). The cells were then incubated with each photosensitizer. In the case of dendrimer porphyrins, the cells were coincubated with Tex-Red dextran (Molecular Probes, U.S.A.) as a neutral endocytosis marker (24). At definite time intervals, the cells were washed two times with sterile PBS, followed by confocal microscopy observation. Confocal microscopy was conducted using LSM 510 (Carl Zeiss Co., Ltd., Germany) at excitation wavelengths of 458 nm (Ar laser) for dendrimers and 543 nm (He-Ne laser) for Tex-Red dextran. Photoirradiation and Cell Viability Assay. The cytotoxicity of each photosensitizer in vitro was assessed against LLC cells. In the darkened room, photosensitizers with different concentration in medium (Dulbecco’s modified eagle’s medium (DMEM) + 10% Fetal Bovine Serum (FBS)) were added to cell solutions in 96-well culture plates (n ) 4). After a defined incubation time (30 min, 3 h, and 8 h) at either 4 or 37 °C, the photosensitizers were removed, and then the plates were photoirradiated for 10 min with broad-band visible light using a Xenon lamp (150 W) equipped with a filter passing light of 377700 nm (Estimated incident light irradiance: 150 mW/ cm2). The viability of photoirradiated and nonphotoirradiated cells was evaluated by using the mitochondrial respiration via the 3-(4,5-dimethyl thiazol-2-yl)-2,5diphenyltetrazolium bromide cleavage assay (MTT) following incubation for 24 h after photoirradiation. Detection of Photodamage by Fluorescent Probes. Photodamaged sites in the cell were detected by three different fluorescent probes, Rhodamine 123 (Rh123; excitation 450-490 nm, emission 520-600 nm), 1-(4trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene iodide (tma-DPH; excitation 330-380 nm, emission 420550 nm) (Dojindo Laboratories, Inc., Japan), and LysoTracker Red DND-99 (LysoTracker; excitation 577 nm, emission 590 nm) (Molecular Probes, U.S.A.), which is used for the staining of the mitochondrion, plasma membrane, and lysosome, respectively, in the living cell (25). Twenty minutes after irradiation, control or photo-

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Figure 2. Near-infrared singlet oxygen luminescence emission spectrum of PIX, 32(+)DPZn and 32(-)DPZn excited by Ar laser light of 514.5 nm with 300 mW output power. Table 1. The Efficiency of the 1O2 Emission at 1268 nm and the Absorption Coefficient at 514.5 nm of Each Photosensitizer  (L/mol) Rose Bengal PIX P32(+)DPZn P32(-)DPZn

31480 8705 6239 4849

1O

2 emission (V L mol-1)

I/ (V)

I/ (rel.)

257 ( 34 32.5 ( 7.7 25.1 ( 3.4 16.6 ( 2.3

8.2 ( 1.1 3.7 ( 0.9 4.0 ( 0.5 3.4 ( 0.5

1.00 0.45 0.49 0.41

damaged cells on fibronectin-coated sterile 35 mm glassbase dishes were incubated with 10 µg/mL Rh123 for 10 min or 25 µM LysoTracker for 40 min at 37 °C before the observation. To detect the plasma membrane photodamage, a drop of tma-DPH in dimethylformamide (DMF) (0.5 mg/mL) was added to control or photodamaged cells detached from the culture dish by cell scraper 20 min after irradiation. Fluorescent images were acquired using confocal microscopy (LSM 510, Carl Zeiss Co, Germany). RESULTS

Quantum Yield for Singlet Oxygen Production. To compare the singlet oxygen quantum yield, direct observation of the O2 (1∆g) f O2 (3Σ g-) transition at 1270 nm ((0,0) vibronic band) was performed for PIX, 32(+)DPZn, and 32(-)DPZn in MeOD excited by Ar laser light bundle of 514.5 nm with 300 mW output power. The emission intensity was linear with output power over the range of at least 0-300 mW. Figure 2 presents the near-

