near-infrared optical imaging of b16 melanoma cells via low-density lipoprotein-mediated uptake and...

Near-Infrared Optical Imaging of B16 Melanoma Cells viaLow-Density Lipoprotein-Mediated Uptake and Delivery of HighEmission 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 ofPennsylvania, 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 thehydrophobic core of the apo form of low-density lipoprotein (apo-LDL). These NIR fluorophores arenotable in that they manifest low energy excited states polarized exclusively along the long axis ofthe supermolecule, broad spectral coverage of the visible and high energy NIR spectral domains, intenseS0fS1 transition moments, and comparably large S1fS0 emission dipole strengths. The reconstitutedLDL(PZn3) proteins can be used to deliver rapidly hundreds of copies of PZn3 to a given murine B16melanoma cell via LDL receptor-mediated endocytosis. PZn3-based NIRFs and their correspondingLDL(PZn3) proteins have been shown to display minimal cytotoxicity. Confocal NIR fluorescencemicroscopy evinces that B16 cells can be imaged at very low doses (∼nM) of NIRF. The highly attractivephotophysical properties of PZn3 and closely related chromophores, coupled with their lack of toxicityand 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 cancercells in vivo.

INTRODUCTION

The utilization of near-infrared (NIR) probes in cancerprevention/detection strategies has attracted attentiondue to the facts that (i) light scattering in tissue decreaseswith the reciprocal of the fourth power of wavelength(λ-4), (ii) the NIR spectral domain provides a substantialoptical window where hemoglobin and water absorptionare minimal, and (iii) such optically based imagingtechnology, if realized, would presumably be inexpensive,mobile, and free of the biological effects associated withradiological probes (1, 2). Key to making such a modalitya viable means to image deep tissue is the developmentof strategies that enable adequate NIR optical signaloutput per cellular recognition event.

Deep tissue optical imaging requires both delineationof drastically superior fluorescent probes and deliveryplatforms that guarantee emissive signatures orders ofmagnitude greater than that provided by a single NIRfluorophore (NIRF). With respect to conventional NIRFs,indocyanine green (ICG), currently the only FDA-ap-proved NIR imaging dye, suffers from both a modestfluorescence quantum yield (3) and a dominance ofnonradiative excited state dynamics in physiologicalenvironments (4, 5). Fluorescence-based optical imagingwithin the NIR spectral window (∼710-950 nm) thusrequires the development of new classes of biologically

compatible chromophores that emit at long wavelengthswith high emission dipole strengths; ideally, such NIRFSshould possess emission maxima (λem

max) g 800 nm topermit maximal photon penetration of living tissues(6-8).

Targeted NIR contrast agent delivery for in vivo opticalimaging has exploited NIRF coupling to (i) peptideconjugates preferentially activated by cancer cells (9, 10),(ii) receptor specific peptides that include somatostatinanalogues (11-14), and (iii) monoclonal antibodies (15,16). While successful targeting and imaging of neoplastictissue via each of these approaches has been demon-strated, these methods typically rely upon cell surfacereceptor recognition events involving conjugates featur-ing a single NIRF.

A promising method for packaging and delivery ofmultiple fluorophores involves reconstitution of NIRFsinto the core of low-density lipoprotein (LDL) (17-20).LDL, an endogenous constituent of blood, is a particle20-30 nm in diameter containing a core of neutralcholesterol esters and triglycerides surrounded by a polarshell of phospholipids, unesterified cholesterol, and apo-lipoprotein B100 that is recognized by the LDL receptor(LDLr) (21). Because of the high cholesterol demand ofrapidly dividing cells, a number of tumor cells, such asacute myeloid leukemia, epidermoid cervical cancer, andsquamous lung tumor, overexpress LDLr (22). This fact,coupled with the observation that LDL, due to itsnanoscale dimensions, penetrates even solid tumors (17),has made it an attractive platform for delivery of a widerange of hydrophobic compounds (23, 24) that includeantitumoral drugs (20, 25-28), photodynamic therapy

* To whom correspondence should be addressed. Tel: 215-898-0087. Fax: 215-898-6242. E-mail: [email protected].

† Department of Chemistry.‡ Department of Radiology.§ Department of Bioengineering.

