Synthesis and Evaluation of a Stable Bacteriochlorophyll-Analog and

Oct 19, 2009 - Kenneth K. Ng , Jonathan F. Lovell , and Gang Zheng ... Susmita Das , Mark Lowry , Bilal El-Zahab , Sayo O. Fakayode , Maxwell L. Geng ...
0 downloads 0 Views 2MB Size
Download PDF
Bioconjugate Chem. 2009, 20, 2023–2031

2023

Synthesis and Evaluation of a Stable Bacteriochlorophyll-Analog and Its Incorporation into High-Density Lipoprotein Nanoparticles for Tumor Imaging Weiguo Cao,†,§,⊥ Kenneth K. Ng,‡,⊥ Ian Corbin,† Zhihong Zhang,†,| Lili Ding,† Juan Chen,† and Gang Zheng*,†,‡ Department of Medical Biophysics, Ontario Cancer Institute, Institute of Biomaterials and Biomedical Engineering, University of Toronto, Canada, Department of Chemistry, Shanghai University, China, and Britton Chance Center for Biomedical Photonics, Huazhong University of Science and Technology, Wuhan, China. Received September 14, 2009

The syntheses of novel near-infrared (NIR) dyes with excellent optical properties in biological tissues have driven the continued improvement of fluorescence imaging of deeply seated tumors. Bacteriochlorophyll a (Bchl), a dye synthesized by the phototrophic bacteria, R. sphaeroids, is particularly suited for deep tissue imaging due to its high absorbance coefficient and good fluorescence quantum yield in the NIR spectrum. However, obstacles that impede the development of this fluorophore are its poor stability and lack of tumor specificity. These issues ultimately limit its utility for tumor detection. Herein we describe a robust synthesis of a novel Bchl analog, bacteriochlorin e6 bisoleate (BchlBOA), which is chemically stable, has excellent photophysical properties (ex, 752 nm; em, 762 nm) and is tailored for the incorporation into a tumor targetable high-density lipoprotein (HDL)like nanoparticle (NP). Incorporating BchlBOA into HDL (HDL-BchlBOA) yielded 12 nm sized particles, corresponding well with the diameter of native HDL. Functional cell uptake studies showed that HDL-BchlBOA was taken up by cells expressing the HDL receptor, scavenger receptor B type I (SR-BI), and was inhibited by 25-fold excess native HDL. Furthermore, the NP was successfully detected in KB cancer cells both in vitro and in tumor xenografts. Taken together, these results demonstrate that we successfully synthesized and formulated a stable analog of Bchl that is capable of being incorporated within HDL-like NPs for tumor-targeted imaging.

INTRODUCTION Near-infrared (NIR) fluorescence imaging of cancerous tumors offers several advantages over other imaging modalities. These advantageous include its noninvasive detection, good sensitivity, and utilization of nonionizing radiation. Since the absorbance coefficients of endogenous absorbers including water and hemoglobin are at a minimum within the NIR window (650-900 nm) (1), NIR fluorescent probes offer optimal spectral properties and can theoretically be used to visualize tissues as deep as 10 cm depending on tissue type and the imaging modality (2). Polymethine cyanine-based dyes are the most commonly used NIR probes. These dyes are very useful in designing probes with adjustable wavelengths because they can be tuned to desired wavelengths by altering the heterocyclic nucleus (indolenine or benzindolenine) and the number of double bonds (n ) 1-3) in the polymethine chain. For example, the water-soluble and bioconjugatable CyDyes (3) such as Cy5 (n ) 2, indolenine nucleus, Exmax, 649 nm; Emmax, 670 nm), Cy5.5 (n ) 2, benzindolenine nucleus, Exmax, 676 nm; Emmax, 694 nm), and Cy7 (n ) 3, indolenine nucleus, Exmax, 743 nm; Emmax, 767 nm) are NIR active and proven to be useful in a wide range of in vivo imaging applications (4-6). Furthermore, the only FDA approved NIR dye, indocyanine green (ICG), also belongs to this family (n ) 3, benzindolenine nucleus, Exmax, 805 nm; Emmax, 830 nm, ξ > 150 000 M-1 · cm-1 with a quantum * To whom correspondence should be addressed. Address: TMDT 5-363, 101 College St, Toronto, ON M5G 1L7, Canada. Fax: 416581-7666. E-mail: [email protected]. † Department of Medical Biophysics, Ontario Cancer Institute, University of Toronto. ‡ Institute of Biomaterials and Biomedical Engineering, University of Toronto. § Shanghai University. | Huazhong University of Science and Technology. ⊥ These authors contributed equally to this research article.

efficiency ∼4-5%) and is approved for imaging of retinal vasculature (7) and monitoring hepatic function (8). Another important class of exogenous probes are the porphyrin-based or porphyrin-like fluorophores (phthalocyanines, expanded porphyrins, porphycenes, etc.). Porphyrins are 18 π-electron aromatic macrocycles, where two of its peripheral double bonds are not required to maintain aromaticity. Thus, reduction of one or both of these bonds respectively leads to chlorins and bacteriochlorins with bathochromically shifted bands with higher extinction coefficients. Generally, the longest wavelength absorptions (and extinction coefficients) for porphyrin, chlorin, and bacteriochlorin are at 630 nm (ε ∼5 000 M-1 · cm-1), 660 nm (ε ∼45 000 M-1 · cm-1) and 760 nm (ε ∼75 000 M-1 · cm-1), respectively (9). Nature uses the optical properties of the reduced porphyrins to harvest solar energy for photosynthesis with chlorophylls and Bchls acting as both antenna and photochemical reaction center pigments. The NIR absorption and fluorescence of these natural dyes clearly make them potential candidates as contrast agents for NIR optical imaging, although they are better known for their applications as photodynamic therapy (PDT) agents due to their high triplet state and singlet oxygen quantum yields. Nevertheless, there are already some reports using NIR-active porphyrin-based fluorophores for cancer imaging (10-13) and the fact they are both NIR fluorophores and photosensitizers make them ideal candidates for image-guided therapy (14-17). Bchl extracted from R. sphaeroids is a NIR fluorophore with excitation and emission profile (750-850 nm) highly suited for imaging deeply seated tumors. However, two issues which hinder the utilization of this agent for tumor imaging include (i) poor stability caused by rapid oxidation and (ii) lack of specificity for cancer tissue. Various investigators have attempted to address the poor stability of the Bchl molecule. These include replacing the central magnesium with other metal ions (e.g., palladium) to form more stable complexes (18), modifying

