Preparation of Poly (ethylene glycol)-Attached Dendrimers

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Bioconjugate Chem. 2007, 18, 663−670

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Preparation of Poly(ethylene glycol)-Attached Dendrimers Encapsulating Photosensitizers for Application to Photodynamic Therapy Chie Kojima, Yoko Toi, Atsushi Harada, and Kenji Kono* Graduate School of Engineering, Osaka Prefecture University, Osaka, Japan. Received August 7, 2006; Revised Manuscript Received January 25, 2007

Photodynamic therapy (PDT) is a noninvasive treatment of some diseases including cancer. We have developed poly(ethylene glycol) (PEG)-attached dendrimers as a drug-carrier candidate. In this study, we prepared nanocapsules of photosensitizers using PEG-attached dendrimers for application to PDT. Two PEG-attached dendrimers derived from poly(amido amine) (PAMAM) and poly(propylene imine) (PPI) dendrimers (PEG-PAMAM and PEGPPI) were synthesized, and rose bengal (RB) and protoporphyrin IX (PpIX) were used as photosensitizers. Results showed that fewer PpIX molecules were encapsulated by both PEG-attached dendrimers than RB, but the complexes were more stable under physiological conditions. Furthermore, we demonstrated that PEG-PPI held photosensitizers in a more stable manner than PEG-PAMAM because of their inner hydrophobicity. We described the cytotoxicity of the complexes of photosensitizers induced by light irradiation in vitro. The complex of PpIX with PEG-PPI exhibited efficient cytotoxicity, compared with free PpIX. It was suggested that the cytotoxicity was caused by the high level of singlet oxygen production and the efficient delivery to mitochondria. Our results suggest that these PEG-attached dendrimers are a promising vehicle for PDT.

INTRODUCTION Photodynamic therapy (PDT) is a new clinical treatment of superficial tumors and age-related muscular degeneration. This technique involves the systemic administration of a photosensitive drug, by which singlet oxygen is generated after light irradiation, to engender oxidative damage to cells (1). This PDT affects only the irradiated areas because singlet oxygen is shortlived, thereby realizing a site-specific and noninvasive treatment. Photosensitizers play a crucial role in generating singlet oxygen in PDT. Porphyrin derivatives have been studied for decades as powerful photosensitizers. One of them, Photofrin, has already been approved for clinical application (2). A second generation of photosensitizers for accumulation at affected areas has also been prepared to enhance the potential of PDT, which is achieved using a drug carrier. Liposomes, with their high loading capacity and long circulation properties, are classically used as a drug vehicle that provides less adverse effects. In fact, a liposomal drug for PDT, Visudyne, has been marketed (3). Recently, many researchers have applied dendrimers to drug delivery systems (DDSs). Dendrimers have monodispersed molecular weight, well-defined morphology, and inner space to hold drug molecules. It has been suggested that dendrimer uniformity, along with their chemical and biological reproducibility, is useful for passive targeting to tissues involved in angiogenesis (4). Novel photosensitizers made from dendrimers have been reported by some groups. Kataoka et al. studied poly(aryl ether) dendrimers with a porphyrin core: so-called dendrimer porphyrins. They also prepared complexes of dendrimer porphyrins with block polymers containing poly(ethylene glycol) (PEG) as photosensitive biocompatible nanoparticles (5). As other types of photosensitive dendrimers, pheophorbide-a and a precursor of porphyrin, 5-aminolevulinic acid (ALA), were * To whom correspondence should be addressed: Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan. Tel.: +81 72 254 9330; Fax: +81 72 254 9330; E-mail: [email protected].

