Polycation Liposome Enhances the Endocytic Uptake of

University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan, and Department of Applied Chemistry, Faculty of. Engineering, Nagoya Institute of Technol...
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Bioconjugate Chem. 2003, 14, 790−796

Polycation Liposome Enhances the Endocytic Uptake of Photosensitizer into Cells in the Presence of Serum Yoshito Takeuchi,† Kohta Kurohane,‡ Kanae Ichikawa,† Sei Yonezawa,† Hidetsugu Ori,§ Takayuki Koishi,§ Mamoru Nango,§ and Naoto Oku†,* Department of Medical Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan, Department of Microbiology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan, and Department of Applied Chemistry, Faculty of Engineering, Nagoya Institute of Technology, Gokiso-cho, Nagoya 466-8555 Japan. Received December 10, 2002; Revised Manuscript Received April 10, 2003

To construct a novel drug delivery carrier that possesses high therapeutic efficacy with low dosage, we designed polyethylenimine-modified liposome (polycation liposome, PCL) and examined the entrapment of photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA), for antiangiogenic photodynamic therapy (PDT). Photosensitizer entrapped in PCLs showed enhanced phototoxicity for a human vascular endothelial cell line, ECV304, in comparison with that for nonmodified control liposome. Interestingly, phototoxicity of control liposomal BPD-MA was suppressed in the presence of serum, but PCL maintained the phototoxicity in the presence of serum following PCL-mediated PDT treatment due to the stability of PCL and the reduced detachment of encapsulated photosensitizer from liposome to serum. In fact, PCL enhanced the uptake level of BPD-MA to ECV304 cells despite the presence or absence of serum. Since polycation modification enhances bioavailability of the liposomal photosensitizer and this property is maintained in the presence of serum, PCL would be useful for antiangiogenic PDT.

INTRODUCTION

Cancer chemotherapy is generally accompanied by severe side effects such as bone marrow suppression. On the contrary, photodynamic therapy (PDT)1 is a modality of cancer treatments without such side effects, since a photosensitizer (such as a porphyrin, chlorin, or phthalocyanine derivatives) induces cytotoxicity only after exposure to laser light with a specific wavelength (1-4). Laser irradiation results in an induction of singlet oxygen to afford tumor destruction. In the case of hydrophobic macrocycles, it would be expected that biodistribution of administered photosensitizers in malignant tissues would be mediated by LDL in blood, selectively taken up in the tumor cells through the highly expressed LDL receptor (5-9). However, administered photosensitizer is bound by LDL and rapidly cleared by the reticuloendothelial system (RES), namely, liver and spleen, affording a reduction of the photosensitization level in the tumor. Therefore, in pursuit of a drug delivery system for such photosensitizers, lipid formulation of photosensitizers has been attempted (10-13). * To whom correspondence should be addressed. Tel: +8154-264-5701. Fax: +81-54-264-5705. E-mail: [email protected]. † Department of Medical Biochemistry, University of Shizuoka. ‡ Department of Microbiology, University of Shizuoka. § Nagoya Institute of Technology. 1 Abbreviations: BPD-MA, benzoporphyrin derivative monoacid ring A; cetyl-PEI, cetylated polyethylenimine; DPPC, dipalmitoylphosphatidylcholine; DPPG, dipalmitoylphosphatidylglycerol; ELS, electrophoretic light scattering; FBS, fetal bovine serum; LDL, low-density lipoprotein; PCL, polycation liposome; PDT, photodynamic therapy; PEI polyethylenimine; RES, reticuloendothelial system.

In the present study, we used BPD-MA (Chart 1) as a photosensitizer. The compound is also known as Verteporfin and Visudyne and is commercialized for the treatment of choroidal neovascularization of age-related macular degeneration. Photoactivation of BPD-MA induces local damage to neovascular endothelium, resulting in vessel occlusion (14). We previously observed that antiangiogenic PDT using BPD-MA caused blood flow stasis and significant regression of tumor in comparison with conventional PDT scheduling (laser irradiation at 3 or 4 h post BPD-MA administration) (15-19). Antiangiogenic PDT was achieved by laser irradiation at 5-15 min post BPD-MA administration, and the PDT treatment damaged angiogenic vascular endothelial cells rather than tumor cells (16). Our interest in antiangiogenic PDT treatment is further motivated by development of a novel liposomal formulation targeted to vasucular endothelial cells. For this purpose, we selected PCL as a carrier for a photosensitizer. Polyethylenimine (PEI) is frequently utilized as an effective nonviral DNA vector, both in vitro (20) and in vivo (21, 22), where compacted DNA is delivered to the cytoplasm via endosome by the proton-sponge effect (23). It would be expected that PEI would adhere to the anionic plasma membrane by electrostatic interaction, even in small doses, due to the concentrated positive charges afforded by the polymerization of amino groups. Furthermore, the polycation can give high tissue selectivity and low toxicity by chemical conjugation to biofunctionalized molecules such as oligopeptides and poly(ethylene glycol). PCL is a cationic polymer-coated liposome; specifically, cetylated PEI (cetyl-PEI, Chart 1) is located on the liposomal surface. Alkyl chains in cetyl-PEI contribute to the stability of the polycation because of the induction