542 Bioconjugate Chem. 2005, 16, 542−550

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

agents (18, 19, 29, 30), and imaging dyes (31, 32) toneoplastic tissues.

We report herein the reconstitution of LDL with meso-to-meso ethyne-bridged tris[(porphinato)zinc(II)] (PZn3)fluorophores. These NIRFs and their structurally relatedanalogues manifest low energy excited states polarizedexclusively along the long axis of the supermolecule (33-42), intense S0fS1 transition moments, and comparablylarge S1fS0 emission dipole strengths (33, 36, 37, 40).We show further via in vitro experiments that PZn3-containing LDL particles deliver the core NIRFs tomurine B16 melanoma cells via the LDLr pathway,which can be subsequently imaged using scanning fluo-rescence confocal microscopy.

EXPERIMENTAL PROCEDURES

Synthesis. All manipulations were carried out undernitrogen previously passed through an O2 scrubbingtower (Schweitzerhall R3-11 catalyst) and a drying tower(Linde 3 Å molecular sieves) unless otherwise stated. Airsensitive solids were handled in a Braun 150-M glovebox.Standard Schlenk techniques were employed to manipu-late 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 andKOH, respectively, and distilled under reduced pressure.Absolute ethanol was used as received from FisherScientific. All NMR solvents were used as received. Thecatalysts tris(dibenzylideneacetone)dipalladium(0) andtriphenylarsine (AsPh3) were purchased from StremChemicals and used as received. 1-Hydroxybenzotriazole(HoBt) and N-methylmorpholine (NMM) (Acros) as wellas (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexa-fluorophosphate (PyBOP) (Nova Biochem) were used asreceived. 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(9-methoxy-1,4,7-trioxanonyl)phenyl]porphinato)-zinc(II) (PZn3a), (5-ethynyl-10,20-bis[3′,5′-bis(3,3-di-methyl-1-butyloxy)phenyl]porphinato)zinc(II) (1), (5,15-dibromo-10,20-bis[3′,5′-bis(3,3-dimethyl-1-butyloxy)-phenyl]-porphinato)zinc(II) (2) (38), [5-ethynyl-10,20-diphenyl-porphinato]zinc(II) (3) (34, 43), and 5-androsten-17â-amino-3â-yl oleate (4) (29) were synthesized accordingto literature procedures.

Chemical shifts for 1H NMR spectra are reportedrelative to residual protium in the deuterated solvents(CDCl3, δ ) 7.24 ppm; DMF-d7, δ ) 8.03 ppm; pyridine-d5, δ ) 8.74 ppm; and DMSO-d6, δ ) 2.50 ppm). Thenumber of attached protons is found in parenthesesfollowing the chemical shift value. Flash and size exclu-sion column chromatography were performed on thebenchtop, 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 inthe University of Pennsylvania Chemistry Mass Spec-trometry Facility. Fast atom bombardment (FAB) massspectrometry was performed at the Mass SpectrometryCenter of Drexel University, while matrix-assisted laserdesorption/ionization time-of-flight (MALDI-TOF) massspectra were obtained using a Perspective Voyager DEinstrument in the Laboratory of Dr. William DeGrado(Department of Biophysics and Biochemistry, Universityof Pennsylvania). Samples for MALDI-TOF mass spectrawere prepared as micromolar solutions in THF; dithranolor 1,4-bis(5-phenyoxazol-2-yl)benzene (Aldrich) was uti-lized as the matrix.

Instrumentation. Electronic spectra were recordedon an OLIS UV-vis/NIR spectrophotometry system that

is based on the optics of a Cary 14 spectrophotometer.Emission spectra were recorded on a SPEX Fluorologluminescence spectrophotometer that utilized a T-chan-nel configuration with a red sensitive R2658 HamamatsuPMT and liquid nitrogen-cooled InGaAs and extendedInGaAs detectors; these spectra were corrected using acalibrated light source supplied by the National Bureauof Standards. NMR spectra were recorded on either 250MHz AC-250 or 360 MHz DMX-360 Bruker spectrom-eters. Confocal microscopy experiments were performedat the Bioengineering Confocal and Multiphoton CoreFacilities at the University of Pennsylvania using aBiorad Radiance 2000-MP laser scanning confocal mi-croscope and a Nikon Eclipse TE-300 CCD camera.