bound to the dendrimers in the periphery (6). It has also been reported that water-soluble dendrimers having a porphyrin core and other dyes in the periphery create two-photon excitation during low-energy light illumination (7). These dendrimers are attached covalently to photosensitizers, so that their photosensitive properties must change from their intrinsic ones. In addition, time and effort are required for synthesis of photosensitive dendrimers. Noncovalent encapsulation of photosensitizers by dendrimers can overcome these obstacles: photosensitizerencapsulating nanoparticles are applicable to a variety of photosensitizers and are also easily prepared. We have studied PEG-attached poly(amido amine) (PAMAM) dendrimers (PEG-PAMAM) as drug capsules of anticancer drugs such as adriamycin and methotrexate (8, 9). This kind of dendrimer cannot be removed from blood immediately, because it has been reported that PEG-modified macromolecules and liposomes are efficient at maintaining circulation in blood (10). These are applicable as vehicles against diseases involved in angiogenesis because of the enhanced permeability of macromolecules (4, 11). In our previous study, we synthesized several PAMAM dendrimers with different generations of dendrimer and different molecular weights of PEG. Results of that study showed that these dendrimers can encapsulate anticancer drugs. However, the drugs were easily dissociated from PEG-attached dendrimers under isotonic conditions (8). Anticancer drugs must be retained until they reach the tumor tissues and must be released to exhibit their action. Therefore, we prepared controlledrelease PEG-attached dendrimers (9). In this study, we prepared PEG-attached dendrimers encapsulating photosensitizers on PDT. They can act without release from carriers because reactive oxygen species generated from them, inside dendrimers, can injure cells. Therefore, photosensitizers are good candidates as guest molecules of PEG-attached dendrimers. We synthesized PEG-attached poly(propylene imine) (PPI) dendrimers (PEG-PPI) in addition to PEGPAMAM, both of which contain many inner tertiary amino groups. Two photosensitizers, rose bengal (RB) and protoporphyrin IX (PpIX), were used because they have acidic moieties

10.1021/bc060244u CCC: $37.00 © 2007 American Chemical Society Published on Web 03/22/2007

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Figure 1. Structures of RB (A), PpIX (B), and PEG-attached PAMAM (C) and PPI (D) dendrimers.

to facilitate interaction with amino groups of dendrimers (Figure 1). We investigated interaction of a couple of photosensitizers with these two PEG-attached dendrimers and compared the complexes’ chemical properties for PDT. We examined cytotoxicity of PEG-attached dendrimers encapsulating these photosensitive drugs after irradiation to evaluate the performance of a carrier of photosensitizers. Efficiency of singlet oxygen production by light irradiation and subcellular localization of photosensitizers influence the phototoxicity. Our results suggest that phototoxicity was affected by the stability, singlet oxygen generation, and the cytological behavior of the complex of PEGmodified dendrimers with photosensitizers.

EXPERIMENTAL PROCEDURES Materials. The PAMAM and PPI dendrimers, poly(ethylene glycol) monomethyl ether, 9,10-anthracene dipropionic acid (ADPA), and PpIX were purchased from Sigma-Aldrich Corp. (St. Louis, MO). From Wako Pure Chemical Industries, Ltd. (Osaka, Japan), RB, 3-(4,5-dimethyl-2-thiazoryl)-2,5-diphenyl2H-tetrazolium bromide (MTT) and paraformaldehyde were purchased. Also, 4-nitrophenyl chloroformate and 5-(dimethylamino)-1-naphthalenesulfonic acid (DNS) were obtained, respectively, from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), and Kanto Chemical Co., Inc. (Tokyo, Japan). MitoTracker Green FM was obtained from Invitrogen Corp. (Carlsbad, CA). Synthesis of PEG-Attached Dendrimers. We synthesized PEG-PAMAM as described in our previous report (8); PEGPPI was prepared according to our previous report, except the starting material. Briefly, poly(ethylene glycol) monomethyl ether with molecular weight of 2000 was reacted with 4-nitrophenyl chloroformate. It was then bound to PPI dendrimer of generation 5 (G5) with 64 amino groups at the periphery. Also, PEG-PPI was purified using a Sephadex G-75 column (Pharmacia) and dialysis. Yield: 577 mg (57.3%). σH (400 MHz, D2O): 1.68 (br, CH2CH2CH2N), 2.83 (br, CH2CH2CH2N, CH2CH2CH2N), 3.03 (br, CH2CH2CH2NHCOO), 3.22 (s, OCH3), 3.55 (m, CH2CH2O), 4.04 (br, NHCOOCH2). Gel Permeation Chromatography (GPC). The GPC system (Shodex SB-2004; Showa Denko K.K., Japan) had differential refractive index detection (RI-930; Jasco, Inc., Japan). Both PEG-PAMAM and PEG-PPI were eluted with 10 mM phosphate buffer containing 0.2 M sodium sulfate (pH 7.4) at 1.0 mL‚min-1 at 25 °C.

