Near-Infrared Optical Imaging of B16 Melanoma Cells via Low-Density

Bioconjugate Chem. 2005, 16, 542−550

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Near-Infrared Optical Imaging of B16 Melanoma Cells via Low-Density Lipoprotein-Mediated Uptake and Delivery of High Emission Dipole Strength Tris[(porphinato)zinc(II)] Fluorophores Sophia P. Wu,† Intae Lee,‡ P. Peter Ghoroghchian,†,§ Paul R. Frail,† Gang Zheng,‡ Jerry D. Glickson,‡ and Michael J. Therien*,† Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, Pennsylvania 19104-6323, Department of Radiology, University of Pennsylvania, 423 Guardian Drive, Philadelphia, Pennsylvania 19104, and Department of Bioengineering, University of Pennsylvania, 3320 Smith Walk, Philadelphia, Pennsylvania 19104-6392. Received October 28, 2004; Revised Manuscript Received March 17, 2005

Meso-to-meso ethyne-bridged tris[(porphinato)zinc(II)] (PZn3) near-infrared (NIR) fluorophores (λemmax ∼800 nm) can be rendered sufficiently amphiphilic to enable their facile incorporation into the hydrophobic core of the apo form of low-density lipoprotein (apo-LDL). These NIR fluorophores are notable in that they manifest low energy excited states polarized exclusively along the long axis of the supermolecule, broad spectral coverage of the visible and high energy NIR spectral domains, intense S0fS1 transition moments, and comparably large S1fS0 emission dipole strengths. The reconstituted LDL(PZn3) proteins can be used to deliver rapidly hundreds of copies of PZn3 to a given murine B16 melanoma cell via LDL receptor-mediated endocytosis. PZn3-based NIRFs and their corresponding LDL(PZn3) proteins have been shown to display minimal cytotoxicity. Confocal NIR fluorescence microscopy evinces that B16 cells can be imaged at very low doses (∼nM) of NIRF. The highly attractive photophysical properties of PZn3 and closely related chromophores, coupled with their lack of toxicity and compatibility with uptake into apo-LDL and subsequent rapid delivery to B16 cells via LDLrmediated endocytosis, suggest the potential utility of this platform for NIR optical imaging of cancer cells in vivo.

INTRODUCTION

The utilization of near-infrared (NIR) probes in cancer prevention/detection strategies has attracted attention due to the facts that (i) light scattering in tissue decreases with the reciprocal of the fourth power of wavelength (λ-4), (ii) the NIR spectral domain provides a substantial optical window where hemoglobin and water absorption are minimal, and (iii) such optically based imaging technology, if realized, would presumably be inexpensive, mobile, and free of the biological effects associated with radiological probes (1, 2). Key to making such a modality a viable means to image deep tissue is the development of strategies that enable adequate NIR optical signal output per cellular recognition event. Deep tissue optical imaging requires both delineation of drastically superior fluorescent probes and delivery platforms that guarantee emissive signatures orders of magnitude greater than that provided by a single NIR fluorophore (NIRF). With respect to conventional NIRFs, indocyanine green (ICG), currently the only FDA-approved NIR imaging dye, suffers from both a modest fluorescence quantum yield (3) and a dominance of nonradiative excited state dynamics in physiological environments (4, 5). Fluorescence-based optical imaging within the NIR spectral window (∼710-950 nm) thus requires the development of new classes of biologically * To whom correspondence should be addressed. Tel: 215898-0087. Fax: 215-898-6242. E-mail: [email protected]. † Department of Chemistry. ‡ Department of Radiology. § Department of Bioengineering.

compatible chromophores that emit at long wavelengths with high emission dipole strengths; ideally, such NIRFS should possess emission maxima (λemmax) g 800 nm to permit maximal photon penetration of living tissues (6-8). Targeted NIR contrast agent delivery for in vivo optical imaging has exploited NIRF coupling to (i) peptide conjugates preferentially activated by cancer cells (9, 10), (ii) receptor specific peptides that include somatostatin analogues (11-14), and (iii) monoclonal antibodies (15, 16). While successful targeting and imaging of neoplastic tissue via each of these approaches has been demonstrated, these methods typically rely upon cell surface receptor recognition events involving conjugates featuring a single NIRF. A promising method for packaging and delivery of multiple fluorophores involves reconstitution of NIRFs into the core of low-density lipoprotein (LDL) (17-20). LDL, an endogenous constituent of blood, is a particle 20-30 nm in diameter containing a core of neutral cholesterol esters and triglycerides surrounded by a polar shell of phospholipids, unesterified cholesterol, and apolipoprotein B100 that is recognized by the LDL receptor (LDLr) (21). Because of the high cholesterol demand of rapidly dividing cells, a number of tumor cells, such as acute myeloid leukemia, epidermoid cervical cancer, and squamous lung tumor, overexpress LDLr (22). This fact, coupled with the observation that LDL, due to its nanoscale dimensions, penetrates even solid tumors (17), has made it an attractive platform for delivery of a wide range of hydrophobic compounds (23, 24) that include antitumoral drugs (20, 25-28), photodynamic therapy

10.1021/bc0497416 CCC: $30.25 © 2005 American Chemical Society Published on Web 04/30/2005