(4-Formyl-phenoxy)acetic Acid Ethyl Ester (5).The reaction conditions for this precursor are analogousto those reported in the literature (44). A 200 mL round-bottom 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 ofacetone. The reaction mixture was brought to a gentlereflux overnight. It was then diluted with water andextracted thrice with ethyl ether. The combined etherlayers were then washed three times with 2 M KOHsolution and once with water. The organic layer was thendried over Na2SO4 and evaporated to yield a yellow oil(15.3 g, 90% yield based on mass of 4-hydroxybenzalde-hyde). 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, 2H), 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]por-phyrin (6). 2,2′-Dipyrrylmethane (45, 46) (1.54 g, 10.5mmol) and 5 (2.19 g, 10.5 mmol) were brought togetherin a 2 L round-bottom flask containing 1.5 L of drymethylene chloride and a magnetic stir bar under N2.Triflouroacetic acid (0.16 mL, 2.1 mmol) was added tothis mixture; the flask was then covered with aluminumfoil and stirred at room temperature for 14 h. 2,3-Dichloro-4,5-dicyanoquinone (3.58 g, 15.75 mmol) wasadded to the methylene chloride solution, and the mix-ture was allowed to stir for 15 min before the solvent wasremoved under reduced pressure. The reaction mixturewas then purified by silica chromatography using 99:1CH2Cl2:methanol as the eluant, to give 1.5 g of theisolated product (43% yield based on 1.54 g of thedipyrrylmethane starting material). Vis (CH2Cl2) λmax nm(log ε): 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, 4H), 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 (calcd667.2537).

5,10-Dibromo-10,20-di[((4-ethyl ester)methyleneox-y)phenyl]porphyrin (7). In a 1.0 L round-bottomedflask, 6 (600 mg, 0.9 mmol) was dissolved in 500 mL ofmethylene chloride. N-Bromosuccinimide (320 mg, 1.8mmol) was then added to the stirring porphyrin solution.The reaction was allowed to proceed for 15 min andquenched by the addition of 30 mL of acetone. Thesolvents were removed, and the recovered reaction mix-ture was purified by silica chromatography using meth-ylene chloride as the eluent. Three bands were isolated;the fastest moving band was isolated as the title com-pound (590 mg, 80% yield based on 600 mg of 6). Vis(CHCl3) λmax (log ε): 415 (5.05), 510 (4.10), 545 (3.78),586 (3.58) 642 (3.18) nm. 1H NMR (250 MHz, CDCl3): δ9.60 (d, 2 H, J ) 4.7 Hz), 8.83 (d, 2H, J ) 4.6), 8.16 (d,

LDLr-Mediated Delivery of NIRFs to B16 Cells Bioconjugate Chem., Vol. 16, No. 3, 2005 543

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)methyl-eneoxy)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 round-bottom flask equipped with a magnetic stir bar and areflux condenser. The reaction was monitored by opticalspectroscopy and was complete within 2 h. Once thesolvents were evaporated, the reaction mixture waspassed down a short silica column with methylenechloride as the eluent. The first band to elute is thedesired product (506 mg, 95% yield based on 500 mg of7). Vis (CHCl3) λmax (log ε): 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 ) 7.95Hz), 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)methylene-oxy)phenyl]porphinato]zinc(II) (9). A 100 mL round-bottom flask was charged with 8 (100 mg, 0.112 mmol)and dissolved in 80 mL of 3:1 THF:EtOH solution. Anaqueous solution of KOH (63 mg, 1.12 mmol, 5 mL) wasadded to the stirred solution. The reaction mixture wasrefluxed for 2 h, following which the solvents wereremoved under reduced pressure. The resulting mixturewas dissolved in ∼20 mL of water and acidified bydropwise addition of concentrated HCl to pH 3. Theresulting purple precipitate was filtered and dried to givethe desired product (86 mg, 92% yield, based on 100 mgof the dihalogenated (porphinato)zinc(II) starting mate-rial). Vis (DMSO) λmax (log ε): 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, 4H, 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-17â-amino-3â-yl oleate)methyleneoxy)phenyl]porphinato]zinc-(II) (10). [5,10-Dibromo-10,20-di[((4-carboxy)methyle-neoxy)phenyl]porphinato]zinc(II) (9) (31 mg, 0.0373 mmol),PyBOP (43 mg, 0.0821 mmol), HoBt (11 mg, 0.0821mmol), and NMM (25 µL, 0.224 mmol) were dissolved in5 mL of DMF at room temperature and stirred underargon for 30 min. 5-Androsten-17â-amino-3â-yl oleate (4)(47 mg, 0.078 mmol), dissolved in approximately 10 mLof DMF, was transferred under Ar to the porphyrin-containing solution. The reaction mixture was stirred atambient temperature and allowed to proceed until theTLC showed that all of the porphyrin starting materialhad been consumed (∼22 h). The mixture was thendiluted with ethyl acetate and washed three times withbrine. The combined organic phases were dried overMgSO4 and evaporated. The residue was chromato-graphed on silica gel using 3:2 hexanes:THF as theeluant, following which it was rechromatographed onsilica gel using 99:1 CH2Cl2:MeOH as the eluant. Theproduct was isolated as a green solid (12 mg, 38% yieldbased on 31 mg of 9). Compound 10 was used im-mediately in the subsequent cross-coupling reactiondescribed below. Vis (CH2Cl2) λmax(log ε): 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, 4H), 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 alkylH) ppm. MALDI-TOF MS (M+): 1936.2 (calcd for C112H152-Br2N6O8Zn, 1935.6).