infrared singlet oxygen luminescence emission spectrum of the photosensitizers in MeOD. The linear relation between the emission intensity and the concentration of photosensitizers in the range of this measurement was confirmed for Rose Bengal, which was used as a reference. The emission efficiency defined as the molar ratio of the 1O2 emission intensity to the absorption coefficient at 514.5 nm of each photosensitizer is listed in Table 1. Small differences in the ratio of the 1O2 emission intensity to the absorption coefficient (I/) were observed between PIX and the dendrimer porphyrins. The observation that PIX and both dendrimers have similar singlet oxygen quantum efficiencies gave basis to directly compare the effects of 3D architecture and surface groups on the photodynamic activity of these sensitizers. Amount of Cellular Associated Photosensitizers. The time-dependent association of PIX and dendrimers with Lewis Lung Carcinoma (LLC) cells at different temperature (4 or 37 °C) was evaluated from their fluorescence and shown in panels A and B, respectively, of Figure 3. The amount of PIX associated with the cells increased linearly with the exposure time for both 4 and 37 °C; yet the slope is remarkably steeper for the latter condition (Figure 3A). The association profile of 32(+)DPZn comprised two-phases: the first temperatureindependent rapid association accomplished within 30 min, and the subsequent slow association particularly observed at 37 °C (Figure 3B). 32(-)DPZn showed a similar trend to 32(+)DPZn, although the associated amount was about one-order of magnitude lower (Figure 3B,C). Intracellular Localization. The intracellular localization of dendrimers in LLC cells was observed by confocal microscopy. Figure 4 shows the fluorescent image of PIX after incubation with LLC cells for 3 h at 37 °C. Diffuse PIX fluorescence was present throughout the cell except for the nuclei, which is consistent with the intracellular distribution of in situ biosynthesized PIX in ∆5-aminolevulinic acid (ALA)-based PDT, as previously reported by Madsen et al. (13). Confocal fluorescent images of Texas-Red dextran (red fluorescence) and 32(+)DPZn (green fluorescence) after incubation with LLC cells for 3 h at 37°C are shown in panels A and B, respectively, of Figure 5. Texas-Red dextran, which is known to be internalized by endocytosis leading to long time location in a lysosomal vesicle, was used as a marker molecule (24). The cellular nuclei appeared not fluorescent for both Texas-Red dextran and 32(+)DPZn, indicating their localization in the extranuclei region of the intracellular compartment. TexasRed dextran revealed the tiny dotted fluorescence around the perinuclear region, resulting from localization in

Figure 3. Amount of photosensitizers associated with LLC cells as a function of incubation time (O: PIX(4 °C); b: PIX(37 °C); 4: 32(+)DPZn(4 °C); 2: 32(+)DPZn(37 °C); 0: 32(-)DPZn(4 °C); 9: 32(-)DPZn(37 °C)).

Light-Harvesting Ionic Dendrimer Porphyrins

Figure 4. Confocal images of PIX in LLC cells after incubation of 3 h at 37 °C.

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Figure 6. The viability of LLC cells treated with PIX (b), 32(+)DPZn (2), and 32(-)DPZn (9) after photoirradiation and at incubation times of 30 min (A) or 8 h (B). Table 2. The in Vitro PDT Effect (1O2-Induced Toxicity) of PIX, 32(+)DPZn, and 32(-)DPZn in LLC Cell Linea IC50 (µM) 4 °C 37 °C

IC50(4 °C)/ IC50(37 °C)

PIX

30 min 3h 8h

2.48 1.02 1.33

1.84 0.980 0.503

1.34 1.04 2.64

P32(+)

30 min 3h 8h

0.0986 0.0330 0.0237

0.0469 0.0280 0.0250

2.10 1.18 0.95

P32(-)

30 min 3h 8h

>80.0 30.1 38.7

35.8 4.31 5.76

6.96 6.72

a All data were presented by 50% inhibitory concentration (IC ) 50 (unit: µM).

Figure 5. Confocal images of Tex-Red dextran (A) and 32(+)DPZn (B) in LLC cells after incubation of 3 h at 37 °C.