LDLr-Mediated Delivery of NIRFs to B16 Cells

agents (18, 19, 29, 30), and imaging dyes (31, 32) to neoplastic tissues. We report herein the reconstitution of LDL with mesoto-meso ethyne-bridged tris[(porphinato)zinc(II)] (PZn3) fluorophores. These NIRFs and their structurally related analogues manifest low energy excited states polarized exclusively along the long axis of the supermolecule (3342), intense S0fS1 transition moments, and comparably large S1fS0 emission dipole strengths (33, 36, 37, 40). We show further via in vitro experiments that PZn3containing LDL particles deliver the core NIRFs to murine B16 melanoma cells via the LDLr pathway, which can be subsequently imaged using scanning fluorescence confocal microscopy. EXPERIMENTAL PROCEDURES

Synthesis. All manipulations were carried out under nitrogen previously passed through an O2 scrubbing tower (Schweitzerhall R3-11 catalyst) and a drying tower (Linde 3 Å molecular sieves) unless otherwise stated. Air sensitive solids were handled in a Braun 150-M glovebox. Standard Schlenk techniques were employed to manipulate air sensitive solutions. CH2Cl2 and tetrahydrofuran (THF) were distilled from CaH2 and K/4-benzoylbiphenyl, respectively, under N2. N,N-Dimethylformamide (DMF) and triethylamine (TEA) were dried over MgSO4 and KOH, respectively, and distilled under reduced pressure. Absolute ethanol was used as received from Fisher Scientific. All NMR solvents were used as received. The catalysts tris(dibenzylideneacetone)dipalladium(0) and triphenylarsine (AsPh3) were purchased from Strem Chemicals and used as received. 1-Hydroxybenzotriazole (HoBt) and N-methylmorpholine (NMM) (Acros) as well as (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP) (Nova Biochem) were used as received. 5,15-Bis[[5′,10′,20′-bis[3,5-di(3,3-dimethyl-1butyloxy)phenyl]porphinato)zinc(II)]ethynyl]-10,20-bis[3,5-di(9-methoxy-1,4,7-trioxanonyl)phenyl]porphinato)zinc(II) (PZn3a), (5-ethynyl-10,20-bis[3′,5′-bis(3,3-dimethyl-1-butyloxy)phenyl]porphinato)zinc(II) (1), (5,15dibromo-10,20-bis[3′,5′-bis(3,3-dimethyl-1-butyloxy)-phenyl]porphinato)zinc(II) (2) (38), [5-ethynyl-10,20-diphenylporphinato]zinc(II) (3) (34, 43), and 5-androsten-17βamino-3β-yl oleate (4) (29) were synthesized according to literature procedures. Chemical shifts for 1H NMR spectra are reported relative to residual protium in the deuterated solvents (CDCl3, δ ) 7.24 ppm; DMF-d7, δ ) 8.03 ppm; pyridined5, δ ) 8.74 ppm; and DMSO-d6, δ ) 2.50 ppm). The number of attached protons is found in parentheses following the chemical shift value. Flash and size exclusion column chromatography were performed on the benchtop, using, respectively, silica gel (EM Science, 230-400 mesh) and Bio-Rad Bio-Beads SX-1 as media. Electrospray ionization (ESI-MS) data were obtained in the University of Pennsylvania Chemistry Mass Spectrometry Facility. Fast atom bombardment (FAB) mass spectrometry was performed at the Mass Spectrometry Center of Drexel University, while matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra were obtained using a Perspective Voyager DE instrument in the Laboratory of Dr. William DeGrado (Department of Biophysics and Biochemistry, University of Pennsylvania). Samples for MALDI-TOF mass spectra were prepared as micromolar solutions in THF; dithranol or 1,4-bis(5-phenyoxazol-2-yl)benzene (Aldrich) was utilized as the matrix. Instrumentation. Electronic spectra were recorded on an OLIS UV-vis/NIR spectrophotometry system that