5,15-Bis[[5′,-10′,20′-bis((4-5-androsten-17â-amino-3â-yl oleate)methyleneoxy)phenyl]porphinato)-zinc(II)ethynyl]-10,20-(diphenyl)porphinato)zinc-(II)(PZn3b). Compound 10 (22 mg, 0.0116 mmol), 3 (14mg, 0.0243 mmol), Pd2dba3 (2.7 mg, 0.0029 mmol), AsPh3(7.1 mg, 0.0232 mmol), and TEA (0.3 mL) were dissolvedin THF (20 mL) in a 50 mL Schlenk tube and subjectedto three freeze-pump-thaw degas cycles. The mixturewas heated at 40 °C under Ar overnight, during whichtime the solution changed from purple to brown. Thesolution was then diluted with ethyl acetate, washedthree times with water, dried over MgSO4, and evapo-rated. The residue was purified by flash chromatographyon silica gel, using 3:2 hexanes:THF as the eluant,following which it was rechromatograhed on silica gelusing CH2Cl2 as the eluant. The isolated product wasrecrystallized from THF/hexanes to give 12 mg of theproduct (38% yield, based on 22 mg of compound 10). Vis(CH2Cl2) λmax (log ε): 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)phe-nyl]porphinato)zinc(II) (PZn3c). [5-Ethynyl-10,20-diphenylporphinato]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 (0.1mL) were brought together in a 50 mL Schlenk tubeunder Ar and stirred overnight at 50 °C. The reactionmixture was diluted with ethyl acetate, washed threetimes with water, dried over MgSO4, and evaporated. Theresidue was chromatographed on silica gel using 3:2hexanes:THF as the eluant, following which the productwas recrystallized from THF/hexanes to yield PZn3c (66mg, 83% yield based on 38 mg of 8). Vis (THF) λmax (logε): 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 (calcdfor C108H68N12O6Zn3, 1826.0).

5,15-bis[[(5′,-10′,20′-bis[3,5-di(3,3-dimethyl-1-buty-loxy)phenyl]porphinato)zinc(II)]ethynyl]-10,20-bis-[(3,5-di(3,3-dimethyl-1-butyloxy)phenyl)porphina-to]zinc(II) (PZn3d). Compounds 1 (84 mg, 0.0885), 2 (46mg, 0.0421 mmol), Pd2dba3 (9.6 mg, 0.0105 mmol), AsPh3(26 mg, 0.0842 mmol), and TEA (0.1 mL) were dissolvedin THF (20 mL) in a 50 mL Schelnk tube and reactedovernight under Ar at 60 °C. The reaction mixture wasthen diluted with CHCl3, washed three times with water,dried over CaCl2, and evaporated. The residue waschromatographed on silica gel using 17:3 hexanes:THFas the eluant. Trace amounts of a butadiyne-bridged bis-[(porphinato)zinc(II)] contaminant were removed via sizeexclusion chromatography using THF as the eluant togive 97 mg of the product (82% yield based on 46 mg of2). Vis (THF) λmax (log ε): 416 (4.85), 493 (4.99), 542(4.00), 563 (3.97), 760 (4.54) nm. 1H NMR (250 MHz, 50:1CDCl3: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),

544 Bioconjugate Chem., Vol. 16, No. 3, 2005 Wu et al.

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). MALDI-TOF MS (M+): 2823.7 (calcd for C112H200N12O12Zn3,2823.4).