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For complex stability assay, we modified the system except the eluent buffer. The system was equipped with three columns, Shodex SB-803HQ, SB-804HQ (Showa Denko K.K., Japan), and TSK-gel G3000PW (Toso Co., Ltd., Japan), and two detectors, differential refractive index detector (RI-930; Jasco Inc., Japan) and fluorescence detector (λex ) 400 nm, λem ) 630 nm; FR-2020Plus; Jasco, Inc., Japan). Samples (10 µL) were injected and eluted at 0.5 mL‚min-1 at 30 °C. Hydrophobicity of PEG-Attached Dendrimers. PEGPAMAM or PEG-PPI was added to 1 µM of DNS dissolved in distilled water. The emission spectra excited at 310 nm were examined (12). Encapsulation of Photosensitive Drugs. Encapsulation assay was done according to our previous report (8). RB and PEGattached dendrimers were dissolved in water (first solvent) and incubated for 30 min at 27 °C. The solutions were lyophilized to remove water; then, chloroform (second solvent) was added. After 6 h incubation, the mixtures were centrifuged to remove free RB aggregation. The supernatants were evaporated and dried under vacuum; then, the residues were dissolved in 10 mM phosphate buffer containing 100 mM NaCl (third solvent). The absorbance was measured at 557 nm, an isosbestic point (9). In the case of PpIX, the treatment was almost identical, except for the solvents and UV-vis measurement. The first, second, and third solvents were replaced, respectively, by N,N′-dimethylformamide (DMF), water, and DMF. The absorbance was measured at 407 nm, the maximum wavelength of PpIX absorption spectra. We confirmed that the spectra in the absence and presence of dendrimers were the same above 350 nm. Complex Stability with PEG-Attached Dendrimers. The release of photosensitizers from PEG-attached dendrimers was estimated according to our previous report (8). Briefly, PEGattached dendrimers encapsulating RB or PpIX at 10 equiv per dendrimer were dissolved in phosphate buffer saline (PBS) and dialyzed (pore size: 120-140 kDa). The residues in the dialysis bag were monitored by measuring the absorbance at 557 nm (RB) or 407 nm (PpIX). The PpIX solutions sampled in the bag were diluted 20 times by DMF to measure the absorbance. The external solution was changed every 3 h. Free RB solution was analyzed as a control, but free PpIX was not attainable because of its poor water solubility. For complex stability assay in the presence of fetal bovine serum (FBS), the complexes of PpIX with PEG-attached dendrimers at 1 equiv per dendrimer were incubated in PBS containing various amounts of FBS for 3 h, and the solutions were centrifuged at 21 800 g for 20 min. The supernatants were used as samples for GPC analysis. Generation of Singlet Oxygen. We prepared PBS solutions containing 36 µM of ADPA and 10 µM of RB or PpIX in the absence or presence of PEG-attached dendrimers. These solutions were irradiated using a Xe lamp (400 W‚m-2, XEF-152S; San-Ei Electric Co., Ltd.) with a 530 nm filter. Then, the timedependent absorbance of ADPA was measured at 379 nm (7). Phototoxicity of PEG-Attached Dendrimers Loading PpIX. The HeLa cells (5 × 104 cells) incubated for 1 day in a 24-well plate were added to free photosensitizers and PEG-PAMAM and PEG-PPI loading them at different equivalents at a wide range of concentrations. After 3 h incubation, cells were washed twice with PBS and irradiated using a Xe lamp (400 W‚m-2, XEF-152S; San-Ei Electric Co., Ltd.) for 10 min, followed by 24 h incubation (6b). Then, surviving cells were evaluated using MTT assay (13). Photosensitizers in the absence and presence of dendrimers were dissolved in PBS, except free PpIX. Free PpIX was dissolved in distilled dimethylsulfoxide (DMSO). Under our conditions, neither DMSO nor light irradiation without photosensitive drugs influenced cell viability.