10.1021/bc025648a CCC: $25.00 © 2003 American Chemical Society Published on Web 05/31/2003

Stability of Polycation Liposome against Serum

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Chart 1. Chemical Structures of Cetyl-PEI (left) and BPD-MA (right)

of hydrophobic and van der Waals interactions between the cetyl group and aliphatic hydrocarbons of lipids. We consider that PCL combines not only the above-mentioned advantages of both liposome and polycation but also enhanced endocytic uptake efficiency in the presence of serum (24, 25). In our recent study, we reported the high efficacy of BPD-MA-entrapped PCL (BPD-MA PCL) as a novel antiangiogenic liposomal drug for PDTmediated in vivo cancer treatment (26). The results indicated that strong suppression of tumor growth was observed by PCL-mediated PDT treatment, and the antitumor activity was induced by destruction of tumorderived neovasculature and subsequent apoptotic doom of tumor cells in comparison with those by BPD-MAentrapped nonmodified control liposomes (BPD-MA liposome). The the present study, we demonstrated the phototoxicity for the vascular endothelial cell line with BPD-MA PCLs and subsequent laser exposure. Furthermore, we described the uptake manner and the stability of liposomal structure against FBS using BPD-MA PCLs in comparison with that using BPD-MA liposomes. EXPERIMENTAL SECTION

Materials. PEI, consisting of 25%, 50%, and 25% for primary, secondary, and tertiary amino groups, respectively, with an average MW of 1800, was kindly provided by Nippon Shokubai Co., Ltd., Osaka, Japan. Polymer purification was performed by an ultrafiltration technique with an ultrafiltration apparatus equipped with a YM-1 ultrafiltration membrane (Amicon Inc., Beverly MA). 1H NMR spectra were recorded in CDCl3with a Valian Gemini-300 instrument with tetramethylsilane as an internal standard. The visible absorption spectra were recorded on a Beckman DU-70 spectrophotometer. The purified polycation was lyophilized and stored in EtOH before the preparation of cetyl-PEI. DC-Plstikfolein Kieselgel 60 F254 (Merck, Darmstadt, Germany) was used for analytical TLC. Dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) were kindly provided by Nippon Fine Chemical Co., Ltd., Takasago, Hyogo, Japan, and cholesterol was purchased from Sigma Chemical Co., St. Louis, MO. BPD-MA and [14C]BPD-MA were generous gifts from QLT PhotoTherapeutics, Inc., Vancouver, British Columbia, Canada. Preparation of Cetylated Polyethylenimine (cetylPEI). Cetyl-PEI was prepared by a similar procedure as described previously (24, 25). In brief, PEI (2.40 g, 1.33 × 10-3 mol) was stirred at 65 °C in EtOH (10.0 mL) for 30 min under reflux conditions and N2 bubbling, and to