LDL Reconstitution. A modified version of the classicKrieger reconstitution procedure (47) was used. HumanLDL was purchased from either Dr. Sissel Lund-Katz,who purified it from serum of patients with familialhypercholesteremia at the Children’s Hospital of Phila-delphia (48), or from Intracel Corporation (Frederick,Maryland). Prior to reconstitution, 1.9 mg of LDL, storedin 150 mM NaCl with 0.01% EDTA at pH 7.2, wasdialyzed at 4 °C against a PBS buffer (137 mM NaCl,2.7 mM KCl, 10 mM phosphate, pH 7.4) containing 0.3mM EDTA, following which it was lyophilized in thepresence of 25 mg of starch in a test tube pretreated withan antiwetting agent (Sigmacote). Endogeneous lipidswere extracted by vortexing the lyophilized apoprotein/starch mixture with 5 mL of heptanes. The LDL-contain-ing suspension was centrifuged at 2000 rpm for 10 min,and the supernatant was discarded. This procedure toextract 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 in200 µL of benzene was added to the LDL pellet andincubated at 4 °C for 90 min. The organic solvents werethen removed under a stream of Ar at -15 °C over a 30min period. The reconstituted LDL was solubilized bybriefly sonicating the residue in 2 mL of tricine buffer(10 mM, pH 8.4); this solution was incubated at 4 °C for24 h. Starch and excess protein were removed by low-speed centrifugation (2000 rpm) at 4 °C for 10 min. Thesupernatant was collected and further clarified by cen-trifuging at 9000 rpm for two 10 min periods. Theconcentration of the reconstituted LDL solutions wasdetermined by the Lowry method (49). ReconstitutedLDL was stored under a nitrogen atmosphere at 4 °C.The integrity of the LDL(PZn3) protein solutions, asdetermined from measurements of fluorescence intensity,remained constant over 8 weeks.

Cytotoxicity Experiments. The in vitro cytotoxicitiesof PZn3a, PZn3b, and their corresponding reconstitutedLDL(PZn3) protein solutions [LDL(PZn3a) and LDL-(PZn3b)] were determined using a clonogenic assay. B16melanoma cells from a cell line that overexpresses LDLr(generously provided by Dr. Theodore van Berkel, LeidenUniversity, The Netherlands) were plated on six well

plates overnight for cell attachment. Cells were exposedto DMSO solutions of PZn3a, PZn3b, and solutions (10mM tricine, pH ) 8.4) containing the analogous recon-stituted LDL(PZn3) proteins for 3 h, 24 h, and 8 days(no rinse). After exposure, the cells were twice rinsed withHBSS and then added to DMEM media supplementedwith 10% fetal bovine serum and 25 mM HEPES buffer.After incubation for 8 days, the media were discardedand the cells were fixed with 5 mL of 99.5% ethanol for10 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 clonogenicassay.

Cellular Uptake and Imaging. B16 murine mela-noma cells (2 × 104) were plated into two well chamberslides and incubated for 3 days at 37 °C prior totreatment with LDL(PZn3a) and LDL(PZn3b). The cellswere incubated with the reconstituted dye solutions (1µg/mL) for 1 h and subsequently washed twice withHBSS prior to fixing with absolute EtOH for 10 min atambient temperature. The slides were then sealed andmounted for confocal imaging.