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is based on the optics of a Cary 14 spectrophotometer. Emission spectra were recorded on a SPEX Fluorolog luminescence spectrophotometer that utilized a T-channel configuration with a red sensitive R2658 Hamamatsu PMT and liquid nitrogen-cooled InGaAs and extended InGaAs detectors; these spectra were corrected using a calibrated light source supplied by the National Bureau of Standards. NMR spectra were recorded on either 250 MHz AC-250 or 360 MHz DMX-360 Bru¨ker spectrometers. Confocal microscopy experiments were performed at the Bioengineering Confocal and Multiphoton Core Facilities at the University of Pennsylvania using a Biorad Radiance 2000-MP laser scanning confocal microscope and a Nikon Eclipse TE-300 CCD camera. (4-Formyl-phenoxy)acetic Acid Ethyl Ester (5). The reaction conditions for this precursor are analogous to those reported in the literature (44). A 200 mL roundbottom flask was charged with of 4-hydroxybenzaldehyde (6.5 g, 53 mmol), ethyl bromoacetate (9.78 g, 58.5 mmol), potassium carbonate (8.08 g, 58.5 mmol), and 100 mL of acetone. The reaction mixture was brought to a gentle reflux overnight. It was then diluted with water and extracted thrice with ethyl ether. The combined ether layers were then washed three times with 2 M KOH solution and once with water. The organic layer was then dried over Na2SO4 and evaporated to yield a yellow oil (15.3 g, 90% yield based on mass of 4-hydroxybenzaldehyde). 1H NMR (250 MHz, CDCl3): δ 9.88 (s, 1 H), 7.82 (d, 2 H, J ) 4.6 Hz), 7.0 (d, 2 H, J ) 4.6 Hz), 4.69 (s, 2 H), 4.25 (q, 2 H, J ) 7.0 Hz), 1.25 (t, 3 H, J ) 7.0 Hz) ppm. HRMS (M+): 208.0734 (calcd 208.0735). 5,15-Di[((4-ethyl ester)methyleneoxy)phenyl]porphyrin (6). 2,2′-Dipyrrylmethane (45, 46) (1.54 g, 10.5 mmol) and 5 (2.19 g, 10.5 mmol) were brought together in a 2 L round-bottom flask containing 1.5 L of dry methylene chloride and a magnetic stir bar under N2. Triflouroacetic acid (0.16 mL, 2.1 mmol) was added to this mixture; the flask was then covered with aluminum foil and stirred at room temperature for 14 h. 2,3Dichloro-4,5-dicyanoquinone (3.58 g, 15.75 mmol) was added to the methylene chloride solution, and the mixture was allowed to stir for 15 min before the solvent was removed under reduced pressure. The reaction mixture was then purified by silica chromatography using 99:1 CH2Cl2:methanol as the eluant, to give 1.5 g of the isolated product (43% yield based on 1.54 g of the dipyrrylmethane starting material). Vis (CH2Cl2) λmax nm (log ): 408 (5.37), 504 (4.17), 539 (3.79), 577 (3.75), 632 (3.37). 1H NMR (250 MHz, CDCl3): δ 10.29 (s, 2 H), 9.37 (d, 4 H, J ) 4.65 Hz), 9.07 (d, 4 H, J ) 4.65 Hz), 8.16 (d, 4 H, J ) 7.95 Hz), 7.28 (d, 4 H, J ) 7.95 Hz) 5.53 (s, 4 H), 4.40 (q, 4 H, J ) 7.28 Hz), 1.42 (t, 6 H, J ) 7.30 Hz), -3.02 (broad s, 2 H) ppm. HRMS (MH+): 667.2573 (calcd 667.2537). 5,10-Dibromo-10,20-di[((4-ethyl ester)methyleneoxy)phenyl]porphyrin (7). In a 1.0 L round-bottomed flask, 6 (600 mg, 0.9 mmol) was dissolved in 500 mL of methylene chloride. N-Bromosuccinimide (320 mg, 1.8 mmol) was then added to the stirring porphyrin solution. The reaction was allowed to proceed for 15 min and quenched by the addition of 30 mL of acetone. The solvents were removed, and the recovered reaction mixture was purified by silica chromatography using methylene chloride as the eluent. Three bands were isolated; the fastest moving band was isolated as the title compound (590 mg, 80% yield based on 600 mg of 6). Vis (CHCl3) λmax (log ): 415 (5.05), 510 (4.10), 545 (3.78), 586 (3.58) 642 (3.18) nm. 1H NMR (250 MHz, CDCl3): δ 9.60 (d, 2 H, J ) 4.7 Hz), 8.83 (d, 2H, J ) 4.6), 8.16 (d,