the solution were added 1-bromohexadecane (3.87 mL, 1.33 × 10-2 mol) and triethylamine (223 µL, 1.60 × 10-3 mol). After a 7-h reaction, the solution was evaporated. The resulting solid was dissolved in 20% EtOH aqueous solution (100 mL), and the solution was ultrafiltered for 2 days, using more 20% EtOH aqueous solution (over 1000 mL). Finally, the solution was lyophilized. The stoichiometric conjugation percentage of the cetyl group, determined by 1H NMR spectra, was 24.1% per total PEI amino groups. 1H NMR (CDCl3): δ 0.82-0.95 (10H, t, cetyl CH3), 1.13-1.44 (101H, br, cetyl CH2), 2.36-3.19 (167H, br, CH2), 4.84-5.26 (32H, br, PEI amine H). Preparation of BPD-MA Liposome and BPD-MA PCL. BPD-MA liposomes that were not modified cetylPEI were prepared as described previously (26). BPDMA liposome consists of DPPC, cholesterol, DPPG, and BPD-MA (20/10/5/0.3 as molar ratio, respectively); on the contrary, BPD-MA PCL additionally contains cetyl-PEI at a molar ratio of 0.175, 0.875, 1.75, and 3.5 (total lipids/ cetyl-PEI molar ratio: 20/0.1, 20/0.5, 20/1, 20/2, respectively). Lipids, BPD-MA, and cetyl-PEI that were dissolved in CHCl3 were evaporated to construct the thin lipid film and hydrated with PBS (300 nM as the final concentration of BPD-MA). The liposomal solution was freeze-thawed by using liquid N2 and sonicated for 15 min at 60 °C. Finally, these liposomes were sized at 100-nm diameter by extrusion through a polycarbonate membrane filter under 15 kgf/cm2 of N2 pressure. The BPDMA and phospholipid concentrations were determined before and after the extrusion step. The result supports the successful encapsulation of BPD-MA (over 98%). BPD-MA concentration was determined following extrusion by measuring the absorbance at 688 nm. Cell Culture. The human vascular endothelial cell line ECV304 was used for in vitro investigations (27, 28). ECV304 cells (1 × 105 cells) were seeded in 35-mm cell culture dishes in 199 medium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 10% heatinactivated FBS (Sigma Chemical Co., St. Louis, MO) and incubated in a 5% CO2 incubator for 48 h at 37 °C. PDT Treatment in Vitro. The medium in the cell culture dish was removed, and 199 medium supplemented with 10% heat-inactivated FBS (990 µL) was added. BPD-MA liposomes or BPD-MA PCLs (10 µL) were added (final 30-500 ng/mL in terms of BPD-MA) and incubated for 60 min. PDT treatment was performed with irradiation from the top-side of the cell culture dish using a diode-laser system, SP689 (Suzuki Motor Co., Ltd., Yokohama, Japan). The cells that were incubated with photosensitizer-entrapped liposomes were exposed

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to the laser light with 2.0 J/cm2 of fluence (0.25 W, 76.9 s) at 689 nm. At 24 h after PDT treatment, the cells were washed with PBS for the removal of destroyed cells. Viable cells were determined by crystal violet dye assay. The cells were soaked in 0.5% crystal violet solution (dissolved in MeOH/H2O ) 1/4 (v/v)) for 10 min following the removal of the redundant dying solution by washing in PBS attentively. The dishes were completely dried, and 33% aqueous AcOH solution (1 mL) was added to elute dye from the stained cells. Survival percentage of the cells was spectroscopically quantified by the absorbance at 630 nm, and the parameters were normalized to a control set of cells without liposomal photosensitizer treatment and laser exposure. ELS Measurement. Preparation of BPD-MA liposomes and BPD-MA PCLs 20/1 were followed as described above. After being sized at 100-nm in diameter, the liposomal solutions were incubated in the presence and absence of an equal volume of FBS at 37 °C for 3 h. Particle sizes and ζ-potentials of BPD-MA liposomes and BPD-MA PCLs 20/1 were recorded on a ELS-800 electrophoretic light scattering spectrophotometer (Otsuka Electronics Co., Ltd., Osaka, Japan). The results supported the assigned liposomal sizes in saline-hydrated liposomes, and the ζ-potentials were +5.06 mV for BPDMA liposome and +32.53 mV for BPD-MA PCL 20/1. Spin Column Assay. The assay was followed Chonn’s method (29). [14C]BPD-MA liposomes and [14C]BPD-MA PCLs that were hydrated with 0.3 M glucose were incubated in the presence of an equal volumes of FBS or HEPES at 37 °C for 30 min. Bio-Gel A-15 m, agarose beads 200-400 mesh (Bio-Rad Laboratories, CA), was eluted with saline three times and filled into a 1-mL cylinder with cotton cap. A spin column was prepared by centrifugation of the cylinder at 800 rpm for 30 s. The incubated liposomal samples (100 µL) were applied to the spin column, and the first fraction was collected by centrifugation at 500 rpm for 30 s (n ) 3). After collection of the first fraction, saline (100 µL) was applied to the spin column, and it was centrifuged at 500 rpm for 30 s. The procedure was repeated until the 12th fraction eluted. Several fractions (50 µL) were mixed with 10 mL of Ultima-Fluor (Packard Japan, Tokyo, Japan), and the radioactivity of the 14C-labeled macrocycles was determined with an Aloka LSC-3500 liquid scintillation counter. Cellular Uptake of BPD-MA. ECV304 cells (3 × 105 cells) were seeded in 60-mm cell culture dishes and incubated in a 5% CO2 incubator for 48 h at 37 °C. The medium in the cell culture dish was changed to fresh 199 medium supplemented with 10% heat-inactivated FBS (2.97 mL). BPD-MA liposomes or BPD-MA PCLs (30 µL) were added (100 ng/mL as final concentration of BPDMA) and incubated for 60 min. After the cells were washed with ice-cold PBS, they were dissolved in 0.1% SDS-containing 5 mM Tris buffer (1.2 mL), and the amount of BPD-MA was fluorometrically determined with an excitation wavelength of 450 nm and emission at 619.4 nm by a Hitachi F-4010 fluorescence spectrophotometer. Quantification of cell protein concentration was determined by BCA protein assay (Pierce Chemical Company, IL). Thus, BPD-MA uptake was quantatively evaluated and represented as the amount of BPD-MA per cell protein. RESULTS