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Scheme 1

Subcellular Localization of PpIX with and without PEGAttached Dendrimers. The HeLa cells (1 × 105 cells) incubated for 1 day in 35 mm glass-bottom dishes were added to 1 µM of free PpIX and PpIX-loading PEG-PAMAM and PEG-PPI at 10 equiv per dendrimer. After 3 h incubation, cells were washed with PBS, fixed with 3.7% paraformaldehyde, and analyzed using laser confocal microscopy with the attached computer software (LSM 510 v 2.5; Carl Zeiss, Inc.) according to a procedure described in a previous report (14).

RESULTS Synthesis and Characterization of PEG-Attached PAMAM and PPI Dendrimers. PAMAM dendrimer of G4 and PPI dendrimer of G5, each with 64 termini, were reacted with PEG chain (molecular weight: 2000) according to Scheme 1. PEGPAMAM was already reported (8). As for PEG-PPI, the integral ratio of a typical signal of PEG chain (OCH3) to PPI dendrimer (NCH2CH2CH2) of the 1H NMR spectrum indicated that every terminal amino group was attached to PEG chains. In addition, a ninhydrin test of PEG-PPI was negative, indicating that these dendrimers have no primary amino groups. These dendrimers were characterized by GPC. Figure 2 showed a single peak, suggesting no contamination of noncovalent PEG chains. The elution volumes of PEG-PAMAM and PEG-PPI were almost equal, indicating that the hydrodynamic sizes are similar, even though intact PAMAM dendrimer of G4 was larger than PPI dendrimer of G5 (15). This fact suggested that these dendrimers extend to similar sizes because of steric hindrance and PEG chain solubility. We analyzed emission spectra of DNS to examine the hydrophobicity of the dendrimer interior (12). Emission spectrum of DNS is affected by the hydrophobicity of the surroundings. The blue shift of the emission maximum and the increase of fluorescence intensity are observed in the hydrophobic environment. The emission maximum of PEG-PPI was blueshifted to a greater extent, although that of PEG-PAMAM was blue-shifted only slightly, as shown in Figure 3A. The fluorescence intensity in the presence of PEG-PPI was also higher than that in PEG-PAMAM (Figure 3B). These facts indicate that PPI dendrimer is more hydrophobic than PAMAM dendrimer, as reported previously (16). Both PAMAM and PPI dendrimers have equal amounts of amino groups, but PAMAM

Figure 2. GPC profiles of PEG-attached PAMAM (top) and PPI (bottom) dendrimers. A peak of PEG with 2 kDa is apparent at 58.5 mL.

Figure 3. Comparison of the hydrophobicity of PEG-PAMAM (triangles) with that of PEG-PPI (squares). (A) Emission maximum. (B) Fluorescence intensity.

Figure 4. Encapsulation of RB (square) and PpIX (triangles) by PEGPAMAM (open) and PEG-PPI (closed) dendrimers.

