Low-Density Lipoprotein Reconstituted by Pyropheophorbide

It was filtered and washed with water three times to give a crude residue. This crude product was then chromatographed on silica column with 10% ethyl...
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Bioconjugate Chem. 2002, 13, 392−396

Low-Density Lipoprotein Reconstituted by Pyropheophorbide Cholesteryl Oleate as Target-Specific Photosensitizer Gang Zheng,*,† Hui Li,† Min Zhang,†,§ Sissel Lund-Katz,| Britton Chance,‡ and Jerry D. Glickson† Department of Radiology, Department of Biochemistry and Biophysics, University of Pennsylvania Medical School, Philadelphia, Pennsylvania 19104; and Department of Chemistry, Shanghai University, Shanghai 200436, China; and Department of Pediatric GI Nutrition, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104. Received December 14, 2001

To target tumors overexpressing low-density lipoprotein receptors (LDLr), a pyropheophorbide cholesterol oleate conjugate was synthesized and successfully reconstituted into the low-density lipoprotein (LDL) lipid core. Laser scanning confocal microscopy studies demonstrated that this photosensitizer-reconstituted LDL can be internalized via LDLr by human hepatoblastoma G2 (HepG2) tumor cells.

INTRODUCTION

Photodynamic therapy (PDT) is a promising cancer treatment that involves the combination of light and a photosensitizer, normally a porphyrin derivative. Each factor is harmless by itself, but when combined with oxygen they can produce lethal reactive oxygen species that kill tumor cells (1). Singlet oxygen (1O2) is a powerful, fairly indiscriminate oxidant that is generally recognized as the key agent of PDT-induced tumor necrosis. Because the diffusion range of 1O2 is much smaller than the diameter of a single cell, the site of the primary generation of 1O2 determines which subcellular structures may be attacked (2). Consequently, if a photosensitizer is preferentially localized in tumor cells, PDT-induced cellular damage will be highly tumor specific. Therefore, target-specific photosensitizers are desirable. The LDL particle consists of a nonpolar core of neutral lipids (cholesterol esters, triglycerides) surrounded by a polar shell of phospholipid, unesterified cholesterol, and apolipoprotein B100 that is recognized by cell surface LDLr (3). A number of tumors overexpress LDLr to meet the high cholesterol demand for membrane synthesis. These include acute myelogenous leukemia (3- to 100fold), colon cancer (6-fold), adrenal adenoma (8-fold), lung carcinoma, brain tumors, and metastatic prostate cancer (4-6). Therefore, numerous efforts have been made to use LDL as a vehicle for selective delivery of photosensitizers and other antitumor agents. Porphyrins are known for their ability to associate with LDL through direct incubation without inhibiting LDLr binding (7) or processing (8). Thus, many PDT agents were incorporated into LDL via this approach and showed improved activity * Address correspondence to Dr. Gang Zheng, University of Pennsylvania, Chemistry Building, Box 66, 231 South 34th Street, Philadelphia, PA 19104. Tel: +1-215-898-3105. Fax: +1215-573-2113. E-mail: [email protected]. † Department of Radiology, University of Pennsylvania Medical School. ‡ Department of Biochemistry and Biophysics, University of Pennsylvania Medical School. § Shanghai University. | Children’s Hospital of Philadelphia.