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4H, J ) 7.95 Hz), 7.28 (d, 4H, J ) 7.95 Hz) 4.92 (s, 4H), 4.40 (q, 4 H, J ) 7.28 Hz), 1.42 (t, 6 H, J ) 7.30 Hz), -2.75 (broad s, 2 H). [5,10-Dibromo-10,20-di[((4-ethyl ester)methyleneoxy)phenyl]porphinato]zinc(II) (8). Compound 7 (500 mg, 0.6 mmol) and zinc acetate (658 mg, 3 mmol) were refluxed in 400 mL of chloroform in a 1 L roundbottom flask equipped with a magnetic stir bar and a reflux condenser. The reaction was monitored by optical spectroscopy and was complete within 2 h. Once the solvents were evaporated, the reaction mixture was passed down a short silica column with methylene chloride as the eluent. The first band to elute is the desired product (506 mg, 95% yield based on 500 mg of 7). Vis (CHCl3) λmax (log ): 420 (5.38), 550 (4.10), 590 (3.33) nm. 1H NMR (250 MHz, CDCl3): δ 9.60 (d, 2H, J ) 4.7 Hz), 8.83 (d, 2H, J ) 4.6 Hz), 8.0 (d, 4H, J ) 7.95 Hz), 7.26 (d, 4H, J ) 7.95 Hz), 4.89 (s, 4H), 4.40 (q, 4 H, J ) 7.28 Hz), 1.39 (t, 6 H, J ) 7.30 Hz). [5,10-Dibromo-10,20-di[((4-carboxy)methyleneoxy)phenyl]porphinato]zinc(II) (9). A 100 mL roundbottom flask was charged with 8 (100 mg, 0.112 mmol) and dissolved in 80 mL of 3:1 THF:EtOH solution. An aqueous solution of KOH (63 mg, 1.12 mmol, 5 mL) was added to the stirred solution. The reaction mixture was refluxed for 2 h, following which the solvents were removed under reduced pressure. The resulting mixture was dissolved in ∼20 mL of water and acidified by dropwise addition of concentrated HCl to pH 3. The resulting purple precipitate was filtered and dried to give the desired product (86 mg, 92% yield, based on 100 mg of the dihalogenated (porphinato)zinc(II) starting material). Vis (DMSO) λmax (log ): 435 (5.44), 571 (4.11), 614 (4.07) nm. 1H NMR (250 MHz, DMF-d6): δ 9.70 ppm (d, 4 H, J ) 4.65 Hz), 8.90 (d, 4 H, J ) 4.63 Hz), 8.17 (d, 4 H, J ) 8.4 Hz), 7.45 (d, 4 H, J ) 8.6 Hz), 5.09 (s, 4 H) ppm. ESI-MS (MH+): 830.3 (calcd for C36H22Br2N4O6Zn, 830.9). FAB-MS (M+): 829.6 (calcd 829.9). [5,10-Dibromo-10,20-di[((4-5-androsten-17β-amino3β-yl oleate)methyleneoxy)phenyl]porphinato]zinc(II) (10). [5,10-Dibromo-10,20-di[((4-carboxy)methyleneoxy)phenyl]porphinato]zinc(II) (9) (31 mg, 0.0373 mmol), PyBOP (43 mg, 0.0821 mmol), HoBt (11 mg, 0.0821 mmol), and NMM (25 µL, 0.224 mmol) were dissolved in 5 mL of DMF at room temperature and stirred under argon for 30 min. 5-Androsten-17β-amino-3β-yl oleate (4) (47 mg, 0.078 mmol), dissolved in approximately 10 mL of DMF, was transferred under Ar to the porphyrincontaining solution. The reaction mixture was stirred at ambient temperature and allowed to proceed until the TLC showed that all of the porphyrin starting material had been consumed (∼22 h). The mixture was then diluted with ethyl acetate and washed three times with brine. The combined organic phases were dried over MgSO4 and evaporated. The residue was chromatographed on silica gel using 3:2 hexanes:THF as the eluant, following which it was rechromatographed on silica gel using 99:1 CH2Cl2:MeOH as the eluant. The product was isolated as a green solid (12 mg, 38% yield based on 31 mg of 9). Compound 10 was used immediately in the subsequent cross-coupling reaction described below. Vis (CH2Cl2) λmax(log ): 420 (5.38), 550 (4.16), 590 (3.69) nm. 1H NMR (250 MHz, CDCl3): 9.71 (d, 4 H, J ) 4.75 Hz), 8.84 (d, 4 H, J ) 4.66 Hz), 8.00 (d, 4 H, J ) 8.41 Hz), 6.80 (d, 4 H, J ) 7.92 Hz), 5.40 (s, 4 H), 5.34 (m, 6 H), 5.05 (m, 2 H), 4.65 (m, 2 H), 2.28 (m, 12 H), 1.98 (m, 22 H), 1.3-0.8 (m, cholesteryl oleate alkyl H) ppm. MALDI-TOF MS (M+): 1936.2 (calcd for C112H152Br2N6O8Zn, 1935.6).

Wu et al.