10.1021/bc900404y CCC: $40.75  2009 American Chemical Society Published on Web 10/19/2009

2024 Bioconjugate Chem., Vol. 20, No. 11, 2009

the isocyclic ring (19), and replacing the phytyl group at the propionyl residue either through transesterification or by conversion to an amide derivative (20). In particular, Pandey et al. have shown that naturally occurring unstable Bchl a can be converted to stable bacteriochlorins, bacteriopurpurin-18, and bacteriopurpurinimide (19). In this approach, converting the fused isocyclic ring to a cyclic imide moiety enhanced the stability and solubility of the Bchl analogs, while attachment of a variable alkyl substituent to the imide moiety allowed the lipophilicity of the molecule to be fine-tuned (21). In another elegant demonstration, Fiedor et al. found that inserting palladium into the Bchl ring significantly improves the stability of this molecule, however fluorescence properties of this complex is compromised (22). Nevertheless, this Bchl analog led to the development of the promising PDT agent Tookad, which is currently in clinical trials (23, 24). So far there have been minimal efforts in developing target-specific Bchls and even fewer attempts in exploring the utility of stable Bchl analogs as NIR fluorescent probes. High-density lipoproteins (HDL) are endogenous carriers used to transport hydrophobic compounds throughout the body. The structural/functional properties of these particles, ultrasmall size (7-12 nm) (25), long circulation time (26, 27), nonimmunogenic, and biodegradable, also make them attractive as delivery vehicles for exogenous compounds. HDL’s hydrophobic core facilitates the incorporation of hydrophobic compounds into this carrier, while the small size and favorable surface properties enable these particles to remain undetected by the reticular endothelial system as well as rendering them accessible to most tissues. Furthermore, some cancer cell lines have been shown to have a high expression of the HDL receptor, formally known as scavenger receptor type B1 receptor (SR-BI). Since HDL’s core lipid transfer is mediated through the SR-BI pathway, targeting this receptor represents a novel way to deliver imaging agents to cancer tumors which overexpress this receptor. This paper outlines (1) a simple and high yield synthesis of a stable Bchl analog; (2) methods of actively incorporating this Bchl analog into HDL-like NPs (HDL BchlBOA); and (3) the utility of HDL-BchlBOA as a novel diagnostic agent for cancer imaging. The overall goal of this work is to combine the advances of both technologies to generate a new tool for the diagnostic imaging of cancers in vivo.

EXPERIMENTAL PROCEDURES Reagents. All chemicals were of reagent grade and used as such. Reactions were carried out under nitrogen atmosphere and were monitored by precoated (0.20 mm) silica TLC plastic sheet (20 × 20 cm) strips (POLYGRAM SIL N-HR) and/or UV-visible spectroscopy. Silica gel 60 (70-230 mesh, Merck) was used for column chromatography. UV-visible spectra were recorded on a Varian (Cary-50 Bio) spectrophotometer. Fluorescence spectra were collected using a FluoroMax-4 (HORIBA Scientific, Kyoto, Japan) spectrofluorometer. Bacteriopheophorbide a (1) was synthesized from R. sphaeroids (Frontier Sciences, Utah). Proton NMR spectra were recorded on a Bruker AMX 400 MHz NMR spectrometer operating at 303 K. Proton chemical shifts (δ) are reported in parts per million (ppm) relative to CDCl3 (7.26 ppm), or TMS (0.00 ppm). MS and purity data were obtained by a Waters Analytical HPLC equipped with a Waters 600 Controller, a Delta 600 pump, and a 996 Photodiode Array Detector and a Waters ZQTM mass detector (Waters Limited, Mississauga, ON). A reverse phase, Symmetry C18, 5 µm, 4.6 × 150 mm column (Waters, Made in Ireland) was used under an isocratic setting of MeCN/H2O for all compounds. The solvent flow rate was kept constant at 1.00 mL/min. All final products were found to be >95% pure. Synthesis of BchlBOA. Synthesis of Bacteriopheophorbide a (Bchl-acid 1). Bacteriopheophorbide a (Bchl-