lysosomes (Figure 5A), and 32(+)DPZn showed a similar fluorescent image. The superposition of both fluorescent images revealed that 32(+)DPZn partially colocalized with Texas-Red dextran, suggesting that 32(+)DPZn is likely to be internalized via endocytosis, followed by localization in membrane-limited organelles such as lysosomes. 32(-)DPZn showed subcellular localization similar to that of 32(+)DPZn (data not shown). In Vitro PDT Effect. The viability of LLC cells upon photoirradiation was evaluated by MTT assay and determined as a function of the concentration and the exposure time of photosensitizers, PIX, 32(+)DPZn, and 32(-)DPZn. In this assay, photosensitizers were incubated with LLC cells for a definite time period (30 min, 3 h, and 8 h), and nonassociated photosensitizers were removed prior to photoirradiation. The cellular viability upon photoirradiation indicates the in vitro photodynamic (PD) effect (1O2 -induced toxicity). Panels A and B of Figure 6 illustrate the PD effect for LLC cells

incubated with an increasing concentration of PIX (circle), 32(+)DPZn (trigon), and 32(-)DPZn (square) for 30 min (A) and 8 h (B), respectively. Obviously, the PD effect observed decreased in the order of 32(+)DPZn, PIX, and 32(-)DPZn. Table 2 summarizes 50% inhibitory concentration (IC50) of each photosensitzer, which is defined as the concentration of photosensitizer at which fifty percent of tumor cells survive after photoirradiation, calculated from the cell viability-concentration curve (Figure 6). At 37 °C with 30 min and 8 h incubation, respectively, 32(+)DPZn has 39 and 20 times lower IC50 values than PIX and 763 and 230 times lower IC50 values than 32(-)DPZn, indicating a remarkably high PD efficiency of 32(+)DPZn. Notably, 32(+)DPZn achieved an extremely high PD effect even after a short exposure period (30 min), whereas the PD effect of 32(-)DPZn was low and appeared in a time-dependent manner particularly at 37 °C, where appreciable decrease in IC50 value by increasing temperature was observed. On the other hand, significant differences in IC50 between 4 and 37 °C were not observed for 32(+)DPZn, especially for the sample with prolonged incubation time, but were observed for 32(-)DPZn, as can be concluded from Table 2. It should be noted that the level of endocytotic activity of cells is greatly diminished at 4 °C. Dark Toxicity. It is of primary importance to evaluate the dark toxicity (inherent toxicity) of photosensitizers in long time exposure from the standpoint of their application in vivo. Therefore, each photosensitizer, PIX, 32(+)DPZn, and 32(-)DPZn, was examined in terms of IC50 values without photoirradiation after 72 h exposure in a dark room, and the results are shown in Table 3. 32(+)DPZn and 32(-)DPZn, respectively, exhibited 113and 157-fold lower dark toxicity than PIX, indicating the remarkably high light-induced toxicity of dendrimer porphyrins, especially for 32(+)DPZn.

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Figure 7. Photodamage from PIX and 32(+)DPZn in LLC cells as detected by three fluorescent probes: Rh123(A-C), LysoTracker (D-F), and tma-DPH (G-I). Control cells with photoirradiation alone are shown in panels A, D, and G, cells photodamaged with PIX in panels B, E, and H, and cells photodamaged with 32(+)DPZn in panel C, F, and I. Table 3. Dark Toxicity of PIX, 32(+)DPZn, and 32(-)DPZn in LLC Cell Line after Incubation of 72 h at 37 °C IC50 (µM)

PIX

P32(+)

P32(-)

0.200

22.6

31.3

Photodamaged Sites in the Cell. Control and photodamaged cells were stained with Rh123, LysoTracker, and tma-DPH to detect the mitochondrial, lysosomal, and plasma membrane photodamage, respectively. In this experiment, LLC cells were incubated with each photosensitizer at the concentration of 10-fold IC50 for a definite time period (30 min and 3 h) at 4 or 37 °C prior to photoirradiation. Note that the fluorescent patterns of every probe in control cells were not altered by photoirradiation alone. Fluorescent images of Rh123, LysoTracker, and tmaDPH-stained LLC cells are shown in panels A-C, D-F, and G-I, respectively, of Figure 7. Irradiated control cells without incubated photosensitizers showed spotted fluorescence of Rh123 and LysoTracker corresponding to the mitochondrion and lysosome, respectively (Figure 7A,D). The fluorescence of tma-DPH was mainly present at the cell surface (Figure 7G). After photodamage by PIX, diffuse fluorescence of Rh123 and LysoTracker (Figure 7B,E) and penetration of tma-DPH into submembrane

loci (Figure 7H) were detected, suggesting a disruption of mitochondrion, lysosome, and plasma membrane, respectively. On the other hand, photodamage by 32(+)DPZn seemed to induce no substantial change in the characteristic fluorescent pattern of each probe, except that a small portion of LysoTracker fluorescence became diffuse and the number of spotted fluorescence of Rh123 evidently decreased (Figure 7C,F,I). Photodamage by 32(-)DPZn rendered the fluorescent pattern of LysoTracker diffusive in small part similar to 32(+)DPZn but kept the characteristic fluorescent pattern of each probe, as observed in control cells (data not shown). DISCUSSION