(9). For example, a benzoporphyrin derivative (BPD) associated with LDL by simple mixing induced photodynamic cell killing 7-fold better than BPD alone (10). However, such a simple incubation method has a major drawback in that porphyrins can exchange between LDL, other phospholipids, and cell membranes, reducing the targeting specificity. One of the most important methods for packaging and delivering LDL-targeting drugs is the LDL reconstitution approach. Kreiger et al. were the first to report that it is possible to reconstitute the core of LDL with exogenous neutral lipids or suitable hydrophobic compounds, and such reconstituted LDL (r-LDL) is essentially identical with native LDL in its ability to bind to LDLr, to be internalized by cells and to be hydrolyzed in lysosomes (11). Since r-LDL can be internalized exclusively by LDLr, many cytotoxic compounds were delivered to cancer cells using this method and showed good antitumor activity (4-6). However, competing with tumor for r-LDL are organs known to overexpress LDLr, particularly the liver and adrenals, leading to undesirable toxicity (12). In this regard, the PDT protocol utilizing photosensitizer based r-LDL will be more suitable because (1) organ selectivity of PDT will come from the ability to confine activation of the photosensitizer to the tumors by restricting the illumination to that specific region; (2) the photosensitizer itself should bear no dark toxicity; and (3) the photosensitizer phototoxicity can be postponed until after non-tumor-bound photosensitizer has been cleared from the body. Goldstein, Brown, and co-workers first introduced a pyrene-based r-LDL for targeted killing of cells by LDLr-mediated photosensitization (irradiation at 300-400 nm) (13). However, this pioneering approach has never been applied using porphyrins, the most useful PDT agents. We report here the design and synthesis of a novel chlorophyll-based photosensitizer containing anchors that render it compatible with LDL’s phospholipid coat and lipophilic core. This new dye conjugate was successfully reconstituted into the LDL core, and such an r-LDL-based photosensitizer was internalized by tumor cells overexpressing LDLr and accumulated in intracellular compartments such as endosomes or lysosomes.

10.1021/bc025516h CCC: $22.00 © 2002 American Chemical Society Published on Web 04/18/2002

Communications EXPERIMENTAL PROCEDURES

Materials and General Methods. Melting points (uncorrected) were measured with a Mel-Temp II apparatus. UV-vis spectra were recorded with a Beckman DU-600 spectrophotometer. 1H NMR and 13C NMR spectra were recorded with Bruker ASPECT 360 MHz instrument. Mass spectrometry analysis and elemental analysis were performed at the Mass Spectrometry Facility of the Department of Chemistry, University of Pennsylvania. 