Communications

acid 1) was synthesized from Bchl which was extracted from R. sphaeroids in an inert atmosphere using 1-propanol following literature methods (28). Synthesis of Bacteriochlorin e6 Di-N-Boc-aminopropylamide (Bchl-2BOC 2). Bchl-acid 1 (330 mg, 0.54 mmol) was first dissolved in 20 mL of dichloromethane. To this solution, 160 mg of 4-N,N-dimethylaminopyridine (DMAP, 1.31 mmol) and 400 mg of N-Boc-1,3-diaminopropane (2.30 mmol) were added. This mixture was stirred under argon for 30 min at room temperature. Subsequently, 260 mg of dicyclohexylcarbodiimide (DCC, 1.26 mmol) was added, and the mixture was stirred for 40 h. The solvent was then removed by vacuum. The crude product was purified by silica gel column chromatography with 8% acetone in dichloromethane, and then with 4% methanol in dichloromethane. The desired product Bchl2BOC 2 (368.3 mg) and a side-product Bchl-BOC-DCC 3 (140.8 mg) were obtained in 72% (0.39 mmol) yield and 26% yield (0.14 mmol), respectively. Bchl-2BOC 2: Mass calculated for C51H72N8O9: 941.17. Found by ESI-MS: 941.8 (M+). 1H NMR (CDCl3, δ ppm): 9.31, 8.65, and 8.56 (each, s, 3H for 5, 10, 20-H), 7.30, 6.42, 6.07, and 5.98 (each, brs, 4H for 4 × NH), 5.21 (s, 2H, 151-CH2), 4.32 (br, 1H for 18-H), 4.25 (brs, 1H, for 7-H), 4.23 (d, 1H, for 17-H), 4.14 (brs, 1H, for 8-H), 3.72 (s, 3H, for 152-CO2CH3), 3.69 (m, 2H, for 133-NCH2), 3.39 (s, 3H, for 12-CH3), 3.38 (m, 2H, 175-NCH2), 3.32 (s, 3H, for 2-CH3), 3.17 (s, 3H, for 3-COCH3), 2.84-2.59 (m, 4H, for 135-CH2N, 177-CH2N), 2.36 (m, 1H, for 171-H), 2.22 (m, 1H, for 171-H), 2.04 (m, 2H, for 172-CH2), 1.94 (m, 2H, for 81-CH2), 1.82 (d, 3H, for 18-CH3), 1.63 (m, 5H, for 7-CH3, 134-CH2), 1.40-1.23 (brs, 20H, for 176-CH2, 139-C(CH3)3, 1711-C(CH3)3), 1.08 (t, 3H, for 82-CH3), -1.21 and -1.25 (each, s, 2H, for 2 × NH). Bchl-BOC-DCC 3: Mass calculated for C56H78N8O8: 991.27. Found by ESI-MS: 991.8 (M+). 1H NMR (CDCl3, δ ppm): 9.29, 8.67, and 8.57 (each, s, 3H for 5, 10, 20-H), 7.14 and 5.16 (each, brs, 2H for 2 × NH), 5.16-5.35 (m, 3H, 151-CH2 + NH), 4.20-4.33 (m, 4H for 18-H, 7-H, 17-H and 8-H), 3.72 (m, 2H, for 133-NCH2), 3.70 (s, 3H, for 152-CO2CH3), 3.58 (s, 3H, for 12-CH3), 3.35 (s, 3H, for 2-CH3), 3.18 (s, 3H, for 3-COCH3), 2.36 (m, 1H, for 171-H), 2.25-2.29 (m, 2H, for 135-CH2N), 1.82-2.08 (m, 7H, for 171-H, 172-CH2, 81-CH2 and 2 × N-CH in cyclohexyl), 1.59 (d, 3H, for 18-CH3), 1.52 (m, 2H, 134CH2), 1.39 (s, 9H, 139-C(CH3)3), 1.32 (m, 3H, for 7-CH3), 0.83-1.25 (m, 23H, for cyclohexyl-H and 82-CH3), -1.23 and -1.27 (each, s, 2H, for 2 × NH). Synthesis of Bacteriochlorin e6 Diaminopropylamide (Bchl-2NH2 4) and Bacteriochlorin e6 Bisoleate (BchlBOA 5). Bchl-2BOC 2 40 mg (0.043 mmol) was dissolved in 1.5 mL of trifluoroacetic acid (TFA). The solution was stirred first in an ice-water bath under argon atmosphere for 1 h, and then at room temperature for 1 h. The TFA was removed under vacuum, the crude product Bchl-2NH2 4 did not require further purification and was used directly for the next reaction. The crude product Bchl-2NH2 4 was dissolved in 5 mL dichloromethane upon which 0.15 mL of diisopropylethylamine (DIPEA) was added to the solution. The mixture was then stirred under argon atmosphere at room temperature for 0.5 h, oleoyl chloride 0.15 mL was added into the mixture and stirred for an additional 4 h at room temperature. The solution was poured into 50 mL of ice water and washed with ice water three times (3 × 20 mL). The organic layer was dried over anhydrous Na2SO4 (100 mg) for 1 h. After the solvent was removed by vacuum, the crude product was purified by silica gel column chromatography with 5% methanol in dichloromethane. The desired product BchlBOA 5 (30 mg, 0.024 mmol) was obtained in 63% yield from two steps.

Communications

Bchl-2NH2 4: Mass calculated for C41H56N8O5: 740.93. Found by ESI-MS: 741.5 (M+). BchlBOA 5: UV-vis in CHCl3 λmax: 357 nm (ε, 8.6 × 104), 386 nm (6.5 × 104), 522 nm (2.4 × 104), and 752 nm (8.4 × 104). Mass calculated for C77H120N8O7: 1269.83. Found by ESIMS: 1270.2 (M+). 1H NMR (CDCl3, δ ppm): 9.31, 8.67, and 8.57 (each s, 3H for 5-H, 10-H, and 20-H), 7.16, 6.36, 5.98, and 5.82 (each brs, 4H for 4 × NH), 5.31-5.15 (m, 6H, for 2 × CHdCH in oleoyl and 151-CH2), 4.32 (brs, 1H, for 18-H), 4.25 (brs, 1H, for 7-H), 4.22 (brs, 1H, for 17-H), 4.13 (brs, 1H, for 8-H), 3.68 (s, 3H, 15-CO2CH3), 3.66 (m, 2H, for 133-NCH2), 3.55 (s, 3H, 12-CH3), 3.47 (m, 2H, 175-NCH2), 3.30 (s, 3H, for 2-CH3), 3.15 (s, 3H, for 3-COCH3), 2.91-2.83 (m, 4H, for 135CH2N, 177-CH2N), 2.37 (m, 1H, 171-H), 1.94-2.08 (m, 25H, 171-H, 1314-CH2-CHd, 1716-CH2-CHd, 1317-dCH-CH2, 1319- dCH-CH2, 138-COCH2, 1710 COCH2, 172-CH2, 1312-CH2, 1313-CH2, 1714-CH2, 1715-CH2, and 81-CH2), 1.82 (d, 3H, 18CH3), 1.61 (d, 3H, 7-CH3), 1.51 (m, 2H, 134-CH2), 1.43 (m, 2H, 176-CH2), 1.23-1.17 (brs, 34H, other H in oleoyl), 1.08 (t, 3H, 82-CH3), 0.85 (m, 6H, 1324-CH3, 1726-CH3), -1.24 and -1.27 (each s, 1H, 2 × NH). Apolipoprotein Preparation. Lipoproteins were isolated by sequential ultracentrifugation of human plasma using standard methods (29). Expired human plasma was obtained from the local blood transfusion services and was approved by the University Health Network’s research ethics board. Apolipoprotein A-1 (ApoA-1) was isolated from HDL by delipidation with organic solvents as reported by Scanu and colleagues (30). All incubation steps were conducted at -20 °C. Briefly, 1 mL of HDL in 0.1 M sodium phosphate buffer (pH 7.5) was pipetted into a 50 mL Falcon tube containing 50 mL of a precooled 3:2 ethanol/diethyl ether mixture. This solution was mixed in a rotating mixer for 6 h. The precipitate was pelleted by centrifugation at 2000 rpm for 10 min and the supernatant was decanted. After this step, the pellet was resuspended in 50 mL of fresh diethyl ether and mixed in a rotating mixer for an additional 18 h. Subsequently, the delipidated protein was subjected to 3 washes of diethyl ether. After the last step, each tube of delipidated ApoA-1 protein was resuspended in 1 mL of fresh diethyl ether, aliquoted and dried using a centrifuge under high vacuum. Formulation of HDL-BchlBOA. HDL-BchlBOA was formulated by combining BchlBOA (0.35 mg), dimyristoylphosphatidylcholine (DMPC) (2.16 mg) and cholesterol oleate (CO) (0.08 mg) in a 5 mL glass tube. The mixture was dried by vortexing the sample under N2 for 5 min. Once the solvent was evaporated, the sample was dried under vacuum for 1 h. The dried film was resuspended with 1 mL Tris buffered saline (10 mM Tris, 0.1 M KCl, 1 mM EDTA; pH 8.0; degassed) and transferred to a sonicator bath (Bransonic Model 5510, Branson, Danbury, CT) where it was sonicated for 1.5 h at 50 °C. The sample was diluted to 8 mL with Tris buffer and returned to the sonication bath at 40 °C. ApoA-1 (4 mg) dissolved in 1 mL of urea (4 M) was added dropwise to the lipid-dye complex over a period of 10 min. The formulation was then sonicated for an additional 50 min. Subsequently, the sample was removed from the bath and allowed to cool to room temperature before storing overnight (approximately 15 h) at 4 °C. The next day, the sample was passed through a 0.22 µm filter and concentrated to 2 mL using a 10 000 MW cutoff Amicon centrifugal filter device (Millipore, Billerica, MA) (980 g for 30 min). The sample was then purified by fast protein liquid chromatography (FPLC) using a Superdex 200pg 26/60 column (GE Healthcare, Piscataway, NJ) and stored at 4 °C until use. Characterization of Formulation. Chemical Composition. The total protein content of the formulation was measured using a