Cetyl-PEI was prepared using PEI and 1-bromohexadecane in EtOH at 65 °C for 6-9 h in the presence of

Takeuchi et al.

Figure 1. Phototoxicity of BPD-MA against ECV304 cells following PDT treatment in the presence of BPD-MA liposomes or BPD-MA PCLs. ECV304 cells were incubated in the presence of BPD-MA liposomes or BPD-MA PCLs 20/1 at selected BPDMA concentrations for 60 min, and irradiated with 689-nm laser light (2.0 J/cm2). Viable cells were determined by crystal violet dye assay at 24 h after laser exposure. Data points represent the mean ( SD of the relative absorbance at 630 nm in comparison with that of PDT-nontreated control in the absence of BPD-MA liposomes or BPD-MA PCLs (n ) 3). Asterisks and crosses indicate P vs control and an equal concentration of BPDMA liposomes, respectively: * and +, P < 0.05; ** and ++, P < 0.01; *** and +++, P < 0.001.

triethylamine. The grafting reaction was performed under N2 atmosphere. After the reaction, the solvent was removed by evaporation and dissolved in 20% EtOH aqueous solution. Ungrafted compounds were removed by ultrafiltration technique for 2 days by filtering with 10 volumes of 20% EtOH aqueous solution. Finally, the unfiltered solution was lyophilized, resulting in a white powder. The resulting solid was soluble in CHCl3 and partly soluble in H2O, so that preparation of PCL is enabled. The grafted percentage of cetyl group was determined by measuring the 1H NMR spectra in CDCl3. The spectra supported the assigned structure, and the stoichiometric grafting of cetyl groups was achieved. Seven kinds of cetyl-PEIs were synthesized with varying MW of PEI (600, 1800, and 25000) and grafted percentage of cetyl group (5 to 24% per ethylenimine unit). To optimize the MW and the grafted percentage of cetyl-PEI, PCLs were prepared using the seven types of cetyl-PEI, and the availability was determined by gene transfer assay to various cells in the presence of serum. In particular, the most effective transfection was shown by using cetyl-PEI with MW 1800 of PEI and 24% grafted percentage of cetyl group (data not shown), so that this cetyl-PEI was used for preparing PCL (24, 25). BPD-MA PCL Enhanced the Phototoxicity against Human Endothelium Cell Line. Initially, the phototoxic action of BPD-MA against a human endothelial cell line, ECV304 cells, was examined by use of BPD-MA liposomes or BPD-MA PCLs 20/1. Figure 1 shows the survival for ECV304 cells in the presence of BPD-MA liposomes or BPD-MA PCLs 20/1 after laser exposure. In the range of 30-300 ng/mL in terms of BPD-MA concentration, the cells that were treated using BPD-MA PCLs 20/1 were efficiently destroyed following PDT treatment in comparison with those using an equal concentration of BPD-MA liposomes: The phototoxicity in the presence of BPD-MA PCLs 20/1 corresponded to approximately twice the dose of BPD-MA liposomes (LD50: 247.7 ng/mL for BPD-MA liposomes and 146.1 ng/ mL for BPD-MA PCLs 20/1). To verify the direct cytotoxic action of BPD-MA liposomes and BPD-MA PCLs, a large amount of photosen-