32(+)DPZn and 32(-)DPZn aryl ether dendrimer zinc porphyrins both absorb light at 415, 434 (Soret bands), and 559 nm (Q-band) and emit fluorescent light at 610 and 660 nm. Due to their well-defined three-dimensional shape, which is morphologically trying to mimic biological light-harvesting antennae, they are of interest as artificial antennae for large distance energy transfer. Recently it has been shown that the efficiency of photoinduced energy transfer to the porphyrin core is enhanced via the rigid dendritic architecture (8, 9). The encapsulation of the reactive porphyrin center into a dendritic architecture

Light-Harvesting Ionic Dendrimer Porphyrins

was also expected to have additional interesting properties, like the possibility to influence the interactions of the ionic groups on the periphery with biocomponents. The above considerations initiate our interest to investigate the potential of porphyrin dendrimers as photosensitizers for PDT. We have chosen third-generation water soluble ionic dendrimers which have an interesting combination of their size (∼5 nm), surface charge, and water solubility (7). The time-dependent profile of 32(+)DPZn association with LLC cells comprised two distinct phases: first, temperature-independent rapid association accomplished within 30 min, followed by temperature-dependent slow association (Figure 3B). The first rapid association appears to correspond to 32(+)DPZn adsorption to the negatively charged plasma membrane through electrostatic interaction, and the second phase, which is apparent at 37 °C, is likely to be internalization of dendrimers by adsorptive endocytosis. Note that the size of dendrimer (∼5 nm) is obviously too large to diffuse through the plasma membrane. On the other hand, 32(-)DPZn showed a very low initial adsorption, which is less than 1/10 of the 32(+)DPZn adsorption. The slow and small increase in the associated amount after the initial adsorption was only observed at 37 °C (Figure 3C), suggesting that 32(-)DPZn is taken up by fluid-phase endocytosis. Both dendrimers internalized into the cells finally appear to localize in endosomal compartments because they colocalized with Tex-Red dextran, a fluidphase endocytic marker (Figure 5). The PD effect (1O2induced cytotoxicity) at 37 °C of 32(+)DPZn was 230 times higher than that of 32(-)DPZn (Table 2), while the associated amount of 32(+)DPZn was estimated to be only 22-25 times higher than that of 32(-)DPZn (Figure 3B), illustrating the higher PD efficiency of 32(+)DPZn compared to 32(-)DPZn. Because both compounds have similar efficiencies of singlet oxygen production, difference in the photoinduced cytotoxicity cannot solely be attributed to the different internalized amounts. The difference in the efficiency of PD effect between 32(+)DPZn and 32(-)DPZn should be due to the different interactions of both compounds with cellular components. 32(+)DPZn is likely to adsorb strongly with negatively charged membrane components, for example, glycoproteins, through electrostatic interactions, whereas negatively charged 32(-)DPZn seems not to be in a close contact with membrane components due to electrostatic repulsion. Consequently, the photodamage to the plasma membranes may be more vigorous for 32(+)DPZn as compared to 32(-)DPZn and may cause lipid peroxidation, protein cross-linking, and loss of ionic homeostasis (26-29). Further, the photodamage to the lysosomal membranes may cause release of hydrolases into the cytoplasm (30). The direct possible interaction of dye radicals with biocomponents can be excluded in the present case because the dendrimers have a relatively dense 3D structure in which the dye molecule (porphyrin) is protected by aryl ether dendrons. Thus, it is likely that only singlet oxygen or other reactive oxygenic species are responsible for the PD effect of dendrimer porphyrins. Singlet oxygen has a quite short lifetime, and the diffusion distance of 1O2 is estimated to be less than 10 nm (10). Nevertheless, the 3D strucuture of dendrimers with the size of 5 nm appears not to be a barrier for the 1 O2-induced PD effect, as 32(+)DPZn showed a remarkably high cytotoxicity upon photoirradiation. The protoporphyrin (PIX) used as a control in this study is one of primitive but effective photosensitizers well-studied by many research groups and is a component