5,15-Bis[[5′,-10′,20′-bis((4-5-androsten-17β-amino3β-yl oleate)methyleneoxy)phenyl]porphinato)zinc(II)ethynyl]-10,20-(diphenyl)porphinato)zinc(II)(PZn3b). Compound 10 (22 mg, 0.0116 mmol), 3 (14 mg, 0.0243 mmol), Pd2dba3 (2.7 mg, 0.0029 mmol), AsPh3 (7.1 mg, 0.0232 mmol), and TEA (0.3 mL) were dissolved in THF (20 mL) in a 50 mL Schlenk tube and subjected to three freeze-pump-thaw degas cycles. The mixture was heated at 40 °C under Ar overnight, during which time the solution changed from purple to brown. The solution was then diluted with ethyl acetate, washed three times with water, dried over MgSO4, and evaporated. The residue was purified by flash chromatography on silica gel, using 3:2 hexanes:THF as the eluant, following which it was rechromatograhed on silica gel using CH2Cl2 as the eluant. The isolated product was recrystallized from THF/hexanes to give 12 mg of the product (38% yield, based on 22 mg of compound 10). Vis (CH2Cl2) λmax (log ): 421 (5.06), 489 (4.93), 768 (4.61) nm. 1H NMR (99:1 CDCl3:pyridine-d5): δ 10.44 (d, 4 H, J ) 4.65 Hz), 10.37 (d, 4 H, J ) 4.65 Hz), 10.09 (s, 2 H), 9.27 (d, 4 H, J ) 4.43 Hz), 9.13 (d, 4 H, J ) 4.63 Hz), 9.01 (d, 4 H, J ) 4.50 Hz), 8.96 (d, 4 H, J ) 4.43 Hz), 8.27 (m, 8 H), 7.78 (m, 12 H), 7.36 (d, 4 H, J ) 8.58 Hz), 6.72 (d, 4 H, J ) 9.18 Hz), 5.31 (br s, 4 H), 4.84 (s, 6 H), 4.10 (m, 2 H), 3.83 (m, 2 H), 2.30 (m, 12 H), 2.0-0.8 (m, cholesteryl oleate alkyl H) ppm. MALDI-TOF MS (M+): 2894.0 (calcd for C180H190N14O8Zn3, 2894.3). 5,15-Bis[(5′,-10′,20′-diphenyl)porphinato)zinc(II)ethynyl]-10,20-bis[(4-ethyl ester)methyleneoxy)phenyl]porphinato)zinc(II) (PZn3c). [5-Ethynyl-10,20diphenylporphinato]zinc (II) (3) (50 mg, 0.0885 mmol), 8 (38 mg, 0.0421 mmol), Pd2dba3 (9.6 mg, 0.0105 mmol), AsPh3 (26 mg, 0.0842 mmol), THF (20 mL), and TEA (0.1 mL) were brought together in a 50 mL Schlenk tube under Ar and stirred overnight at 50 °C. The reaction mixture was diluted with ethyl acetate, washed three times with water, dried over MgSO4, and evaporated. The residue was chromatographed on silica gel using 3:2 hexanes:THF as the eluant, following which the product was recrystallized from THF/hexanes to yield PZn3c (66 mg, 83% yield based on 38 mg of 8). Vis (THF) λmax (log ): 412 (5.42), 494 (5.51), 548 (4.34), 576 (4.33), 776 (5.07) nm. 1H NMR (360 MHz, 50:1 CDCl3:pyridine-d5): 10.46 (d, 2H, J ) 4.7 Hz), 10.36 (d, 2H, J ) 4.5 Hz), 10.05 (s, 2H), 9.26 (d, 2H, J ) 4.2 Hz), 9.13 (d, 2H), 9.04 (d, 2H, J ) 4.5 Hz), 8.96 (d, 2H, J ) 4.8 Hz), 8.27 (m, 10H), 7.78 (m, 8H), 4.95 (s, 2H), 4.43 (q, 4H, J ) 7.2 Hz), 1.43 (t, 6H, J ) 7.1 Hz). MALDI-TOF MS (M+): 1826.0 (calcd for C108H68N12O6Zn3, 1826.0). 5,15-bis[[(5′,-10′,20′-bis[3,5-di(3,3-dimethyl-1-butyloxy)phenyl]porphinato)zinc(II)]ethynyl]-10,20-bis[(3,5-di(3,3-dimethyl-1-butyloxy)phenyl)porphinato]zinc(II) (PZn3d). Compounds 1 (84 mg, 0.0885), 2 (46 mg, 0.0421 mmol), Pd2dba3 (9.6 mg, 0.0105 mmol), AsPh3 (26 mg, 0.0842 mmol), and TEA (0.1 mL) were dissolved in THF (20 mL) in a 50 mL Schelnk tube and reacted overnight under Ar at 60 °C. The reaction mixture was then diluted with CHCl3, washed three times with water, dried over CaCl2, and evaporated. The residue was chromatographed on silica gel using 17:3 hexanes:THF as the eluant. Trace amounts of a butadiyne-bridged bis[(porphinato)zinc(II)] contaminant were removed via size exclusion chromatography using THF as the eluant to give 97 mg of the product (82% yield based on 46 mg of 2). Vis (THF) λmax (log ): 416 (4.85), 493 (4.99), 542 (4.00), 563 (3.97), 760 (4.54) nm. 1H NMR (250 MHz, 50:1 CDCl3:pyridine-d5): 10.41 (d, 4H, J ) 4.75 Hz), 10.34 (d, 4H, J ) 4.25 Hz), 10.03 (s, 2H), 9.23 (d, 8H, J ) 4.5 Hz),

LDLr-Mediated Delivery of NIRFs to B16 Cells

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Scheme 1. Synthetic Route to NIRFs

9.13 (d, 4H, J ) 4.5 Hz), 9.05 (d, 4H, J ) 4.0 Hz), 7.45 (m, 12H), 6.87 (s, 6H), 4.19 (t, 24H, J ) 7.4 Hz), 4.16 (s, 6H), 1.83 (t, 24H, J ) 7.4 Hz), 0.96 (s, 108H). MALDITOF MS (M+): 2823.7 (calcd for C112H200N12O12Zn3, 2823.4). LDL Reconstitution. A modified version of the classic Krieger reconstitution procedure (47) was used. Human LDL was purchased from either Dr. Sissel Lund-Katz, who purified it from serum of patients with familial hypercholesteremia at the Children’s Hospital of Philadelphia (48), or from Intracel Corporation (Frederick, Maryland). Prior to reconstitution, 1.9 mg of LDL, stored in 150 mM NaCl with 0.01% EDTA at pH 7.2, was dialyzed at 4 °C against a PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM phosphate, pH 7.4) containing 0.3 mM EDTA, following which it was lyophilized in the presence of 25 mg of starch in a test tube pretreated with an antiwetting agent (Sigmacote). Endogeneous lipids were extracted by vortexing the lyophilized apoprotein/ starch mixture with 5 mL of heptanes. The LDL-containing suspension was centrifuged at 2000 rpm for 10 min, and the supernatant was discarded. This procedure to extract LDL core lipids was repeated two more times. After the heptanes layer was removed for the final time, 6 mg of the appropriate PZn3-based NIRF dissolved in 200 µL of benzene was added to the LDL pellet and incubated at 4 °C for 90 min. The organic solvents were then removed under a stream of Ar at -15 °C over a 30 min period. The reconstituted LDL was solubilized by briefly sonicating the residue in 2 mL of tricine buffer (10 mM, pH 8.4); this solution was incubated at 4 °C for 24 h. Starch and excess protein were removed by lowspeed centrifugation (2000 rpm) at 4 °C for 10 min. The supernatant was collected and further clarified by centrifuging at 9000 rpm for two 10 min periods. The concentration of the reconstituted LDL solutions was determined by the Lowry method (49). Reconstituted LDL was stored under a nitrogen atmosphere at 4 °C. The integrity of the LDL(PZn3) protein solutions, as determined from measurements of fluorescence intensity, remained constant over 8 weeks. Cytotoxicity Experiments. The in vitro cytotoxicities of PZn3a, PZn3b, and their corresponding reconstituted LDL(PZn3) protein solutions [LDL(PZn3a) and LDL(PZn3b)] were determined using a clonogenic assay. B16 melanoma cells from a cell line that overexpresses LDLr (generously provided by Dr. Theodore van Berkel, Leiden University, The Netherlands) were plated on six well