5-Androsten-17β-amino-3β-ol was purchased from Steraloid Inc., Newport, RI. Methyl pheophorbide a was obtained from Spirulina pacifica algae available from Cyanotech Corp., Hawaii. Other chemicals were purchased from Aldrich. Where necessary, solvents were dried before use. For TLC, EM Science TLC plates (silica gel 60 F254) were used. Chlorophyll derivatives were visible without staining, and other compounds were stained with phosphomolybdic acid reagent. Reverse phase (RP) analytical HPLC was performed using a Zorbax RX-C8 (9.4 mm × 250 mm) column eluting at 1.0 mL/min with MeCN/phosphate buffer (25% to 95% MeCN gradient) with UV-vis detection at 414 nm. 5-Androsten-17β-Boc-amino-3β-ol (2). Di-tert-butyl dicarbonate (420 g, 1.90 mmol) was added to a solution containing 5-androsten-17β-amino-3β-ol (475 mg, 1.64 mmol) and triethylamine (0.27 mL, 1.94 mmol) in dichloromethane (50 mL). The reaction mixture was stirred at room temperature for 2 days. Evaporation of the solvent gave a white residue. This crude product was purified by silica gel column chromatography (30% ethyl acetate in hexanes) to give 2 as a white solid in 95% yield (610 mg, 1.57 mmol). Mp: 168-172 °C; exact mass calcd: 389.3; found by ESI-MS: 390.4 (MH+). Anal. Calcd for C24H39NO3: C, 73.99; H, 10.09; N, 3.60. Found: C, 73.52; H, 10.39; N, 3.19. 1H NMR (CDCl3): δ 5.34 (m, 1H, 6-H), 4.42 (brs, 1H, N-H), 3.53 (m, 2H, 3-H + 17-H), 2.27 (m, 2H), 2.17-1.92 (m, 2H), 1.90-1.70 (m, 3H), 1.69-1.49 (m, 6H), 1.48-1.32 (m, 3H), 1.44 (s, 9H for tert-butyl), 1.31-1.15 (m, 2H), 1.15-0.95 (m, 2H), 1.00 (s, 3H, 19CH3), 0.67 (s, 3H, 18-CH3); 13C NMR (CDCl3): δ 156.2, 141.1, 121.5, 79.2, 71.9, 60.5, 53.0, 50.4, 42.8, 42.5, 37.5, 37.2, 36.8, 32.3, 31.8, 31.7, 29.0, 28.6, 28.6, 28.6, 23.7, 20.9, 19.6, 12.0. 5-Androsten-17β-Boc-amino-3β-yl Oleate (3). Oleoyl chloride (710 mg, 2.35 mmol) was slowly added into a 20 mL pyridine solution of 2 (610 mg, 1.57 mmol). After 2 h, the reaction mixture was poured into 40 mL of icewater. It was filtered and washed with water three times to give a crude residue. This crude product was then chromatographed on silica column with 10% ethyl acetate in hexanes to afford the title compound as sticky solid in 60% yield (615 mg, 0.94 mmol). Exact mass calcd: 653.5; found by ESI-MS: 654.6 (MH+). Anal. Calcd for C42H71NO4: C, 77.13; H, 10.94; N, 2.14. Found: C, 77.35; H, 11.41; N, 1.76. 1H NMR (CDCl3): δ 5.32 (m, 3H, 6-H + 2 × vinyl-H of oleate), 4.58 (m, 1H, 3-H), 4.41 (brs, 1H, N-H), 3.52 (m, 1H, 17-H), 2.28 (m, 4H), 2.17-1.70 (m, 10H), 1.61 (m, 8H), 1.50-1.38 (m, 2H), 1.43 (s, 9H for tert-butyl), 1.37-0.95 (m, 23H), 1.00 (s, 3H, 19-CH3), 0.85 (t, 3H, terminal CH3 of oleate), 0.65 (s, 3H, 18-CH3); 13C NMR (CDCl3): δ 173.3, 139.4, 130.1, 129.9, 122.4, 78.1, 73.7, 60.5, 52.8, 50.2, 42.7, 38.3, 37.1, 37.0, 36.8, 34.8, 32.2, 32.1, 31.7, 29.9, 29.8, 29.7, 29.5, 29.5, 29.3, 29.3, 29.2, 29.2, 28.9, 28.6, 28.6, 28.6, 27.9, 27.4, 27.3, 25.2, 23.7, 22.8, 20.7, 19.5, 14.3, 11.9. 5-Androsten-17β-amino-3β-yl Oleate (4). Compound 3 (340 mg, 0.52 mmol) was dissolved in trifluoroacetic acid (5 mL), and the mixture was stirred at room temperature under argon atmosphere for 2.5 h. The acid