Bioconjugate Chem., Vol. 20, No. 11, 2009 2025

modified Lowry Protein Assay (Sigma-Aldrich, St. Louis, MO). BchlBOA concentration was measured by extracting the dye twice using a CHCl3/MeOH mixture (2:1). The organic layer was dried by vacuum and redissolved in 1 mL CHCl3. The concentration of BchlBOA in the sample was calculated from the molar extinction coefficient by measuring the absorbance at 752 nm. Dynamic Light Scattering (DLS). The volume size distribution of the HDL-BchlBOA particles was obtained by DLS (Nanosizer ZS90, Malvern, Worcestershire, UK). Transmission Electron Microscopy (TEM). TEM (Hitachi 7000, Japan) in combination with negative staining was used to visualize the morphology of the HDL-like NPs. Briefly, carbon film 400 mesh copper grids (Lakefield, QC, Canada) were glow discharged prior to addition of the sample. Ten microliters of purified HDL formulations diluted 1000-2000 times using distilled and deionized H2O was pipetted onto the copper grid such that a droplet was formed. The droplet was allowed to stand on the copper grid for 1 min. Subsequently, 6 µL of 1% uranyl acetate was added to the droplet and allowed to stand for 1 min. All liquid was then removed by whisking the droplet away using filter paper. The sample was allowed to dry for 1 min before mounting into the apparatus. The acceleration voltage of the TEM was set to 75 kV. Samples were viewed at 100 000-150 000 times magnification. Cell Culture. All cells were cultured in 5% CO2/95% air at 37 °C. KB cells were grown as a monolayer in a 125 cm2 culture flask using modified Eagle’s medium (ATCC 30-2003), supplemented with 10% fetal bovine serum (FBS). SR-BI positive ldlA[mSR-BI] cells were cultured in RPMI 1640 medium supplemented with 10% FBS. SR-BI negative ldlA7 cells were cultured in Ham’s F-12 medium, supplemented with 2 mM L-glutamine, 1% penicillin/streptomycin, and 25% FBS. Western Blot Analysis. ldlA[mSR-BI], ldlA7, and KB cells were cultured in a 6-wellplate. After confluence, protein was extracted from the cells using lysis buffer (20 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerolphosphate) with a protease inhibitor cocktail (SigmaAldrich, St. Louis, MO) on ice for 10 min and centrifuged (11 000 g for 15 min, 4 °C). Supernatant protein levels were assayed using the Bradford method (Biorad Laboratories Inc., Hercules, CA). Twenty micrograms of each sample was separated on a 10% SDS-polyacrylamide gel and then transferred onto polyvinylidene difluoride membranes for standard Western blot analysis using monoclonal antibodies specific for SR-BI receptor (1:2000× dilution; AbCam, Cambridge, MA). Cell Uptake Studies. ldlA[mSR-BI], KB, ldlA7 cells were seeded into 8-well Lab-tek chamber slides (0.8 cm2/well) at a density of 3.0 × 104 cells per well. After 24 h, cells were washed with PBS followed by the addition of either cell media alone, cell media containing HDL-BchlBOA (8 µM), or cell media containing HDL-BchlBOA with 25-fold excess of HDL. The cells were allowed to incubate at 37 °C for 4 h after which the cells were washed twice with PBS and reincubated with 300 µL of incubation medium for imaging. Cell uptake was visualized using the Olympus FluoView 1000 laser scanning confocal microscope equipped with a Multiline Argon Laser and a 60× objective lens (oil; NA, 1.4). Fluorescence in the near-infrared was monitored from 700-800 nm using a tunable filter with a 515 nm excitation laser source. Animal Studies. All animal experiments were conducted in accordance to the animal use protocol approved by University Health Network’s animal care committee. Female athymic nude mice (7-8 weeks; 25 g) were purchased from Harlan Laboratories (Mississauga, ON). Tumor xenografts were generated in the right flank by inoculating mice subcutaneously with KB cells (3 × 106

2026 Bioconjugate Chem., Vol. 20, No. 11, 2009

Communications

Scheme 1. Synthesis of BchlBOA

cells) three weeks prior to the study. In order to decrease the background signal during fluorescence imaging, animals were fed a chlorophyll-free diet (TD.97184, Harlan Laboratories) 2 days before the imaging study. In Vivo Fluorescence Imaging. Spectral reflectance fluorescence imaging was conducted on KB tumor-bearing mice using the Maestro in vivo imaging system (Cambridge Research and Imaging Inc., Woburn, MA). The NIR fluorescence reflectance

system is comprised of a halogen light source with an excitation bandpass filter in the range of 641-681 nm and a 700 nm longpass emission filter connected to a CCD camera. The image cube was taken from 700-800 nm in 10 nm steps, while the exposure time was set to 2000 ms. Animals were prescanned to determine background autofluorescence. After prescanning, animals received an intravenous tail vein injection of 37 µM HDL-BchlBOA (dye concentration;

Figure 1. Schematic illustration of the formulation process for the incorporation of BchlBOA within HDL-like NPs. The size change of the particles in each step of the formulation is shown by DLS.