plates overnight for cell attachment. Cells were exposed to DMSO solutions of PZn3a, PZn3b, and solutions (10 mM tricine, pH ) 8.4) containing the analogous reconstituted LDL(PZn3) proteins for 3 h, 24 h, and 8 days (no rinse). After exposure, the cells were twice rinsed with HBSS and then added to DMEM media supplemented with 10% fetal bovine serum and 25 mM HEPES buffer. After incubation for 8 days, the media were discarded and the cells were fixed with 5 mL of 99.5% ethanol for 10 min, rinsed three times with water, stained with 2% crystal violet for 10 min, and rinsed again with water. Clones with more than 50 cells were counted as survivors. The surviving fraction was calculated for the clonogenic assay. Cellular Uptake and Imaging. B16 murine melanoma cells (2 × 104) were plated into two well chamber slides and incubated for 3 days at 37 °C prior to treatment with LDL(PZn3a) and LDL(PZn3b). The cells were incubated with the reconstituted dye solutions (1 µg/mL) for 1 h and subsequently washed twice with HBSS prior to fixing with absolute EtOH for 10 min at ambient temperature. The slides were then sealed and mounted for confocal imaging. RESULTS AND DISCUSSION

Synthesis. The synthesis of PZn3-based NIRFs is outlined in Scheme 1 (33, 38, 50). Because of the oxidative instability of cholesteryl oleate, reactions involving 5-androsten-17β-amino-3β-yl oleate-modified (porphinato)zinc(II) species proceeded in lower yields relative to analogous syntheses of PZn3 NIRFs lacking ancillary steroidal groups (PZn3a, PZn3c, and PZn3d). Because endogeneous LDL lipids are completely displaced during its reconstitution, the NIRFs utilized for LDL reconstitution reactions must be sufficiently lipophilic in order to circumvent nonspecific leakage and aggregation of the reconstituted LDL (22). Furthermore, solubility of the PZn3-based NIRFs in apolar organic solvents is also crucial for incorporation into the LDL interior. All the PZn3 ancillary substituents highlighted in Scheme 1 satisfy these constraints. Note that the 4′-arylester groups of PZn3c provide functionality for conjugation to moieties that engender augmented lipophilic character. PZn3b, which bears two 5-androsten-17β-amino-3β-yl oleate esters, was synthesized from synthetic precursors to PZn3c to determine the extent to which such modifications impact LDL

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Figure 1. Electronic absorption and emission spectra of tris[(porphinato)zinc(II)] fluorophores. (A) Cholesteryl oleateconjugated NIRF PZn3b in benzene solvent. (B) Reconstituted LDL(PZn3a) in tricine buffer (10 mM, pH 8.4). Corrected emission spectra, labeled with the emission wavelength maximum, are shown in the insets.

reconstitution with these tris[(porphinato)zinc(II)] species and affect subsequent cellular uptake by B16 murine melanoma cells. Previous studies demonstrated the utility of this approach for driving incorporation of cytotoxic and phototoxic agents into the LDL interior and facilitating their respective release into cells via LDLr-mediated endocytosis (17, 25, 29, 31, 51, 52), consistent with the fact that oleoyl residues are abundant components of native LDL (53). LDL Reconstitution. Following the modified Krieger protocol for LDL reconstitution outlined above (see Experimental Procedures) (47), NIRFs PZn3a and PZn3b were successfully incorporated into the LDL lipid core. Neither PZn3c nor PZn3d was taken up into apo-LDL under these conditions. These experiments suggest that without the cholesteryl oleate lipid anchor, PZn3c lacks the hydrophobicity necessary to drive LDL incorporation; furthermore, it is apparent that the increased PZn3d hydrophobicity relative to that of successfully-incorporated PZn3a neither enhances nor guarantees successful reconstitution. This result may indicate that PZn3d amphiphilicity has decreased below the threshold required for LDL compatibility; qualitatively similar observations emerge from structure-activity relationships delineated for amphiphilic drugs, which often show that potency is parabolically related to lipophilicity (54-56). Alternatively, disparate NIRF solvational environments that trace their genesis to the nature of the PZn3 mesoaryl-pendant solubilizing groups may serve as a key determinant of the extent to which PZn3a and PZn3d are taken up into apo-LDL under these conditions (53). Figure 1 displays representative optical spectra of PZn3-based NIRFs in low dielectric strength solvent and for reconstituted LDL(PZn3) proteins. These optical spectra display absorptive and emissive signatures characteristic of extensive π conjugation and exciton coupling, similar to those elucidated for closely related compounds in higher dielectric strength media (33-42). These fea-