dendrimer has amide groups. Amide groups of PAMAM dendrimer are expected to contribute to the interior hydrophilicity. Complex of Photosensitive Drugs with PEG-Attached Dendrimers. To evaluate PEG-attached dendrimers as a drug carrier of photosensitizers, we examined the binding property to photosensitizers such as RB and PpIX. Photosensitizers can be soluble even in a poor solvent, through complexation with dendrimers. We extracted complexes from poor solvents to estimate the numbers of photosensitizers bound to a dendrimer (8). We found that 182 and 177 molecules of RB and 20 and 30 molecules of PpIX were bound, respectively, per PEGPAMAM and PEG-PPI at the maximum (Figure 4). We reported that an important driving force of that binding is the electrostatic interaction between inner amino groups of dendrimers and carboxyl groups of guest molecules (8). This fact suggested that carboxyl groups of RB and PpIX interacted with inner tertiary amino groups of PEG-PPI and PEG-PAMAM via electrostatic interaction. Our results indicated that more RB molecules were encapsulated by PEG-attached dendrimers than

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by PpIX. Experimental conditions might contribute to this phenomenon: The complexes with RB were extracted using chloroform; PpIX was extracted using water. In a hydrophobic environment, electrostatic interaction between dendrimers and guest molecules is more effective: RB was complexed easily with dendrimers. In water, on the other hand, hydrophobic interaction among guest molecules is efficient; consequently, hydrophobic PpIX tends to interact with other PpIX molecules to engender their aggregation. We also found that PEG-PPI encapsulated more PpIX molecules than PEG-PAMAM. This result suggests that the association of PpIX with dendrimers in water is driven by hydrophobic interaction, consistent with the difference of hydrophobicity of PEG-attached dendrimers (Figure 3). We next investigated the release behavior of photosensitizers from these dendrimers by dialysis. Free drug molecules are permeable through a dialysis bag, but the complexes are not. Residual photosensitizers in the dialysis bag were detected using UV-vis absorbance (8). The RB complexes showed similar profiles to those of free RB, indicating that water-soluble RB molecules were released immediately in PBS solution (Figure 5A). In contrast, more than 50% of hydrophobic PpIX remained in PEG-attached dendrimers after 24 h (Figure 5B). Dialysis assay of PpIX did not obviate the possibility that aggregates of released PpIX remained in the dialysis bag. We performed another release assay to validate this release behavior of PpIX. Absorption of the supernatant of PpIX-loading PEG-attached dendrimers after centrifugation was measured at different time incubations because free PpIX molecules aggregate easily for removal. These results were almost identical to those of the dialysis assay (see Supporting Information). These release experiments suggest that the complexes of PpIX with PEGattached dendrimers were stable. Our results indicate that hydrophilic RB was released more rapidly from PEG-attached dendrimers in PBS than hydrophobic PpIX. As described previously, the complex formation was involved in electrostatic and hydrophobic interaction. Electrostatic interaction is suppressed under isotonic conditions. Therefore, hydrophobicity might influence the complexes’ stability. Figure 5 also shows that photosensitizers were released more easily from PEG-PAMAM than from PEG-PPI. That result is consistent with our results, which indicated that PEGPPI is more hydrophobic than PEG-PAMAM (Figure 3). The complex of PpIX with PEG-PPI was the most stable one assessed in this study. Therefore, we suggest that the hydrophobicity of host and guest molecules mainly influenced the complex stability in PBS. The influence of serum proteins on the complex stability is indispensable for application to PDT. We investigated the complex stability in the presence of serum proteins. The complexes were incubated in the serum-containing PBS for 3 h. Since it is well-known that PpIX molecule binds to serum proteins (17), PEG-attached dendrimers were separated from serum proteins using GPC, and then fluorescence intensity of PpIX associated with the PEG-attached dendrimer was measured. We found that serum proteins hardly promoted the dissociation of PpIX from PEG-PPI in the presence of 10% serum. A large fraction of PpIX molecules were still retained in the dendrimer after 3 h incubation in 50% serum. In contrast, PpIX molecules were readily released from the PEG-PAMAM even in the presence of 10% serum (Figure 5C). These results suggest that the complexes with PEG-PPI were much more stable than those with PEG-PAMAM in the presence of serum proteins. This stability difference might be related to the hydrophobicity of their interior: binding of PpIX to PEGPAMAM was too weak to maintain the complex due to its relatively hydrophilic interior. Therefore, we established stable