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of protoheme (28, 29, 31-33). Excessive levels of PIX in circulating red blood cells cause erythropoietic protoporphyria, leading to cutaneous photosensitivity (33). The PIX, which is rather unpolar and only slightly soluble in water, may penetrate into cells through the plasma membrane by simple diffusion (Figure 3A) and partitions into the plasma as well as subcellular membrane components, including the mitochondrial, lysosomal, endoplasmic reticulum, and nuclear membranes (31, 32). Indeed, PIX showed a diffuse pattern of fluorescence throughout the cell except the nuclei. It seems that the mitochondrion is most likely susceptible to the PIXinduced photodamage (32), since PIX can be ligands for the mitochondrial peripheral benzodiazepine receptor (PBR) (10). The reason many PD efficient photosensitizers are hydrophobic appears to be because the plasma as well as subcellular membranes are critical sites for the 1O2-induced photodamage to the cells (25, 27, 33, 34). Worth noting is that 32(+)DPZn showed a 20 times higher PD effect than PIX (Table 2) even though the former cell association after 8 h incubation was 4.5 times lower (Figure 3A,B). These results suggest that 32(+)DPZn has a markedly enhanced efficiency of PD effect even compared with PIX, which should be explained by the their different localization in the plasma and subcellular membranes such as plasma membrane proteins, including transporters, ion channels, enzymes or cytoskeleton, lipids, etc. (26, 27, 35), as well as the differences in photodamaged sites in the organelles in the cells (25, 30, 31, 33, 34, 36, 37). Kochevar et al. demonstrated that the photodamaged sites and functions vary with the localization or distribution of the dye molecules in the cell membranes (27). PIX is known to accumulate primarily in the lipophilic compartments of the cell membranes (31, 32), while 32(+)DPZn appears to interact electrostatically with cell membranes, leading to different localization and/or distribution on the membranes as compared to PIX. Dendrimer porphyrins with a size of 5 nm are likely too large to penetrate into the cytoplasm, so that their internalization does occur in membranelimited organelles (Figure 5). Therefore, the PD effect of 32(+)DPZn may also be different from that of low molecular weight photosensitizers with a cationic charge, which have been shown to localize selectively within mitochondria leading to efficient cell death (10). Furthermore, 32(+)DPZn may overcome the drawback of low molecular weight, cationic photosensitizers, as they clear very rapidly from tumor tissues (38, 39). Macromolecules retain in tumor tissue at higher levels due to the EPR effect (21, 22). Recently, several studies have demonstrated that nucleic acid compounds can be an alternative potential target of 1O2-induced photodamage (40, 41). However, this mechanism seems to be unlikely for the PD effect of 32(+)DPZn because, as seen in Figure 5, fluorescence of 32(+)DPZn was hardly detectable in cellular nuclei. Although the exact molecular targets of 32(+)DPZn are an issue to be clarified in future, it is apparently important to control the intracellular disposition of the dye molecules based on the nature of the interaction forces to achieve an efficient PD effect. Photodamage by 32(+)DPZn maintained the characteristic structure of membrane and intracellular organelles (the plasma membrane, mitochondria, and lysosomes), whereas such organelles were severely photodamaged by PIX (Figure 7). Thus, the question is raised how photodamage by 32(+)DPZn nevertheless leads to the most efficient cell death. In this regard, the number of spotted fluorescence of the mitochondrion marker Rh123 decreased due to photodamage by 32(+)DPZn even