Communications

Bioconjugate Chem., Vol. 20, No. 11, 2009 2027

200 µL) and imaged at 1, 3, 6, and 48 h. After 48 h, animals were sacrificed and tissues were removed for tissue biodistribution characterization. Fifteen to 55 mg of tissue were weighed and homogenized in 1.5 mL phosphate-buffered saline. This homogenate then underwent organic extraction using CHCl3:MeOH (2:1). The organic layer was removed and measured by fluorescence (excitation, 742 nm; emission, 762 nm; slit size, 5 nm).

RESULTS AND DISCUSSION

Figure 2. Purification and characterization of HDL-BchlBOA. (A) Purification of the formulated sample by size exclusion FPLC yielded three major size populations. The largest peak was collected as it corresponded with the size of native HDL (filled in blue). (B) The purified HDL-BchlBOA was sized for DLS (inset) and imaged by TEM at 100 000× magnification with 75 kV acceleration voltage. Scale bar equals 100 nm.

The synthesis of BchlBOA is outlined in Scheme 1. The final product, BchlBOA 5 was synthesized via the following 4-step procedure: (1) Bchl was extracted from R. sphaeroids (15), which was further hydrolyzed in dilute HCl to generate the key starting material, Bchl-acid 1. (2) Bchl-acid was then reacted with N-Boc-1,3-diaminopropane in the presence of DMAP and DCC to yield the desired product Bchl-2BOC 2 in 72% yield as well as a minor side product, Bchl-BOC-DCC 3 in 26% yield. These products were obtained via (i) N-Boc-1,3-diaminopropane attack at the 13-C position of the β-keto ester, resulting in ringopening to form an amide bond and (ii) a DCC-activated amidation reaction of the carboxylic acid located on the 17-C side chain. (3) Deprotection of the BOC group was carried out in TFA, and the product Bchl-2NH2 4 was used directly in the next step. (4) The desired final product, BchlBOA 5, was synthesized through the reaction of Bchl-2NH2 with oleoyl chloride in the presence of DIPEA in dichloromethane. The purity of the product was confirmed to be 96% by analytical HPLC and the overall yield for deprotection and acylation from Bchl-2BOC was 63%. Native HDL particles transport lipophilic molecules such as triacylglycerides and cholesterol esters throughout the body by incorporating these molecules into its hydrophobic core. Introducing exogenous compounds to HDL particles can be challenging as the right conditions must be met to successfully incorporate compounds into the core of the particle. In general, the HDL core-loading works best with compounds containing a long unsaturated hydrocarbon chain (e.g., oleic or linoleic acid) or branched side chains (e.g., polyisoprenes such as phytol). Our strategy of incorporating the Bchl derivative into HDL was to attach dual unsaturated lipid “anchors” to the molecule. This strategy has been used in our lab and others to incorporate

Figure 3. Representative fluorescence microscope images of cells treated with HDL-BchlBOA. Wells were seeded with ldlA[mSR-BI] (A-C) or ldlA7 (D-F) cells 24 h before imaging at a density of 3 × 104 cells per well. Final well volume was 200 µL. Cells either had no treatment (A,D), 8 µM HDL-BchlBOA (dye concentration) (B,E), or 8 µM HDL-BchlBOA (dye concentration) with 25-fold molar excess of HDL protein (C,F). Cells were excited at 515 nm and emission was captured from 700-800 nm. Scale bars correspond to 20 µm.

2028 Bioconjugate Chem., Vol. 20, No. 11, 2009

compounds such as bisoleoyl fluorescein (31), oleoyl-conjugated 5-fluorodeoxyuridine (32), bisoleoyl derivatives of silicon phthalocyanine, (33) and naphthalocyanine (34) into low density lipoproteins. Furthermore, attempts to incorporate oleoyl conjugated molecules into HDL have also been successful (35). By utilizing this strategy in the development of a novel HDL compatible fluorophore, we synthesized Bchl with bisoleate chains at the 13 and 17-C positions and successfully incorporated it into the HDL-BchlBOA. The formulation of HDL-BchlBOA first involved preparing a thin film comprised of BchlBOA, CO, and DMPC. We found that a molar ratio of 22:1:2:1 (DMPC/CO/BchlBOA/ApoA-1) generated the desired HDL-sized NPs. In the initial steps, the addition of TRIS buffer and vortexing created a suspension of large emulsion particles, this is observed by the large and broad size distribution in DLS measurements (Figure 1). In order to prepare the sample for the addition of ApoA-1, we subsequently sonicated the formulation in a temperature-controlled bath sonicator at 50 °C for 1 h. Mechanical disruption of the lipid emulsion at this elevated temperature allowed for the generation of emulsion particles approximately 35 nm in diameter. At this point, the solution changed from an opaque appearance to a clear brown color. In the next step, ApoA-1 dissolved in 4 M urea, which served to maintain the protein in an unfolded state, was added over a period of 10 min to the diluted 8 mL emulsion. The concentration of urea decreased to 0.4 M upon addition to the sample, which allowed the protein to renature and interact with the lipid emulsion (36). Confirmation of this interaction and the formation of the desired HDL particles were validated by DLS size measurements as the diameter of the particle dropped to an average of 12 nm. This result corresponded well with the size of native HDL which ranges from 7-12 nm (25, 37). Lastly, purification by FPLC allowed us to isolate a narrow size distribution of NPs and remove any remaining unbound ApoA-1 (Figure 2A). We next calculated the payload of BchlBOA within the HDL and found that there were on average 3 dyes per ApoA-1 protein, while the incorporation efficiency was calculated to be 40%. With the assumption that each HDL particle had between 2 to 3 molecules of ApoA-1 (37), this results in 6-9 molecules of dye per particle and represents a significant improvement on previous HDL-like NP formulations (35). We next visualized our formulations by TEM to observe the morphology of our NP. Negative stain showed HDL-BchlBOA overwhelming possessed a spherical shape (Figure 2B). This shape was expected as neutral lipids (i.e., cholesterol oleate, BchlBOA) loaded into HDL particles will partition to the core and transform it into a spherical particle under favorable conditions (38). To validate that HDL-BchlBOA retained similar functional properties as the native particle, cell uptake studies were performed. ApoA-1, the major protein found in HDL, is responsible for both the structural and functional properties of the native particle. Functionally, ApoA-1 is able to bind to the SR-BI receptor, which mediates the transfer of its cholesterol content into the host cell (39). Two Chinese hamster ovary (CHO) mutant clones, ldlA[mSR-BI] (ldlA7 with stably transfected mSR-BI receptor) and ldlA7 were used to delineate the high and low SR-BI receptor expression groups, respectively (39). In this study, we monitored the uptake of HDL-BchlBOA in these two cell lines. Under identical treatment conditions, HDL-BchlBOA was taken up exclusively by ldlA[mSR-BI] cells (Figure 3B) but was not taken up by ldlA7 cells (Figure 3E). Furthermore, the degree of uptake of HDL-BchlBOA by ldlA[mSR-BI] cells was successfully inhibited by a 25-fold