Bioconjugate Chem., Vol. 13, No. 3, 2002 393

was removed by rotavapor. Saturated aqueous Na2CO3 (5 mL) was added to the residue and then extracted with CH2Cl2 (3 × 10 mL), and the combined extracts were dried with Na2SO4. After the solvent was removed, the residue was dried under high vacuum and used for the conjugation step without further purification (260 mg, 0.47 mmol). Mass calcd for C37H63NO2: 553.5; found by ESI-MS: 554.8 (MH+). Pyropheophorbide a (5). Methyl pheophorbide a (500 mg, 0.82 mmol) isolated from Spirulina algae was heated under refluxed temperature in collidine (100 mL) for 3 h under nitrogen atmosphere. The solution was evaporated under high vacuum, and the residue so obtained was chromatographed over an alumina column (Grade III) and eluted with CH2Cl2. Pyropheophorbide a methyl ester was crystallized from CH2Cl2/hexanes in 91% yield (411 mg, 0.75 mmol). This intermediate (250 mg, 0.46 mmol) was dissolved in THF (65 mL) and mixed with a solution containing LiOH (500 mg), methanol (7 mL), and water (3 mL). The mixture was stirred under argon atmosphere for 24 h. After workup, the crude product was chromatographed on a silica column with 10% methanol in CH2Cl2 to give the title compound in 82% yield (200 mg, 0.37 mmol). The spectral and physical data of compound 5 are consistent with the literature (14). Mp: 220-223 °C. UV-vis in CH2Cl2: 411 nm ( 1.1 × 105), 509 (1.1 × 104), 537 (9.6 × 103), 611 (8.2 × 103), and 669 (4.5 × 104). Mass calcd for C33H34N4O3: 534.5; found by ESI-MS: 535.6 (MH+) and 557.6 (M + Na+). 1H NMR (CDCl ): 9.47, 9.35, and 8.53 (each s, 1H, 5-H, 3 10-H, and 20-H); 8.00 (dd, J ) 17.7, 11.4 Hz, 1H, 31-CHd CH2); 6.27 (d, J ) 17.7 Hz, 1H, trans-32-CHdCH2); 6.15 (d, J ) 11.4 Hz, 1H, cis-32-CHdCH2); 5.18 (ABX, 2H, 132CH2); 4.47 (q, J ) 7.1, 1.9 Hz, 1H for 18-H); 4.29 (m, J ) 7.8 Hz, 1H for 17-H); 3.68 (q, J ) 7.4 Hz, 2H, 8-CH2CH3); 3.64, 3.39, and 3.22 (each s, 3H, 12-CH3, 2-CH3 and 7-CH3); 2.65 and 2.32 (each m, 2H, for 2 × 171-H and 2 × 172-H); 1.81 (d, J ) 7.2 Hz, 3H, 18-CH3); 1.70 (t, J ) 8.3 Hz, 3H, 8-CH2CH3), 0.87 and -1.35 (each brs, 1H, 2 × N-H). Pyropheophorbide Cholesterol Oleate (6). Compound 5 (45 mg, 0.084 mmol) was activated with DCC (20 mg, 0.097 mmol) and 1-hydroxybenzotriazole (14 mg, 0.103 mmol) in CH2Cl2 (2 mL) and added to a solution of the cholesterol oleate amine 4 (50 mg, 0.090) in CH2Cl2 (3 mL). After being stirred for 24 h, solvent was removed, and the crude product was purified by silica gel plate chromatography (3% methanol in CH2Cl2) to afford the desired conjugate in 20% yield (18 mg, 0.017 mmol). Analytical RP HPLC: tR 35.81 min, 99%. UV-vis in CH2Cl2: 414 nm ( 9.8 × 104), 510 (1.1 × 104), 541 (1.1 × 104), 610 (5.4 × 103), and 667 (4.5 × 104). Mass calcd for C70H95N5O4: 1070.7462 (MH+); found by HRMS: 1070.7412 (MH+); 1H NMR (CDCl3): 9.37, 9.33, and 8.55 (each s, 1H, 5-H, 10-H, and 20-H of pyro); 8.00 (dd, J ) 17.7, 11.4 Hz, 1H, 31-CHdCH2 of pyro); 6.28 (d, J ) 17.7 Hz, 1H, trans-32-CHdCH2 of pyro); 6.17 (d, J ) 11.4 Hz, 1H, cis-32-CHdCH2 of pyro); 5.31 (m, 3H, 2 × vinyl-H and 6-H of oleate), 5.23 (ABX, 2H, 132-CH2 of pyro); 5.02 (m, 1H, N-H of cholesterol), 4.55 (m, 2H, 18-H of pyro, 3-H of cholesterol); 4.35 (m, J ) 7.8 Hz, 1H for 17-H of pyro); 3.75 (m, 1H, 17-H of cholesterol), 3.63 (q, J ) 7.4 Hz, 2H, 8-CH2CH3 of pyro); 3.47, 3.46 and 3.23 (each s, 3H, 12-CH3, 2-CH3 and 7-CH3 of pyro); 2.71 and 2.46 (each m, 2H, for 2 × 171-H and 2 × 172-H of pyro); 2.22 (m, 6H of cholesterol oleate), 2.10-1.75 (m, 11H of cholesterol oleate), 1.70-0.95 (m, 39H of cholesterol oleate), 1.82 (d, J ) 7.2 Hz, 3H, 18-CH3 of pyro); 1.65 (t, J ) 8.3 Hz, 3H, 8-CH2CH3 of pyro), 0.88 (m, 6H, 19-CH3

394 Bioconjugate Chem., Vol. 13, No. 3, 2002

Zheng et al.