RESULTS AND DISCUSSION

Synthesis. The synthesis of PZn3-based NIRFs isoutlined in Scheme 1 (33, 38, 50). Because of the oxidativeinstability of cholesteryl oleate, reactions involving 5-an-drosten-17â-amino-3â-yl oleate-modified (porphinato)-zinc(II) species proceeded in lower yields relative toanalogous syntheses of PZn3 NIRFs lacking ancillarysteroidal groups (PZn3a, PZn3c, and PZn3d). Becauseendogeneous LDL lipids are completely displaced duringits reconstitution, the NIRFs utilized for LDL reconstitu-tion reactions must be sufficiently lipophilic in order tocircumvent nonspecific leakage and aggregation of thereconstituted LDL (22). Furthermore, solubility of thePZn3-based NIRFs in apolar organic solvents is alsocrucial for incorporation into the LDL interior. All thePZn3 ancillary substituents highlighted in Scheme 1satisfy these constraints.

Note that the 4′-arylester groups of PZn3c providefunctionality for conjugation to moieties that engenderaugmented lipophilic character. PZn3b, which bears two5-androsten-17â-amino-3â-yl oleate esters, was synthe-sized from synthetic precursors to PZn3c to determinethe extent to which such modifications impact LDL

Scheme 1. Synthetic Route to NIRFs


reconstitution with these tris[(porphinato)zinc(II)] speciesand affect subsequent cellular uptake by B16 murinemelanoma cells. Previous studies demonstrated the util-ity of this approach for driving incorporation of cytotoxicand phototoxic agents into the LDL interior and facilitat-ing their respective release into cells via LDLr-mediatedendocytosis (17, 25, 29, 31, 51, 52), consistent with thefact that oleoyl residues are abundant components ofnative LDL (53).

LDL Reconstitution. Following the modified Kriegerprotocol for LDL reconstitution outlined above (seeExperimental Procedures) (47), NIRFs PZn3a and PZn3bwere successfully incorporated into the LDL lipid core.Neither PZn3c nor PZn3d was taken up into apo-LDLunder these conditions. These experiments suggest thatwithout the cholesteryl oleate lipid anchor, PZn3c lacksthe hydrophobicity necessary to drive LDL incorporation;furthermore, it is apparent that the increased PZn3dhydrophobicity relative to that of successfully-incorpo-rated PZn3a neither enhances nor guarantees successfulreconstitution. This result may indicate that PZn3damphiphilicity has decreased below the threshold re-quired for LDL compatibility; qualitatively similar ob-servations emerge from structure-activity relationshipsdelineated for amphiphilic drugs, which often show thatpotency is parabolically related to lipophilicity (54-56).Alternatively, disparate NIRF solvational environmentsthat trace their genesis to the nature of the PZn3 meso-aryl-pendant solubilizing groups may serve as a keydeterminant of the extent to which PZn3a and PZn3dare taken up into apo-LDL under these conditions (53).

Figure 1 displays representative optical spectra ofPZn3-based NIRFs in low dielectric strength solvent andfor reconstituted LDL(PZn3) proteins. These opticalspectra display absorptive and emissive signatures char-acteristic of extensive π conjugation and exciton coupling,similar to those elucidated for closely related compoundsin higher dielectric strength media (33-42). These fea-

tures include substantial B-state (S0fS2 absorption)domain spectral breadths, high oscillator strength, lowenergy Q-derived S0fS1 transitions, and correspondinglyintense S1fS0 fluorescence emission bands. Assumingthat the electronic absorption extinction coefficients ofthese NIRFs within the LDL hydrophobic core are similarto those determined previously in hydrophobic solvents,we estimate that ∼30 chromophores are incorporated perLDL protein; note that this result is consistent withprevious data that show that similar copy numbers of arange of amphipathic drugs are internalized into LDLusing analogous reconstitution methodologies (25, 27, 52,57).

With respect to other classes of NIRFs that have beenincorporated into the LDL core (29, 31), note that theelectronic absorption characteristics of these PZn3-basedchromophores include unusually comprehensive coverageof the UV, visible, and high energy NIR regions of thesolar spectrum. Because PZn3 and related chromophoresexhibit rapid S2fS1 internal conversion rate constants(τic ∼ 150 fs), which are 1 order of magnitude faster thanthat manifested by conventional porphyrin monomers,formation of the low energy emitting state thus occurswithin the ultrafast time domain, regardless of whetherthese species are excited within their lowest energyabsorption band or within the higher lying B-statemanifold (36, 40); hence, optical excitation of the dyescould be performed at any wavelength between 400 and800 nm.