Communications

Figure 4. Fluorescence microscopic images of cells treated with HDL-BchlBOA. Cells were seeded with KB cells at a density of 3 × 104 cells per well 24 h before imaging. Final well volume was 200 µL. Cells either received no treatment (top), 8 µM HDL-BchlBOA (dye concentration) (middle), or 8 µM HDL-BchlBOA (dye concentration) with 25-fold molar excess of HDL protein (bottom). Cells were excited at 515 nm and fluorescence was captured from 700-800 nm. Scale bar corresponds to 20 µm.

excess native HDL (Figure 3C). These results indicate that the SR-BI mediated lipid transfer activity of HDL-BchlBOA was retained. The next question that needed to be addressed was whether HDL-BchlBOA can be taken up by cancer cells. Since KB cells express the SR-BI receptor, evidenced by the Western blot analysis of SR-BI expression (Supporting Information Figure S1), it was expected that HDL-BchlBOA should bind and transfer lipids to KB cells. KB cells were cultured and treated with the same conditions as the ldlA[mSR-BI] and ldlA7 cells. As expected, we observed a weaker but similar pattern of uptake as ldlA[mSR-BI] (Figure 4B). Furthermore, this uptake was also successfully inhibited by a 25-fold excess of HDL (Figure 4C). These results demonstrate that KB cancer cells are able to bind and take up lipids from HDL-like NPs through the SR-BI receptor. To assess the tumor targeting capacity of HDL-BchlBOA, we employed a mouse tumor xenograft model. Athymic nude mice bearing KB tumors in the right flank were injected

Communications

Bioconjugate Chem., Vol. 20, No. 11, 2009 2029

Figure 5. Spectral reflectance fluorescence images of tumor-bearing mice dosed with HDL-BchlBOA. Mice were dosed with 7.4 nmoles (200 uL) HDL-BchlBOA (dye concentration) and imaged at 1 h (A), 3 h (B), 6 h (C), and 48 h (D). Pseudo color images were captured with 2000 ms exposure.

intravenously with HDL-BchlBOA. Whole body distribution of the dye was monitored over 48 h by multispectral reflectance fluorescence imaging (Figure 5). We observed rapid accumulation of the probe in the chest area as early as 1 h. This fluorescence decreased over time, with a concomitant increase in signal intensity in the tumor vicinity. After 6 h, the signal was visualized intensely at the tumor site. Extraction of BchlBOA from various tissues after 48 h revealed that the liver and adrenal glands had the greatest accumulation of BchlBOA, followed by the spleen and tumor (data not shown). This pattern of fluorescence accumulation in the liver and adrenal glands was consistent with immunoblot studies conducted by Acton et al. which showed that the greatest SR-BI receptor expression in mice was found to be in these tissues (39). The fact that the HDL-BchlBOA signal remained visible in the tumor after 48 h can be explained by the long plasma circulation half-life of HDL (approximately 12 h) (26) as well as the enhanced permeability and retention (EPR) effect (40). The EPR effect allows NPs of a given size (10-100 nm) to penetrate through fenestrated vasculature into the tumor microenvironment (41). Furthermore, since the tumors generally do not have an effective lymphatic drainage system, NPs will tend to penetrate and accumulate around the tumor (42). Both of these effects enhance the probability of interactions between the particles and surface SRBI receptors found on the KB cells. Taken together, these results demonstrate that we successfully synthesized an imaging agent derived from Bchl that is stable and has excellent NIR fluorescence properties. We have also shown that this dye can be inserted into HDL-like NPs that retained its endogenous structure and function. More importantly, these NPs were delivered and visualized in cancer cells and implanted tumors. While the ability of the particle to distribute to cancer cells and tumor tissues are mediated by the SR-BI pathway, it is conceivable that a wide range of tumor-specific targets can be applied to HDL-like NPs such as the folate receptor or the prostate-specific membrane antigen. This will largely be dependent on the types of targeting ligands as well as the conjugation strategies. Examples of targeting ligands that may be conjugated to HDL-like NPs include folate (43, 44), peptides, (45) and aptamer-based (46) moieties. Further work will be required to validate whether these targeting strategies can improve the tumor specificity of HDL-BchlBOA. In addition to its NIR fluorescence properties, BchlBOA also has important implication for PDT. Currently, TOOKAD, a derivative of Bchl with similarities to BchlBOA is being investigated in phase II/III clinical trials as a PS for PDT in patients with recurrent prostate cancer (24). However, BchlBOA differs from TOOKAD in that (i) it is stabilized through modifications to the isocyclic ring, (ii) it is fluorescent, and (iii) can be incorporated into HDL-like NPs. These differences may

be significant as we predict that HDL-BchlBOA may also potentially be used for NIR fluorescence-guided PDT of prostate cancer. In conclusion, the present study outlines the synthesis of a novel Bchl-analog that is stable, fluorescent, and has dual lipid anchors for its efficient incorporation into HDL-like NPs. The resulting HDL-BchlBOA has similar characteristics to HDL in size and morphology. Furthermore, these particles can interact specifically with SR-BI receptors on both ldlA[mSR-BI] and KB cancer cell lines. This uptake was also observed in KB tumor xenografts. Our results demonstrate HDL-BchlBOA is a useful tool for in vivo molecular imaging. With this knowledge in hand, future work will continue investigating the applicability of this NP for cancer imaging and therapy. Emphasis will be placed on functionalizing the NP surface with ligands for specific targeting to tumor cells and tissues. The PDT efficiency of BchlBOA delivered in HDL will also be examined to determine the suitability of HDL-BchlBOA for image-guided therapy.