Scheme 1. Synthesis of Cholesteryl Oleate Amine 4 and Its Corresponding Pyropheophorbide Conjugate 6

a Reagents (yields in parentheses): (a) (BOC) O, Et N, CH Cl , 24 h (95%); (b) oleoyl chloride, pyridine, 2 h (60%); (c) TFA, 2.5 h 2 3 2 2 (90%); (d) DCC, 1-hydroxybenzotriazole, CH2Cl2, 24 h (20%).

of cholesterol and terminal CH3 of oleate), 0.32 (s, 3H, 18-CH3 of cholesterol). LDL Reconstitution. LDL (1.9 mg) was lyophilized with 25 mg starch and then extracted three times with 5 mL of heptane at -5 °C. Following aspiration of the last heptane extract, 6 mg of pyropheophorbide cholesteryl oleate (6) was added in 200 µL of benzene. After 90 min at 4 °C, benzene and any residual heptane were removed under a stream of N2 in an ice-salt bath for about 45 min. The r-(Pyro-CE)-LDL was solubilized in 10 mM Tricine, pH 8.2, at 4 °C for 24 h. Starch was removed from the solution by low-speed centrifugation (500g) and followed by a 20 min centrifugation (6000g). The reconstituted LDL was stored under an inert gas at 4 °C. The protein content of the specimen was determined by the Lowry method (15), and the fluorescence was measured using an LS-50B Fluorometer. Cell Preparations. HepG2 tumor cells, which were obtained from Dr. Theo van Berkel’s laboratory from the University of Leiden in The Netherlands, were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin-streptomycin. Cells were grown at 37 °C in an atmosphere of 5% CO2 in a humidified incubator. Confocal Microscopic Studies. For confocal microscopic studies, HepG2 cells were grown for 5 days in fourwell Lab-Tek chamber slides (Naperville, IL). Before the cell experiments, the culture medium was replaced by preincubation medium (medium with 1% (w/v) BSA instead of FBS). The cells were washed three times with preincubation medium (for 15, 15, and 30 min) and cultured in this medium for a further 20 h. Experiments were started, after two quick washes with preincubation medium, by the addition of preincubation medium containing the indicated amounts of r-(Pyro-CE)-LDL and/

or unlabeled LDL. After 3 h incubation at 37 °C, the cells were washed five times with ice-cold PBS containing 0.8% BSA, two times with PBS alone, and fixed for 20 min with 2% formaldehyde in PBS at room temperature. Then the chamber slides were mounted and sealed for confocal microscopic analysis. RESULTS AND DISCUSSION

Our choice of the photosensitizer, pyropheophorbide a, is a stable analogue of natural chlorophyll a. It has excellent photophysical properties with long wavelength absorption of 667 nm. One of its derivatives, the hexyl ether analogue of pyropheophorbide a (Photochlor) is currently in phase I/II clinical trial at the Roswell Park Cancer Institute (Buffalo, NY) (16). As for the anchor, a cholesteryl oleate containing a primary amine linker was designed for conjugation with a variety of PDT agents. The oleate moiety contains one cis-double bond leading to a bent form of the long hydrocarbon chain that may facilitate the reconstitution process, whereas the cholesterol moiety will serve to anchor lipids in the core, thus eliminating nonspecific leakage. To synthesize the desired cholesteryl oleate amine, we developed a very short and efficient pathway. As shown in Scheme 1, a commercially available cholesterol amine (1), 5-androsten-17β-amino-3β-ol (Steraloid Inc., Newport, RI), was first Boc-protected and esterified with oleoyl chloride. Deprotection of the amine at C-17 gave the corresponding cholesteryl oleate amine 4 in 60% overall yield. Coupling of amine 4 to pyropheophorbide a 5 (Scheme 1), obtained from Spirulina algae in short steps (14), yielded the desired conjugate 6 in sufficient quantities for LDL reconstitution studies. All new compounds were characterized by mass and NMR spectro-