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after incubation of 30 min (Figure 7C). The fluorescent intensity of Rh123 is known to be correlated with the amount of adenosine triphosphate (ATP) in the cell (42). Therefore, photodamage by 32(+)DPZn may have clipped the ATP production in the cell, resulting in the efficient cell death. In general, the eliminated ATP production is characteristic for necrosis or caspase-independent programmed cell death (43, 44) (oncosis (45)). Similar necrosis-like cell death was previously observed when the plasma membrane was targeted as the photodamaged site (25, 34, 46). It was suggested that such necrotic process could be induced by photodynamically stimulating some critical targets on the plasma membrane, which results in a delayed or arrested apoptotic signaling pathway. The target sites are thought to be transporters, ion channels, or lipids rather than direct disruption of the outer cell membrane (25, 34, 46). This hypothesis appears to be consistent with our observation that the efficient PD-induced cell death combined with extremely low dark toxicity and the maintained characteristic organella structure after photoirradiation of 32(+)DPZntreated LLC cells. However, the mechanism of necrosislike cell death following photodamage to the plasma membrane remains to be studied. Elucidation of the PD mechanism of dendrimer-based photosentitizers is ongoing in our laboratory. PIX exhibited remarkably high dark toxicity after prolonged incubation of 72 h (Table 3). On the other hand, both dendrimers, 32(+)DPZn and 32(-)DPZn showed extremely low dark toxicity even after 72 h incubation. Upon prolonged incubation, PIX tends to localize largely in the mitochondrial membrane (32), causing mitochondrial dysfunction. Further, it can diffuse to every other organelle in the cytoplasm as shown in Figure 4 (13, 33). The high dark toxicity of PIX may result from such complexity in the intracellular localization. In contrast, the extremely low dark toxicity of dendrimer porphyrins may result from the distinctive intracellular disposition characteristics due to their relative large size and anionic or cationic periphery. Note that a low dark toxicity is one of the important criteria for assessing the usefulness of photosensitizers because unwanted toxicity to normal tissues is one of the major side effects in clinical PDT (33). The low dark toxicity of 32(+)DPZn is consistent with previous results obtained for cationic dendrimers used for gene transfection, which are known to have lower inherent cytotoxicity than other cationic linear polymers (5). The inherent low toxicity of dendrimers may originate from their monodispersed spherical nature as compared with conventional synthetic polymers having a three-dimensional configuration as well as chemical heterogenuity. Thus, the dendrimers are of great interest as the materials for drug delivery system (DDS). The porphyrin dendrimers used in this study have a maximum optical absorption at approximately 559 nm (Q-band), which has only limited tissue penetration efficiency. Therefore, the development of dendrimers having increased absorbance of light in the higher wavelength region may be desirable for clinical use. In this regard, synthesis of dendrimers encapsulating a metallo-phthalocyanine, which has an optical absorption at 675 nm where the light can penetrate the tissues approximately 2 times deeper than Photofrin (630 nm) (18), is in principle feasible in our laboratory (47). Alternatively, two-photon excitation by using nearinfrared lasers may have great potential for dendrimer porphyrin-based PDT of diseases in the deeper tissue (e.g., brain tumors) (48). The possibility of two photon absorption is known to depend linearly on the intrinsic

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two-photon cross section of the compounds, δ, and quadratically on the incident light intensity (48). Although the two-photon cross section of molecules is typically small compared with one-photon absorption, neodymium: YAG (Nd:YAG) laser emitting at 1,064 nm or tunable solid state lasers generating far-red/near-infrared light could significantly increase the possibility of two photon absorption of the compounds due to high laser output (49). On the other hand, substituting peripheral ionic groups of dendrimers with alternative ligands to target particular types of tumor cells is likely a key to performing the most efficient PDT (36, 50). This has wide-reaching implications for achieving efficient cell type-specific PDT because it will enable the active dose of dyes administered to the patients to be reduced. The development of appropriate in vivo carrier system of 32(+)DPZn is of primary importance for systemic PDT of solid tumor with suppression of side effects (17-20). The desirable properties of such in vivo carrier system are (i) improved watersolubility, (ii) specific accumulation in tumor, (iii) safety to the body, and (iv) prompt excretion from the body following PDT. To achieve these requirements, we have developed poly-ion complex (PIC) micelles entrapping dendrimer porphyrins in their core through the selfassociation of ionic dendrimer porphyrins with oppositely charged poly(ethylene glycol) PEG-poly(amino acid) block copolymers (51). In this way, PIC micelle carriers of dendrimer porphyrins, surrounded by a hydrophilic palisade of PEG strands, were prepared. As previously reported, several types of polymeric micelles with PEG shell were demonstrated to accumulate effectively and specifically in solid tumor (52-54). Thus, we expect PIC micelles with dendrimer porphyrins in the core may hold a promise for tumor-directed targeting of photosensitizers, and research in this direction is now ongoing in our laboratory. ACKNOWLEDGMENT

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