ACKNOWLEDGMENT This work was supported by the Canadian Institutes for Health Research, the Ontario Institute for Cancer Research through funding provided by the Government of Ontario, the Canadian Cancer Society Research Institute, and the Joey and Toby Tanenbaum/Brazilian Ball Chair in Prostate Cancer Research. Supporting Information Available: Western blot analysis of SR-BI receptor expression in various cancer cell lines versus SR-BI control cell lines. This material is available free of charge via the Internet at http://pubs.acs.org.

LITERATURE CITED (1) Klohs, J., Wunder, A., and Licha, K. (2008) Near-infrared fluorescent probes for imaging vascular pathophysiology. Basic Res Cardiol 103, 144–51. (2) Weissleder, R. (2001) A clearer vision for in vivo imaging. Nat. Biotechnol. 19, 316–7. (3) Ernst, L. A., Gupta, R. K., Mujumdar, R. B., and Waggoner, A. S. (1989) Cyanine dye labeling reagents for sulfhydryl groups. Cytometry 10, 3–10. (4) Achilefu, S., Jimenez, H. N., Dorshow, R. B., Bugaj, J. E., Webb, E. G., Wilhelm, R. R., Rajagopalan, R., Johler, J., and Erion, J. L. (2002) Synthesis, in vitro receptor binding, and in vivo evaluation of fluorescein and carbocyanine peptide-based optical contrast agents. J. Med. Chem. 45, 2003–15. (5) Licha, K., Riefke, B., Ntziachristos, V., Becker, A., Chance, B., and Semmler, W. (2000) Hydrophilic cyanine dyes as contrast agents for near-infrared tumor imaging: synthesis, photophysical

2030 Bioconjugate Chem., Vol. 20, No. 11, 2009 properties and spectroscopic in vivo characterization. Photochem. Photobiol. 72, 392–8. (6) Becker, A., Hessenius, C., Licha, K., Ebert, B., Sukowski, U., Semmler, W., Wiedenmann, B., and Grotzinger, C. (2001) Receptor-targeted optical imaging of tumors with near-infrared fluorescent ligands. Nat. Biotechnol. 19, 327–31. (7) Wei, X., Runnels, J. M., and Lin, C. P. (2003) Selective uptake of indocyanine green by reticulocytes in circulation. InVest. Ophthalmol. Visual Sci. 44, 4489–96. (8) Caesar, J., Shaldon, S., Chiandussi, L., Guevara, L., and Sherlock, S. (1961) The use of indocyanine green in the measurement of hepatic blood flow and as a test of hepatic function. Clin. Sci. 21, 43–57. (9) Stefflova, K., Chen, J., and Zheng, G. (2007) Killer beacons for combined cancer imaging and therapy. Curr. Med. Chem. 14, 2110–25. (10) Ghoroghchian, P. P., Frail, P. R., Susumu, K., Blessington, D., Brannan, A. K., Bates, F. S., Chance, B., Hammer, D. A., and Therien, M. J. (2005) Near-infrared-emissive polymersomes: self-assembled soft matter for in vivo optical imaging. Proc. Natl. Acad. Sci. U.S.A. 102, 2922–7. (11) Koyama, T., Tsubota, A., Nariai, K., Mitsunaga, M., Yanaga, K., and Takahashi, H. (2007) Novel biomedical imaging approach for detection of sentinel nodes in an experimental model of gastric cancer. Br. J. Surg. 94, 996–1001. (12) Akers, W., Lesage, F., Holten, D., and Achilefu, S. (2007) In vivo resolution of multiexponential decays of multiple nearinfrared molecular probes by fluorescence lifetime-gated wholebody time-resolved diffuse optical imaging. Mol. Imaging 6, 237– 46. (13) Zhang, Z., Li, H., Liu, Q., Zhou, L., Zhang, M., Luo, Q., Glickson, J., Chance, B., and Zheng, G. (2004) Metabolic imaging of tumors using intrinsic and extrinsic fluorescent markers. Biosens. Bioelectron. 20, 643–50. (14) Pichlmeier, U., Bink, A., Schackert, G., and Stummer, W. (2008) Resection and survival in glioblastoma multiforme: an RTOG recursive partitioning analysis of ALA study patients. Neuro-Oncology 10, 1025–34. (15) Pandey, S. K., Gryshuk, A. L., Sajjad, M., Zheng, X., Chen, Y., Abouzeid, M. M., Morgan, J., Charamisinau, I., Nabi, H. A., Oseroff, A., and Pandey, R. K. (2005) Multimodality agents for tumor imaging (PET, fluorescence) and photodynamic therapy. A possible “see and treat” approach. J. Med. Chem. 48, 6286– 95. (16) Stefflova, K., Chen, J., Li, H., and Zheng, G. (2006) Targeted photodynamic therapy agent with a built-in apoptosis sensor for in vivo near-infrared imaging of tumor apoptosis triggered by its photosensitization in situ. Mol. Imaging 5, 520–32. (17) Stefflova, K., Li, H., Chen, J., and Zheng, G. (2007) Peptidebased pharmacomodulation of a cancer-targeted optical imaging and photodynamic therapy agent. Bioconjug. Chem. 18, 379– 88. (18) Fiedor, J., Fiedor, L., Kammhuber, N., Scherz, A., and Scheer, H. (2002) Photodynamics of the bacteriochlorophyll-carotenoid system. 2. Influence of central metal, solvent and beta-carotene on photobleaching of bacteriochlorophyll derivatives. Photochem. Photobiol. 76, 145–52. (19) Chen, Y., Graham, A., Potter, W., Morgan, J., Vaughan, L., Bellnier, D. A., Henderson, B. W., Oseroff, A., Dougherty, T. J., and Pandey, R. K. (2002) Bacteriopurpurinimides: highly stable and potent photosensitizers for photodynamic therapy. J. Med. Chem. 45, 255–8. (20) Rosenbach-Belkin, V., Chen, L., Fiedor, L., Tregub, I., Paviotsky, F., Brumfeld, V., Salomon, Y., and Scherz, A. (1996) Serine conjugates of chlorophyll and bacteriochlorophyll: photocytotoxicity in vitro and tissue distribution in mice bearing melanoma tumors. Photochem. Photobiol. 64, 174–81. (21) Chen, Y., Potter, W. R., Missert, J. R., Morgan, J., and Pandey, R. K. (2007) Comparative in vitro and in vivo studies on long-