Communications

scopic studies, and the purity of these compounds was confirmed by either elemental analysis or HPLC studies. The pyropheophorbide cholesteryl oleate (6) so obtained was then used for reconstituting LDL. Following a modified Krieger’s procedure as reported by Craig et al. (17), we were able to extract the native cholesteryl esters from the LDL lipid core and successfully replace them with our newly synthesized conjugate. The success of reconstitution was determined by a protein recovery assay following Lowry’s method. Our pyropheophorbide cholesteryl oleate reconstituted LDL, r-(Pyro-CE)-LDL, yielded a 45% protein recovery comparable to the published data of 48% protein recovery (17) for other hydrophobic molecules. This LDL-based photosensitizer has long wavelength absorption of 667 nm and produces a fluorescence signal in the near-infrared (NIR) range (720 nm). Therefore, this photosensitizer can also be used for NIR detection of early cancers by optical imaging techniques. To visualize LDLr-mediated internalization into the tumor cells, we performed laser scanning confocal microscopy studies on our newly synthesized r-(Pyro-CE)LDL in HepG2 tumor cells. Four sets of experiments were performed on a Leica TCS SPII laser scanning confocal microscope (Heidelberg, Germany). Figure 1 shows the confocal microscopic images of HepG2 cells incubated with/without fluorescent probe (B, D, F, H) as well as corresponding bright field images (A, C, E, G). In a control experiment, HepG2 cells were incubated with native LDL but without the photosensitizer at 37 °C for 3 h to determine any possible background fluorescence. As expected, no fluorescence signal could be detected without the photosensitizer (Figure 1B). After the incubation with 20 µg/mL of r-(Pyro-CE)-LDL, an intense fluorescence signal was observed distributing throughout the whole cell except for the nucleus, presumably in intracellular compartments such as endosomes or lysosomes (Figure 1D). To determine the specificity of this LDL-based photosensitizer toward LDL receptors, HepG2 cells were incubated with either a 25-fold excess of unlabeled LDL in addition to the 20 µg/mL r-(Pyro-CE)LDL or the same amount of Pyro-CE (compound 6) without reconstitution into LDL. No fluorescence could be observed in both experiments (Figure 1F, 1H), which clearly indicates specific binding of the reconstituted LDL to the LDL receptors. In summary, we have demonstrated that pyropheophorbide, a stable photosensitizer derived from chlorophyll a, can be covalently linked to the primary amine functional group of the cholesteryl oleate, which serves as a double anchor for the LDL lipid core and can be synthesized in short steps. We were able to successfully incorporate this dye conjugate into the LDL lipid core via the LDL reconstitution approach. By using laser scanning confocal microscopy, we have demonstrated that photosensitizer-reconstituted LDL can be internalized by HepG2 tumor cells and, therefore, can be used as a PDT agent directed at LDLr-overexpressing tumors. ACKNOWLEDGMENT

We are grateful to Dr. Ponzy Lu of the Chemistry Department for accommodating our organic laboratory, to Ms. Jasmine X.-Y. Zhao of the Microscopy Core Facility of the Wistar Institute for taking confocal microscope images, to Mr. David Nelson and Mr. Dong Xin for technical assistance, and to Ms. Lida Gifford for critical reading of the manuscript. This research was supported by NIH contract BAA NO1-CM97065-02 and an Onco-

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Figure 1. Fluorescence confocal microscopic images of HepG2 tumor cells incubated with unlabeled LDL as control (B), 20 µg/ mL of r-(Pyro-CE)-LDL for binding assay (D), 20 µg/mL of r-(Pyro-CE)-LDL with 25-fold excess of unlabeled LDL for competition assay (F), 20 µg/mL non-LDL-reconstituted PyroCE for comparison (H), as well as the corresponding bright field images (A, C, E, G).

logic Foundation of Buffalo award. Partial support by NIH grants RO1 CA 83105-02, P20 CA86255-01, and HL 56083 are also acknowledged. LITERATURE CITED (1) (a) Dougherty, T. J., Gomer, C., Henderson, B. W., Jori, G., Kessel, D., Korbelik, M., Moan, J., and Peng, Q. (1998) Photodynamic therapy. J. Natl. Cancer Inst. 90, 889-905. (b) Sternberg, E. D., Dolphin, D., and Bruckner, C. (1998) Porphyrin-based photosensitizers for use in photodynamic therapy. Tetrahedron 54, 4151-4202. (c) Pandey, R. K., and Zheng, G. (2000) Porphyrins as Photosensitizers in Photodynamic Therapy. Porphyrin Handbook (Kadish, K., Smith, K. M., and Guilard, R., Eds.) pp 157-230, Vol. 6, Academic Press, New York. (2) Moan, J. (1990) On the diffusion length of singlet oxygen in cells and tissues. J. Photochem. Photobiol. B: Biol. 6, 343347. (3) Brown, M. S., Kovanen, P. T., and Goldstein, J. L. (1980) Evolution of the LDL receptor concept - cultured cells to intact animals. Ann. N.Y. Acad. Sci. 348, 48-68. (4) Lundberg, B. (1987) Preparation of drug-low-density lipoprotein complexes for delivery of antitumoral drugs via the low-density lipoprotein pathway. Cancer Res. 47, 4105-4108.