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Figure 5. Release of RB (A) and PpIX (B) from PEG-PAMAM (triangles) and PEG-PPI (squares) dendrimers in PBS. Residual photosensitizer (%) was calculated from absorbance of the complex solution in the dialysis bag. Profile for dendrimer-free RB is also shown (rhombi). (C) Effect of serum on release of PpIX from PEG-PAMAM (open) or PEG-PPI (closed). The complexes were incubated in PBS containing 0%, 10%, or 50% FBS for 3 h and were separated using GPC. Relative fluorescence intensity (%) of the complex was calculated by comparison with fluorescence intensity of the complex incubated in serum-free PBS.

complexes under physiological conditions using the combination of hydrophobic PpIX and PEG-PPI with hydrophobic interiors. Generation of Singlet Oxygen Induced by Light Irradiation. Photosensitive drugs generate singlet oxygen after light irradiation. We examined whether the complex formation affected the photosensitive properties of RB and PpIX using ADPA as a chemical trap. Photobleaching of anthracene moiety of ADPA is useful to monitor singlet oxygen generation (7, 18a). We found that RB encapsulated by PEG-attached dendrimers suppressed the decrease of the absorbance, indicating that the complex formation inhibited the production of singlet oxygen (Figure 6A). RB molecules might be quenched because of high local concentration inside dendrimers (6a). In contrast, free PpIX and the complex with PEG-PAMAM generated less singlet oxygen than the complex with PEGPPI (Figure 6B). Free PpIX molecules readily aggregate in PBS to impair the ability of photosensitizers, as described in previous reports (18). PpIX molecules in the complexes with PEG-PPI, however, are dispersible at the molecular level to maintain the

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Figure 6. Generation of singlet oxygen induced by RB (A) and PpIX (B) at various irradiation times. The free photosensitizer (rhombi) data and those of complexes with PEG-PAMAM (triangles) or PEG-PPI (squares) at 10 equiv per dendrimer are shown. Degradation of ADPA alone by irradiation is also shown as dotted lines.

photosensitive activity. Figure 6B might indicate that small PpIX aggregates existed in the presence of PEG-PAMAM. It is inferred that PpIX molecules interact with each other, even inside, because PAMAM dendrimers are more hydrophilic than PPI dendrimers (Figure 3). This suggestion is supported by some reports that porphyrin derivatives can aggregate in the complex with PAMAM dendrimers (19). We also performed ADPA analysis, by which PEG-attached dendrimers encapsulating PpIX at different equivalents were used. The result showed that greater amounts of PpIX per dendrimer generated less singlet oxygen, which might be attributable to quenching of the PpIX molecules inside dendrimers (see Supporting Information). Cytotoxicity of the Photosensitizers Encapsulated by PEGAttached Dendrimers. As an in vitro model of PDT, we examined the cytotoxicity of HeLa cells induced by photosensitizers after light irradiation (20). The HeLa cells were treated with photosensitizers encapsulated by PEG-attached PAMAM or PPI dendrimer as well as free ones for 3 h. Both PEG-attached dendrimers themselves exhibited no cytotoxicity under our conditions (data not shown). We found that free RB and the complex with PEG-attached dendrimers exhibited almost equal cytotoxicity to HeLa cells (see Supporting Information). These results are explained using the release profiles that RB molecules were easily released from PEG-attached dendrimers (Figure 5A). We also examined the cytotoxicity of PpIX in the absence and presence of PEG-attached dendrimers. First, we optimized the equivalent of PpIX molecules per dendrimer for cytotoxicity after light irradiation. As shown in panels A and B of Figure 7, the most effective phototoxicity was obtained for 10 molecules of PpIX encapsulated for each PEG-PAMAM and for each PEG-PPI. That result suggests that the balance of positive and negative factors on complex formation is important to exhibit efficient phototoxicity. The PEG-attached dendrimers with more

Figure 7. Phototoxicity of PpIX in HeLa cells. The complexes of PpIX with PEG-PAMAM (A) or PEG-PPI (B) were prepared at different equivalents (1 equiv (dash line), 10 equiv (closed), and 20 equiv (open)). The most effective complex with PEG-PAMAM (triangles) or PEGPPI (squares) dendrimers are shown in panel C. Free photosensitizer (rhombi) was also shown as a control. Cell viability was expressed as the percentage of the untreated control cells.