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tures include substantial B-state (S0fS2 absorption) domain spectral breadths, high oscillator strength, low energy Q-derived S0fS1 transitions, and correspondingly intense S1fS0 fluorescence emission bands. Assuming that the electronic absorption extinction coefficients of these NIRFs within the LDL hydrophobic core are similar to those determined previously in hydrophobic solvents, we estimate that ∼30 chromophores are incorporated per LDL protein; note that this result is consistent with previous data that show that similar copy numbers of a range of amphipathic drugs are internalized into LDL using analogous reconstitution methodologies (25, 27, 52, 57). With respect to other classes of NIRFs that have been incorporated into the LDL core (29, 31), note that the electronic absorption characteristics of these PZn3-based chromophores include unusually comprehensive coverage of the UV, visible, and high energy NIR regions of the solar spectrum. Because PZn3 and related chromophores exhibit rapid S2fS1 internal conversion rate constants (τic ∼ 150 fs), which are 1 order of magnitude faster than that manifested by conventional porphyrin monomers, formation of the low energy emitting state thus occurs within the ultrafast time domain, regardless of whether these species are excited within their lowest energy absorption band or within the higher lying B-state manifold (36, 40); hence, optical excitation of the dyes could be performed at any wavelength between 400 and 800 nm. Cytotoxicity of PZn3a, PZn3b, and Their Corresponding Reconstituted LDL(PZn3) Proteins. The in vitro cytotoxicities of PZn3a, PZn3b, and their corresponding reconstituted LDL(PZn3) proteins [LDL(PZn3a) and LDL(PZn3b)] were determined using a clonogenic assay. These data show that these conjugates display no apparent toxicity to murine B16 cells, as evident from the respective cellular survival rates determined as a function of time following exposure (Figure 2). Murine B16 melanoma cells have been shown to bind LDL with affinity similar to human parenchymal cells and are thus good models for evaluating the effectiveness of LDLr-mediated delivery (58, 59). Note that the Figure 2 data do not allow determination of IC50 values for PZn3a, PZn3b, LDL(PZn3a), or LDL(PZn3b) at 3 or 24 h of exposure, due to their apparent relative nontoxicity. The IC50 value determined for PZn3a at 8 days of exposure was ∼70 µg/mL, while that for PZn3b was 45 µg/mL. The cytotoxicities for both PZn3a and PZn3b increased slightly as a function of exposure time. Note that the toxicity profiles of PZn3a and PZn3b contrast with that determined for tetrakis[(4-sulfonatophenyl)porphinato]zinc(II) on G361 human melanoma cells (IC50 > 125 µg/mL, texposure ) 60 min) (60). Furthermore, the 50% inhibitory concentrations for both LDL(PZn3a) and LDL(PZn3b) were greater than 1000 ng/mL at 8 days of exposure. The benign nature of these fluorophorereconstituted lipoproteins is similar to that established previously for in vitro toxicity studies of 3,3′-dioctadecylindocarbocyanine-labeled LDL (DiI-LDL) on MRC5 human fetal fibroblasts (61). Recent cytotoxicity studies of the FDA-approved NIR contrast agent, ICG, on human retinal pigment epithelium cells (RPE) showed that exposure to ICG at 100 µg/ mL for 3 h resulted in approximately 20% cell death (62). After exposure to ICG at 5 mg/mL for the same amount of time, no cells survived. Even at a very short exposure time of 3 min at 0.5 mg/mL of ICG, RPE cell survival rate was modestly reduced to 92.8% (63). In contrast, no

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Figure 2. Time-dependent cytotoxicity of PZn3-based NIRFs on B16 melanoma cells determined by a clonogenic assay for (A) PZn3a, (B) PZn3b, (C) LDL(PZn3a), and (D) LDL(PZn3b). Clones with more than 50 cells were counted as survivors. Data points represent means ( standard errors and were obtained from at least four independent experiments. Table 1. Qualitative Comparison of in Vitro Experimental Conditions Required to Generate NIR Confocal Fluorescence Microscope Images of Tumor Cells Qualitatively Similar to that Depicted in Figure 3B as a Function of NIRF, Delivery Platform, Dose, Exposure Time, and Emission Wavelength

a Calculated from a cell culture treatment using [LDL(dye)] ) 20 µg/mL, in which each reconstituted LDL protein is calculated to contain ∼75 dye molecules (29, 64). b Somatostatin receptor subtype 2 transfected rat insulinoma pancreatic tumor. c Incubation of dyepeptide conjugate was performed at 4 °C. d Fluorescence signal generated through the action of proteolytic enzymes preferentially expressed in tumor tissues that release cyanine dyes from a protein/polymer matrix in which NIR fluorescence was previously quenched. e XG human lung carcinoma.

significant toxicity can be detected for either PZn3a or PZn3b on B16 cells even after 24 h of exposure at 100 and 80 µg/mL concentrations, respectively. These data suggest that the PZn3 family of fluorophores, as well as related structural analogues of these species, may prove suitable for in vivo studies.