Stability of Polycation Liposome against Serum

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Table 1. Stability of BPD-MA Liposome and BPD-MA PCL 20/1 in the Presence of Serum recovery % by spin column assayb BPD-MA condition

serum

liposome liposome PCL 20/1 PCL 20/1

none 50% FBS none 50% FBS

ELS measurementa liposomal serum-bound particle size (nm) fraction fraction 113.7 ( 17 228.3 ( 35 103.3 ( 17 189.9 ( 30

100 010 0091c 0074c

89 21

a All liposomes were sized at 100-nm diameter by extrusion and subsequent incubation in saline with or without an equal volume of FBS for 3 h at 37 °C. b All liposomes were sized at 100-nm diameter by extrusion and subsequent incubation in 0.15 M glucose with an equal volume of 5 mM HEPES buffer or FBS for 30 min at 37 °C. To gain the accurate recovery of liposomal and serum-bound photosensitizers, percentage of the fractions were determined by calculating the Gaussian distribution on the basis of the elution profile. c Accurate quantification was not possible due to the strong adsorption of PCLs to agarose beads.

sitizers entrapped in PEI-nonmodified liposomes or PCLs (500 ng/mL in terms of BPD-MA) were applied to ECV304 cells without subsequent laser exposure. The result showed that survival for ECV304 cells was not affected, indicating no cytotoxicity of BPD-MA and PCL (data not shown). Furthermore, cytotoxicity was also not observed by laser exposure alone (2 J/cm2). Stability of Liposomal Photosensitizers in the Presence of Serum. We considered that serum protein binding to liposomes resulting in aggregation of liposomes and detachment of entrapped hydrophobic drugs may occur in the presence of serum. To ascertain the stability of BPD-MA liposomes or BPD-MA PCLs in the presence of serum, electrophoretic light scattering (ELS) measurement and a spin column assay29 were performed. Initially, particle size changes of BPD-MA liposomes or BPD-MA PCLs 20/1 were evaluated after presizing at 100-nm diameter and subsequent incubation with or without 50% FBS at 37 °C for 3 h. As shown in Table 1, the ELS result indicated that the sizes of BPD-MA liposomes were determined as 228.3 ( 35 and 113.7 ( 17 nm in the presence and absence of FBS, respectively. Similarly, those of BPD-MA PCLs 20/1 were 103.3 ( 17 nm in the absence and 189.9 ( 30 nm in the presence of FBS: The aggregation of PCLs in the presence of serum was only control level. The transfer of BPD-MA from liposome or PCL was examined by a spin column assay (Table 1). The spin column chromatogram of two types of liposomal photosensitizers preincubated in HEPES-buffered glucose solution indicated that the liposomal [14C]BPD-MA was eluted only in the void volume (data not shown). Recovery of [14C]BPD-MA was 99% for [14C]BPD-MA liposomes, although that was only 21% for [14C]BPD-MA PCLs 20/ 1. The latter evidence indicates that PCLs absorbed strongly to agarose gel, and [14C]BPD-MA entrapped in PCLs was strongly associated to PCL, association of which might be mediated by PEI in PCLs; on the contrary, after incubation of [14C]BPD-MA liposomes or [14C]BPD-MA PCLs with 50% FBS for 30 min, 80% or 19% of [14C]BPD-MA was eluted at the lipoprotein fractions, respectively. Cellular Uptake of BPD-MA. Since the cellular uptake of liposomal BPD-MA is a potent factor for susceptibility to PDT, BPD-MA uptake level was determined by fluorescence emission spectroscopy. Fluorescence intensity of BPD-MA in the cellular fraction was proportionally increased with increasing BPD-MA concentration in the presence of either BPD-MA liposomes

Figure 2. BPD-MA uptake level against ECV304 cells following addition of BPD-MA. ECV304 cells (3 × 105 cells) were incubated with BPD-MA liposomes or BPD-MA PCLs 20/1 (100 ng/mL in terms of BPD-MA) for 60 min at 37 °C in serum-free, 10% heat-inactivated FBS-containing, and 1.92 mg/mL of BSAcontaining 199 media (A). BPD-MA uptake was also determined after addition of BPD-MA liposomes or BPD-MA PCLs in the presence of 10% heat-inactivated FBS at different incubation temperature (B). BPD-MA fluorescence levels in cell lysate (mean ( SD) were monitored by the fluorescence intensity at 691.4 nm with 450-nm excitation and normalized by the cell protein concentration determined by BCA assay.