PpIX were best for the efficient delivery of PpIX into cells. On the other hand, more PpIX per dendrimer induced quenching of PpIX inside dendrimers, thereby suppressing the generation of singlet oxygen (see Supporting Information). Our data indicated that the complex at 10 equiv generated the greatest amount of singlet oxygen in cells. The most effective PpIX complexes with PEG-PAMAM and PEG-PPI were compared with free PpIX (Figure 7C). Results showed that free PpIX and the complex with PEG-PPI were more cytotoxic than the complex with PEG-PAMAM. The PpIX molecules were complexed stably with PEG-attached dendrimers, different from RB. Especially, 80% of PpIX molecules were still associated with PEG-PPI after 3 h (Figure 5B,C). In addition, PpIX molecules with PEG-PPI can generate more singlet oxygen (Figure 6B), suggesting that the complex with PEG-PPI can function as a photosensitive drug without releasing PpIX. Toxicity in the dark was also investigated. Without irradiation, 10 µM of RB solutions with and without PEG-attached dendrimers exhibited no toxicity (data not shown). However, PpIX exhibits some cytotoxicity (Table 1) even in the presence of PEG-attached dendrimers, consistent with previous reports (5a). For PDT, it is important to reduce cytotoxicity in dark conditions.

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Table 1. Viability (%) of Cells Treated with PpIX in the Dark complex PpIX

free

PEG-PAMAM

PEG-PPI

1 µM 10 µM

66.1 ((3.9) 27.4 ((1.1)

75.9 ((5.0) 40.1 ((0.1)

48.2 ((4.8) 30.5 ((2.4)

Subcellular Localization of PpIX. Intracellular localization of PpIX is a crucial feature of PDT. Singlet oxygen at mitochondria is known to induce apoptotic cell death (21). We observed localization to mitochondria in HeLa cells by staining with MitoTracker. As shown in Figure 8, PpIX molecules complexed with PEG-PPI were mostly merged in MitoTrackerpositive dots. On the other hand, for free molecules and the complex with PEG-PAMAM, PpIX molecules were partially localized at mitochondria. These data suggest that PpIX was localized to mitochondria efficiently by PEG-PPI. PpIX, a precursor of heme, is known to localize at mitochondria. However, it was reported that fewer exogenous PpIX molecules localize there in comparison to ALA-mediated endogenous PpIX (22). Moreover, few exogenous PpIX were accumulated at mitochondria after 3 h (23). It has also been reported that pegylation of a photosensitizer can be efficient for targeting mitochondria (24). These observations suggest that stable PpIX complexes with PEG-attached dendrimers were localized to mitochondria, but that free or released PpIX were not. The PpIX molecules complexed with PEG-PPI were more stable than with PEG-PAMAM, as shown in Figure 5B,C. Therefore, PEG-PPI can contribute to localization of PpIX molecules to mitochondria.

DISCUSSION We used two types of photosensitizers (RB and PpIX) and PEG-attached dendrimers (PEG-PAMAM and PEG-PPI) to produce complexes to apply to PDT. For RB, the complexes with PEG-attached PAMAM and PPI dendrimers were unstable under isotonic conditions. Figure 6A indicated that high local concentrations inside both PEG-attached dendrimers provide less