Cytotoxicity of PZn3a, PZn3b, and Their Corre-sponding Reconstituted LDL(PZn3) Proteins. Thein vitro cytotoxicities of PZn3a, PZn3b, and their corre-sponding reconstituted LDL(PZn3) proteins [LDL(PZn3a)and LDL(PZn3b)] were determined using a clonogenicassay. These data show that these conjugates display noapparent toxicity to murine B16 cells, as evident fromthe respective cellular survival rates determined as afunction of time following exposure (Figure 2).

Murine B16 melanoma cells have been shown to bindLDL with affinity similar to human parenchymal cellsand are thus good models for evaluating the effectivenessof LDLr-mediated delivery (58, 59). Note that the Figure2 data do not allow determination of IC50 values forPZn3a, PZn3b, LDL(PZn3a), or LDL(PZn3b) at 3 or 24h of exposure, due to their apparent relative nontoxicity.The IC50 value determined for PZn3a at 8 days ofexposure was ∼70 µg/mL, while that for PZn3b was 45µg/mL. The cytotoxicities for both PZn3a and PZn3bincreased slightly as a function of exposure time. Notethat the toxicity profiles of PZn3a and PZn3b contrastwith that determined for tetrakis[(4-sulfonatophenyl)-porphinato]zinc(II) on G361 human melanoma cells (IC50

> 125 µg/mL, texposure ) 60 min) (60). Furthermore, the50% inhibitory concentrations for both LDL(PZn3a) andLDL(PZn3b) were greater than 1000 ng/mL at 8 days ofexposure. The benign nature of these fluorophore-reconstituted lipoproteins is similar to that establishedpreviously for in vitro toxicity studies of 3,3′-dioctade-cylindocarbocyanine-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 epithe-lium 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 amountof time, no cells survived. Even at a very short exposuretime of 3 min at 0.5 mg/mL of ICG, RPE cell survivalrate was modestly reduced to 92.8% (63). In contrast, no

Figure 1. Electronic absorption and emission spectra of tris-[(porphinato)zinc(II)] fluorophores. (A) Cholesteryl oleate-conjugated NIRF PZn3b in benzene solvent. (B) ReconstitutedLDL(PZn3a) in tricine buffer (10 mM, pH 8.4). Correctedemission spectra, labeled with the emission wavelength maxi-mum, are shown in the insets.


significant toxicity can be detected for either PZn3a orPZn3b on B16 cells even after 24 h of exposure at 100and 80 µg/mL concentrations, respectively. These datasuggest that the PZn3 family of fluorophores, as well asrelated structural analogues of these species, may provesuitable for in vivo studies.

Confocal Microscopy. Cellular uptake of the LDLreconstituted dye solutions was visualized using laserscanning confocal fluorescence microscopy (Figure 3).Figure 3A,C,E shows transmission images of B16 mela-noma cells, while panels B, D, and F illustrate theircorresponding cellular NIR fluorescence images. As

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 pointsrepresent 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 FluorescenceMicroscope Images of Tumor Cells Qualitatively Similar to that Depicted in Figure 3B as a Function of NIRF, DeliveryPlatform, 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 tocontain ∼75 dye molecules (29, 64). b Somatostatin receptor subtype 2 transfected rat insulinoma pancreatic tumor. c Incubation of dye-peptide conjugate was performed at 4 °C. d Fluorescence signal generated through the action of proteolytic enzymes preferentially expressedin tumor tissues that release cyanine dyes from a protein/polymer matrix in which NIR fluorescence was previously quenched. e XGhuman lung carcinoma.


Figure 3 clearly demonstrates, both LDL(PZn3a) andLDL(PZn3b) were internalized by B16 cells within 1 h;intense fluorescence signals were detected throughout thecellular membranes of the B16 cells. When B16 cells wereincubated in culture medium to which aliquots of DMSOsolutions of PZn3a and PZn3b (1 µg/mL) were added, noemission was detected from the cells (Figure 3E,F),indicating that the NIRF uptake highlighted by theFigure 3 data is mediated solely via LDLr-mediatedendocytosis.