396 Bioconjugate Chem., Vol. 13, No. 3, 2002 (5) Firestone, R. A. (1994) Low-density lipoprotein as a vehicle for targeting antitumor compounds to cancer cells. Bioconjugate Chem. 5, 105-113. (6) Shaw, J. M., Shaw, K. V., Yanovich, S., Iwanik, M., Futch, W. S., Rosowsky, A., and Schook, L. B. (1987) Delivery of lipophilic drugs using lipoproteins. Ann. N.Y. Acad. Sci. 507, 252-271. (7) Candide, C., Morliere, P., Maziere, J. C., Goldstein, S., Santus, R., Dubertret, S., Reyftmann, J. P., and Polonovski, J. (1986) In vitro interaction of the photoactive anticancer porphyrin derivative photofrin II with low-density lipoprotein, and its delivery to cultured human fibroblasts. FEBS Lett. 207, 133-138. (8) de Smidt, P. C., Versluis, A. J., and van Berkel, T. J. C. (1993) Properties of incorporation, redistribution and integrity of porphyrin-low-density lipoprotein complexes. Biochemistry 32, 2916-2922. (9) Reddi, E. (1997) Role of delivery vehicles for photosensitizers in the photodynamic therapy of tumors. J. Photochem. Photobiol. B: Biol. 37, 189-195. (10) Allison, B. A., Waterfield, A. M., Richter, A. M., and Levy, J. G. (1991) The effects of plasma-lipoproteins on in vitro tumor cell killing and in vivo tumor photosensitization with benzoporphyrin derivative. Photochem. Photobiol. 54, 709715. (11) Krieger, M., McPhaul, M. J., Goldstein, J. L., and Brown, M. S. (1979) Replacement of neutral lipids of low-density lipoprotein with esters of long chain unsaturated fatty acids. J. Biol. Chem. 254, 3845-3853.

Zheng et al. (12) Versluis, A. J., van Geel, P. J., Oppelaar, H., van Berkel, T. J. C., and Bijsterbosch, M. K. (1996) Receptor-mediated uptake of low-density lipoprotein by B16 melanoma cells in vitro and in vivo in mice. Br. J. Cancer 74, 525-532. (13) Mosley, S. T., Goldstein, J. L., Brown, M. S., Falck, J. R., and Anderson, R. G. W. (1981) Targeted killing of cultured cells by receptor-mediated photosensitization. Proc. Natl. Acad. Sci. U.S.A. 78, 5717-5721. (14) Pandey, R. K., Sumlin, A. B., Constantine, S., Aoudia, M., Potter, W. R., Bellnier, D. A., Henderson, B. W., Rodgers, M. A., Smith, K. M., and Dougherty, T. J. (1996) Alkyl ether analogues of chlorophyll-a derivatives 0.1. Synthesis, photophysical properties and photodynamic efficacy. Photochem. Photobiol. 64, 194-204. (15) Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. (16) Henderson, B. W., Bellnier, D. A., Greco, W. R., Sharma, A., Pandey, R. K., Vaughan, L., Weishaupt, K., and Dougherty, T. J. (1997) A quantitative structure-activity relationship for a congeneric series of pyropheophorbide derivatives as photosensitizers for photodynamic therapy. Cancer Res. 57, 4000-4007. (17) Craig, I. F., Via, D. P., Mantulin, W. W., Pownall, H. J., Gotto, A. M., Jr., and Smith, L. C. (1981) Low-density lipoproteins reconstituted with steroids containing the nitrobenzoxadiazole. J. Lipid Res. 22, 687-696.

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