singlet oxygen, suggesting that the photosensitive action is controllable by regulating the release behavior of RB from dendrimers. Therefore, PEG-attached dendrimers with the ability of controlled release by bearing a disulfide and polymer network can be more useful as vehicles of RB for PDT (9). In the case of PpIX, we observed different phototoxicity of free dye from that of the complexes. Figure 7C indicated that the PpIX complex with PEG-PPI is more toxic after light irradiation than the complex with PEG-PAMAM. These results probably arose from the complex stability in serum-containing solutions, the productive rate of singlet oxygen and the subcellular localization of photosensitizers. As shown in Figure 5C, the complexes with PEG-PPI were much more stable than those with PEG-PAMAM in the presence of FBS. Because PpIX molecules were released from PEG-PAMAM during the incubation with cells, encapsulation of PpIX in PEG-PAMAM might not be effective for PDT. Demonstrably, encapsulation in PEG-PPI enhanced the generation of singlet oxygen (Figure 6B) and the localization of PpIX molecules at mitochondria (Figure 8) to engender effective phototoxicity. Although these dendrimers have the same numbers of primary and tertiary amino groups, their properties such as hydrophobicity and complex formation are different. Differences of their properties such as hydrophobicity and pKa might be attributable to the efficiency for PDT, which remains to be investigated in detail. The complex with PEG-PPI exhibited almost equal phototoxicity to that of free PpIX. It has already been reported that association of photosensitizers with cells is enhanced by hydrophobicity (23). In addition, it is well-understood that PEG chains abrogate the association with many biomolecules. These reports suggest that PEGylation negatively affects association with cells (10). Consistent with this notion, we found that the fluorescence intensity of free PpIX in HeLa cells was approximately 2-fold higher than that of the complex with PEGPPI (data not shown). However, free PpIX molecules generated less singlet oxygen than the complex with PEG-PPI (Figure 6B). In addition, they were not localized efficiently at mito-

Figure 8. Subcellular localization of free PpIX (A-C) and complexes of PpIX with PEG-PAMAM (D-F) or PEG-PPI (G-I). Here, PpIX is a red fluorescent dye (A,D,G), and mitochondria are labeled using MitoTracker green (B,E,H). Merged images of the left two columns are shown (C,F,I). These are representative images from more than 50 cells examined in each experiment. Bar, 10 µm.

Dendrimers for Photodynamic Therapy

chondria (Figure 8). Taken together, these results suggested that PpIX molecules complexed with PEG-PPI have phototoxicity attributable to the localization at mitochondria, along with more singlet oxygen, in spite of lower amounts of PpIX associated with cells. Although the cytotoxicity of PpIX in dark conditions was observed, toxicity might be reduced using ALA instead of PpIX (2, 6b, 22, 25), which remains to be improved. In conclusion, we synthesized and characterized two PEGattached dendrimers: PEG-PAMAM and PEG-PPI. We prepared complexes using those dendrimers and photosensitizers such as RB and PpIX. Results showed that PEG-PPI can encapsulate more photosensitizers and hold them inside under physiological conditions. Results also indicated that a hydrophobic photosensitizer, PpIX, can form a stable complex with PEG-PPI under physiological conditions, which suggests that the stable complexes were obtained through hydrophobic interaction rather than electrostatic interaction. Properties of complexes influenced the generation of singlet oxygen, subcellular localization, and the cytotoxic effect. Especially, we demonstrated the efficient phototoxicity of PpIX complexed with PEG-PPI via effective production of singlet oxygen and delivery to mitochondria. We demonstrated photosensitive activity of the complex of PpIX with PEG-attached dendrimers at the same level of free PpIX in vitro. This behavior is important for in vivo application because this kind of nanocapsule can be targeted to the affected tissues. Therefore, we propose that our PEG-attached dendrimers are a promising carrier for PDT.

ACKNOWLEDGMENT We are grateful to Hisataka Sabe (Osaka Bioscience Institute, Japan) for generous support in microscopic analysis. We thank Yasuhiro Haba for support in synthesis. This work was supported in part by Grants-in-aid from the Ministry of Education, Science, Sports and Culture of Japan, and Grants from Iketani Science and Technology Foundation and Saneyoshi Scholarship Foundation. Supporting Information Available: Additional experimental information. This material is available free of charge via the Internet at http://pubs.acs.org/BC.

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