The low doses of dye ([LDL(PZn3)] ) 1 µg/mL ) 2 nMprotein; [NIRF] ) 60 nM) used to obtain the confocalimages highlight the emissive characteristics of thePZn3-based NIRFs and the utility of LDL as an efficientvehicle for delivery of multiple copies of these fluoro-phores to a single cell. The short incubation time requiredfor fluorophore uptake in these experiments demon-strates the rapid internalization of LDL through the B16cells’ overexpressed LDLrs, a characteristic displayed bymany tumor types (22).

Table 1 highlights treatment conditions required forpreviously studied biomolecule-dye conjugates to obtain

qualitatively similar confocal NIR fluorescence imagesof in vitro-targeted cancer cells. In vitro cellular deliveryof the NIRF pyropheophorbide via the glucose transporter(Table 1, entry E) requires 20-fold greater concentrationof fluorophore to generate a confocal image similar inquality to that obtained for delivery of the same dye viaLDL reconstitution and subsequent LDLr-mediated en-docytosis (Table 1, entry B) (50). Note that the amountof LDL-reconstituted pyropheophorbide necessary togenerate NIR fluorescence confocal images similar to thatshown in Figure 3B,D is at least 50 times higher thanthat used in this study (Table 1, entry A) (29). Because(i) dye doses cited in Table 1 do not necessarily cor-respond to the minimal amount necessary for obtaininga suitable fluorescence image and (ii) parameters suchas instrument sensitivity, imaging acquisition times, andcamera settings cannot be directly compared, we empha-size that the experimental data comparisons highlightedTable 1 are qualitative in nature. Nonetheless, the smallconcentration of PZN3 fluorophore required to obtain thein vitro images shown in Figure 3B,D underscore thepromise of this NIR emissive platform for imagingapplications.

CONCLUSIONS

We have shown that meso-to-meso ethyne-bridged tris-[(porphinato)zinc(II)] (PZn3) NIRFs can be renderedsufficiently amphiphilic to enable their facile incorpora-tion into the hydrophobic core of apo-LDL. These recon-stituted LDL(PZn3) proteins can be used to deliverrapidly hundreds of copies of PZn3 to a given B16melanoma cell via LDLr-mediated endocytosis. PZn3-based NIRFs and their corresponding LDL(PZn3) pro-teins display minimal cytotoxicity. Confocal NIR fluores-cence microscopy evinces that B16 cells can be imagedat very low doses (∼nM) of NIRF. The highly attractivephotophysical properties of PZn3 and closely relatedchromophores, coupled with their lack of toxicity andcompatibility with uptake into apo-LDL and subsequentrapid delivery to B16 cells via LDLr-mediated endocy-tosis, suggest the potential utility of this platform for NIRoptical imaging of cancer cells in vivo.

ACKNOWLEDGMENT

This research was supported by the National CancerInstitute (N01-CO-29008). J.D.G. acknowledges supportfrom the National Cancer Institute (R24-CA83105-05 andP20-CA86255), and G.Z. thanks NASA and NCI (N01-CO-37119) for funding. S.P.W. is grateful to the NationalScience Foundation for an Access Science PredoctoralFellowship. P.P.G. acknowledges predoctoral fellowshipsupport from the NIH Medical Scientist Training Pro-gram (MSTP) and the Whitaker Foundation. We areindebted to Gladys Gray-Board for her assistance withthe confocal microscopy experiments, to Dr. Sissel Lund-Katz of the Children’s Hospital of Philadelphia for LDL,to Dr. Hui Li for guidance in the LDL reconstitutionexperiments, and to Dr. Theo van Berkel of LeidenUniversity for providing the B16 melanoma cell line thatoverexpresses LDLr.

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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 microscopyimage of B16 cells treated with LDL(PZn3b). (D) Fluorescenceimage of panel C. (E) Transmission microscopy image of B16cells incubated in culture medium to which aliquots of DMSOsolutions of PZn3a (1 µg/mL) were added. (F) Fluorescenceimage of panel E. Experimental conditions: B16 cells (2.5 ×104) were incubated at 37 °C for 3 days and then treated withsolutions of LDL(PZn3) (1 µg/mL; 10 mM tricine buffer, pH 8.4)for 1 h at ambient temperature. Following cellular washing andfixing with EtOH (see Experimental Procedures), slides weresealed and mounted. All images were taken at the samemagnification using λex ) 488 nm and a 700 nm long pass filter.


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