or BPD-MA PCLs 20/1 (data not shown). Quantitative data of the incorporated photosensitizer in the presence or absence of 10% FBS is shown in Figure 2. As is apparent from Figure 2A, PCLs enhanced the BPD-MA uptake in comparison with PEI-nonmodified liposome regardless of the presence of FBS: The uptake level was increased approximately 1.5-fold in the absence of serum and 3.1-fold in the presence of 10% FBS. Furthermore, inhibition of BPD-MA uptake by FBS was reduced by using PCLs: 81.5% inhibition for BPD-MA liposomes and 61.5% inhibition for BPD-MA PCLs 20/1 in comparison with the level in the absence of serum. It is also clarified that BSA, the most abundant protein in FBS, suppressed the photosensitizer uptake to some extent. We next examined the mechanism of BPD-MA uptake by the cells. The percentage of incorporated BPD-MA per initial amount of BPD-MA was 10.9% for PCLs 20/1 and 3.4% for PEI-unmodified liposomes after incubation at 37°C in the presence of 10% FBS, indicating that BPDMA uptake was improved about 3-fold by PCL formulation (Figure 2B); on the contrary, a remarkable reduction of uptake was observed in both BPD-MA liposomes or BPD-MA PCLs at the incubation temperature of 20 and 4 °C. Influence of Serum on the Phototoxicity by BPDMA PCLs. Finally we examined the phototoxicity of BPD-MA in the formulations of liposomes or PCLs in the presence of serum while changing the modified cetyl-PEI molar ratio. As shown in Figure 3, the assay revealed

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Figure 3. Persistence of phototoxic efficiency using BPD-MA liposomes or BPD-MA PCLs in the presence of serum. ECV304 cells were incubated in the presence of BPD-MA liposomes or BPD-MA PCLs 20/1 for 60 min in the presence and absence of 10% heat-inactivated FBS, and irradiated with 689-nm laser light (2.0 J/cm2). Viable cells were determined by crystal violet dye assay following 24 h after laser exposure. Data points represent the mean ( SD of the relative absorbance at 630 nm in comparison with that of PDT-nontreated control in the absence of BPD-MA liposomes or BPD-MA PCLs 20/1 (n ) 3). Asterisks and crosses indicate P vs control and BPD-MA liposomes, respectively: *, P < 0.05; ** and ++, P < 0.01; *** and +++, P < 0.001.

that approximately 70% phototoxicity was observed following PDT treatment in the absence of serum regardless of the cetyl-PEI molar ratio in PCLs. However strong suppression of phototoxicity was observed in the presence of 10% FBS when liposomal formulation was used. Interestingly, the suppression of phototoxicity was gradually canceled with increasing cetyl-PEI molar ratio. DISCUSSION

We developed BPD-MA PCL as an angiogenic vasculature-targeted drug carrier for PDT usage. Our previous in vivo study indicated that PCL enhances strong suppression of tumor growth following PDT treatment (26). In this paper, we described the effect of serum on the phototoxicity of liposomal photosensitizer-mediated PDT treatment to vascular endothelial cells. We designed PCL to be taken up by the target cells efficiently: The cationic liposomal surface of PCL enhances the adherence to plasma membrane of vascular endothelial cells and accelerates the entry of PCL into cytosol via endocytic pathway (24, 25). In fact, BPD-MA PCL-mediated PDT caused strong cytotoxicity against a vascular endothelial cell line, ECV304 cells, in comparison with the nonmodified liposomes-mediated one (Figure 1). Since BPD-MA PCLs without laser exposure did not affect the cell viability, the cytotoxic action is not due to the toxicity of BPD-MA or PCL but due to the photoreaction of BPD-MA. Uptake of liposomal drugs is frequently inhibited in the presence of serum. It is possible that administered liposomes encountered serum protein binding (30), resulting in aggregation of liposomes and detachment of entrapped hydrophobic drugs. Therefore, the aggregation of liposomes or PCLs in the presence of serum was determined by ELS measurement, and detachment level of entrapped photosensitizers was examined by a spin column assay (24). Generally, serum-mediated aggregation of liposomes induces an enormous size of concretions. As is apparent from Table 1, no significant change of liposomal size was observed, regardless of polycation modification and presence of serum. When the aggregation manner is taken into account, undesirable aggrega-

Takeuchi et al.