Confocal Microscopy. Cellular uptake of the LDL reconstituted dye solutions was visualized using laser scanning confocal fluorescence microscopy (Figure 3). Figure 3A,C,E shows transmission images of B16 melanoma cells, while panels B, D, and F illustrate their corresponding cellular NIR fluorescence images. As

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qualitatively similar confocal NIR fluorescence images of in vitro-targeted cancer cells. In vitro cellular delivery of the NIRF pyropheophorbide via the glucose transporter (Table 1, entry E) requires 20-fold greater concentration of fluorophore to generate a confocal image similar in quality to that obtained for delivery of the same dye via LDL reconstitution and subsequent LDLr-mediated endocytosis (Table 1, entry B) (50). Note that the amount of LDL-reconstituted pyropheophorbide necessary to generate NIR fluorescence confocal images similar to that shown in Figure 3B,D is at least 50 times higher than that used in this study (Table 1, entry A) (29). Because (i) dye doses cited in Table 1 do not necessarily correspond to the minimal amount necessary for obtaining a suitable fluorescence image and (ii) parameters such as instrument sensitivity, imaging acquisition times, and camera settings cannot be directly compared, we emphasize that the experimental data comparisons highlighted Table 1 are qualitative in nature. Nonetheless, the small concentration of PZN3 fluorophore required to obtain the in vitro images shown in Figure 3B,D underscore the promise of this NIR emissive platform for imaging applications. CONCLUSIONS

Figure 3. Confocal microscope images of B16 cells. (A) Transmission image of B16 cells treated with LDL(PZn3a). (B) Fluorescence image of panel A. (C) Transmission microscopy image of B16 cells treated with LDL(PZn3b). (D) Fluorescence image of panel C. (E) Transmission microscopy image of B16 cells incubated in culture medium to which aliquots of DMSO solutions of PZn3a (1 µg/mL) were added. (F) Fluorescence image of panel E. Experimental conditions: B16 cells (2.5 × 104) were incubated at 37 °C for 3 days and then treated with solutions of LDL(PZn3) (1 µg/mL; 10 mM tricine buffer, pH 8.4) for 1 h at ambient temperature. Following cellular washing and fixing with EtOH (see Experimental Procedures), slides were sealed and mounted. All images were taken at the same magnification using λex ) 488 nm and a 700 nm long pass filter.

Figure 3 clearly demonstrates, both LDL(PZn3a) and LDL(PZn3b) were internalized by B16 cells within 1 h; intense fluorescence signals were detected throughout the cellular membranes of the B16 cells. When B16 cells were incubated in culture medium to which aliquots of DMSO solutions of PZn3a and PZn3b (1 µg/mL) were added, no emission was detected from the cells (Figure 3E,F), indicating that the NIRF uptake highlighted by the Figure 3 data is mediated solely via LDLr-mediated endocytosis. The low doses of dye ([LDL(PZn3)] ) 1 µg/mL ) 2 nM protein; [NIRF] ) 60 nM) used to obtain the confocal images highlight the emissive characteristics of the PZn3-based NIRFs and the utility of LDL as an efficient vehicle for delivery of multiple copies of these fluorophores to a single cell. The short incubation time required for fluorophore uptake in these experiments demonstrates the rapid internalization of LDL through the B16 cells’ overexpressed LDLrs, a characteristic displayed by many tumor types (22). Table 1 highlights treatment conditions required for previously studied biomolecule-dye conjugates to obtain

We have shown that meso-to-meso ethyne-bridged tris[(porphinato)zinc(II)] (PZn3) NIRFs can be rendered sufficiently amphiphilic to enable their facile incorporation into the hydrophobic core of apo-LDL. These reconstituted LDL(PZn3) proteins can be used to deliver rapidly hundreds of copies of PZn3 to a given B16 melanoma cell via LDLr-mediated endocytosis. PZn3based NIRFs and their corresponding LDL(PZn3) proteins display minimal cytotoxicity. Confocal NIR fluorescence microscopy evinces that B16 cells can be imaged at very low doses (∼nM) of NIRF. The highly attractive photophysical properties of PZn3 and closely related chromophores, coupled with their lack of toxicity and compatibility with uptake into apo-LDL and subsequent rapid delivery to B16 cells via LDLr-mediated endocytosis, suggest the potential utility of this platform for NIR optical imaging of cancer cells in vivo. ACKNOWLEDGMENT

This research was supported by the National Cancer Institute (N01-CO-29008). J.D.G. acknowledges support from the National Cancer Institute (R24-CA83105-05 and P20-CA86255), and G.Z. thanks NASA and NCI (N01CO-37119) for funding. S.P.W. is grateful to the National Science Foundation for an Access Science Predoctoral Fellowship. P.P.G. acknowledges predoctoral fellowship support from the NIH Medical Scientist Training Program (MSTP) and the Whitaker Foundation. We are indebted to Gladys Gray-Board for her assistance with the confocal microscopy experiments, to Dr. Sissel LundKatz of the Children’s Hospital of Philadelphia for LDL, to Dr. Hui Li for guidance in the LDL reconstitution experiments, and to Dr. Theo van Berkel of Leiden University for providing the B16 melanoma cell line that overexpresses LDLr. LITERATURE CITED (1) Hawrysz, D. J., and Sevick-Muraca, E. M. (2000) Developments toward diagnostic breast cancer imaging using nearinfrared optical measurements and fluorescent contrast agents. Neoplasia 2, 388-417. (2) Weissleder, R. (2001) A clearer vision for in vivo imaging. Nat. Biotechnol. 19, 316-317.

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