tion of liposomes does not occur in the presence of serum. The spin column assay revealed that PCLs decreased the detachment level of entrapped [14C]BPD-MA and almost all photosensitizers entrapped in PCLs retained on the agarose beads; on the contrary, photosensitizer clearance occurred primarily with nonmodified liposomes (Table 1). This result suggests that entrapped macrocycles are easily transferred from liposome to lipoproteins and that polycation modification of liposome suppresses the transfer. It is possible that PEI in PCL directly interacted with BPD-MA and suppressed the dissociation. Alternatively, polycation coating of the liposomal surface may increase the stability of liposomes, which caused stable entrapment of the photosensitizer in PCL. It is considered that adsorption of BPD-MA on the agarose beads results in the strong interaction between photosensitizer and polycation. From the results of the BPD-MA uptake assay, the uptake level was enhanced by using BPD-MA PCLs. The uptake efficiency decreased in the presence of serum regardless of the type of liposomes. To elucidate the serum factors that suppress the uptake of BPD-MA, we examined the effect of albumin, a major serum protein, and observed that albumin also suppressed the uptake (Figure 2A). Therefore, albumin is one of the possible factors affecting the uptake. Interestingly, the suppression level of photosensitizer uptake by both BSA and FBS was reduced in BPD-MA PCLs in comparison with BPDMA entrapped in nonmodified liposomes. The strong electrostatic interaction of polycation with the plasma membrane of vascular endothelial cells might overcome the suppression of the uptake by the serum factor(s). Besides the albumin, serum lipoprotein would affect the uptake of BPD-MA in liposomes, since a part of BPDMA was transferred to lipoproteins especially in the presence of serum. However, the spin column data suggested that the transfer of BPD-MA to lipoprotein is suppressed by PCL formulation. To examine the mechanism of incorporation of BPDMA entrapped in nonmodified liposomes or PCLs into the cells, BPD-MA uptake was monitored at various temperatures. Figure 2B shows that enhanced photosensitizer uptake was observed at 37 °C; on the contrary, those at 20 and 4 °C were significantly suppressed. The results suggested that the BPD-MA was mainly taken up in the cells via the endocytic pathway. The amount of BPD-MA in the cellular fraction after incubation at 4 °C might reflect the bound BPD-MA on the cellular surface. From the quantitative results of Figure 2B, the endocytic BPDMA uptake was 2.6% for BPD-MA liposomes and 9.6% for BPD-MA PCLs, and the adhered BPD-MA on the plasma membrane was calculated as 0.8% for BPD-MA liposomes and 1.2% for BPD-MA PCLs per injected BPDMA concentration. The result also supported the enhancement of BPD-MA uptake by use of PCL. The phototoxicity following BPD-MA PCL-mediated PDT treatment was on the level of that using polycationnonmodified control liposomes until 30 min, but significant enhancement of phototoxicity by using BPD-MA PCLs was observed after over a 30-min incubation (data not shown). The results indicated that the enhanced phototoxicity by using BPD-MA PCLs contributes to the adherence of BPD-MA PCLs to plasma membrane by electrostatic interaction and subsequent effective uptake of photosensitizer (31). The reduced phototoxicity for ECV304 cells in the presence of serum was canceled with increasing amount of cetyl-PEI modified on the liposomal surface (Figure 3). The result indicates that stability of BPD-MA en-

Stability of Polycation Liposome against Serum

trapped in PCLs against serum is dependent on the modification of polycation, and that is strongly reflected in the phototoxicity following laser exposure. Taken together, BPD-MA PCL enhances the phototoxicity for ECV304 cells. We considered that the efficacy is induced by not only the facilitation of endocytic BPDMA uptake by the strong electrophoretic interaction with the plasma membrane but also the persistence of the liposomal structure against serum factor(s) in comparison with nonmodified control liposome: PCL inhibits the detachment of encapsulated photosensitizer in the presence of serum. From the results of cellular uptake of BPD-MA, PCL may maintain the BPD-MA entrapment and the strong interaction with the cellular membrane. Since the cellular uptake of BPD-MA is strongly related to the phototoxicity, the phototoxicity of BPD-MA in the PCL formulation is not abolished in the presence of serum. Leunig et al. (32) have reported that the high uptake of Photofrin by endothelial cells compared with several different tumor cell lines may indicate that the vascular endothelium is a major target for PDT. Therefore, PCL may be a useful formulation of photosensitizers for practical PDT. ACKNOWLEDGMENT

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