Communications wavelength photosensitizers derived from bacteriopurpurinimide and Bacteriochlorin p6: fused imide ring enhances the in vivo PDT efficacy. Bioconjug. Chem. 18, 1460–73. (22) Drzewiecka-Matuszek, A., Skalna, A., Karocki, A., Stochel, G., and Fiedor, L. (2005) Effects of heavy central metal on the ground and excited states of chlorophyll. J. Biol. Inorg. Chem. 10, 453–62. (23) Trachtenberg, J., Bogaards, A., Weersink, R. A., Haider, M. A., Evans, A., McCluskey, S. A., Scherz, A., Gertner, M. R., Yue, C., Appu, S., Aprikian, A., Savard, J., Wilson, B. C., and Elhilali, M. (2007) Vascular targeted photodynamic therapy with palladium-bacteriopheophorbide photosensitizer for recurrent prostate cancer following definitive radiation therapy: assessment of safety and treatment response. J. Urol. 178, 1974–9; discussion 1979. (24) Trachtenberg, J., Weersink, R. A., Davidson, S. R., Haider, M. A., Bogaards, A., Gertner, M. R., Evans, A., Scherz, A., Savard, J., Chin, J. L., Wilson, B. C., and Elhilali, M. (2008) Vascular-targeted photodynamic therapy (padoporfin, WST09) for recurrent prostate cancer after failure of external beam radiotherapy: a study of escalating light doses. BJU Int. 102, 556–62. (25) Lund-Katz, S., Liu, L., Thuahnai, S. T., and Phillips, M. C. (2003) High density lipoprotein structure. Front Biosci. 8, d1044– 54. (26) Eisenberg, S., Windmueller, H. G., and Levy, R. I. (1973) Metabolic fate of rat and human lipoprotein apoproteins in the rat. J. Lipid Res. 14, 446–58. (27) Daerr, W. H., Pethke, W., Windler, E. T., and Greten, H. (1990) Biotinyl-high-density lipoproteins as a probe for the determination of high-density lipoprotein turnover in humans. Biochim. Biophys. Acta 1043, 311–7. (28) Kozyrev, A. N., Chen, Y., Goswami, L. N., Tabaczynski, W. A., and Pandey, R. K. (2006) Characterization of porphyrins, chlorins, and bacteriochlorins formed via allomerization of bacteriochlorophyll a. Synthesis of highly stable bacteriopurpurinimides and their metal complexes. J. Org. Chem. 71, 1949– 60. (29) Havel, R. J., Eder, H. A., and Bragdon, J. H. (1955) The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J. Clin. InVest. 34, 1345– 53. (30) Scanu, A. (1966) Forms of human serum high density lipoprotein protein. J. Lipid Res. 7, 295–306. (31) Krieger, M., Smith, L. C., Anderson, R. G., Goldstein, J. L., Kao, Y. J., Pownall, H. J., Gotto, A. M., Jr., and Brown, M. S. (1979) Reconstituted low density lipoprotein: a vehicle for the delivery of hydrophobic fluorescent probes to cells. J. Supramol. Struct. 10, 467–78. (32) Rensen, P. C., de Vrueh, R. L., Kuiper, J., Bijsterbosch, M. K., Biessen, E. A., and van Berkel, T. J. (2001) Recombinant lipoproteins: lipoprotein-like lipid particles for drug targeting. AdV. Drug DeliV. ReV. 47, 251–76. (33) Li, H., Marotta, D. E., Kim, S., Busch, T. M., Wileyto, E. P., and Zheng, G. (2005) High payload delivery of optical imaging and photodynamic therapy agents to tumors using phthalocyanine-reconstituted low-density lipoprotein nanoparticles. J. Biomed. Opt. 10, 41203. (34) Song, L., Li, H., Sunar, U., Chen, J., Corbin, I., Yodh, A. G., and Zheng, G. (2007) Naphthalocyanine-reconstituted LDL nanoparticles for in vivo cancer imaging and treatment. Int. J. Nanomed. 2, 767–74. (35) Corbin, I. R., Chen, J., Cao, W., Li, H., Lund-Katz, S., and Zheng, G. (2007) Enhanced Cancer-Targeted Delivery Using Engineered High-Density Lipoprotein-Based Nanocarriers. J. Biomed. Nanotechnol. 3, 367–376. (36) Pittman, R. C., Glass, C. K., Atkinson, D., and Small, D. M. (1987) Synthetic high density lipoprotein particles. Application to studies of the apoprotein specificity for selective uptake of cholesterol esters. J. Biol. Chem. 262, 2435–42.

Communications (37) Silva, R. A., Huang, R., Morris, J., Fang, J., Gracheva, E. O., Ren, G., Kontush, A., Jerome, W. G., Rye, K. A., and Davidson, W. S. (2008) Structure of apolipoprotein A-I in spherical high density lipoproteins of different sizes. Proc. Natl. Acad. Sci. U.S.A. 105, 12176–81. (38) Sparks, D. L., Davidson, W. S., Lund-Katz, S., and Phillips, M. C. (1995) Effects of the neutral lipid content of high density lipoprotein on apolipoprotein A-I structure and particle stability. J. Biol. Chem. 270, 26910–7. (39) Acton, S., Rigotti, A., Landschulz, K. T., Xu, S., Hobbs, H. H., and Krieger, M. (1996) Identification of scavenger receptor SRBI as a high density lipoprotein receptor. Science 271, 518–20. (40) Maeda, H., Sawa, T., and Konno, T. (2001) Mechanism of tumor-targeted delivery of macromolecular drugs, including the EPR effect in solid tumor and clinical overview of the prototype polymeric drug SMANCS. J. Controlled Release 74, 47–61. (41) Roberts, W. G., and Palade, G. E. (1997) Neovasculature induced by vascular endothelial growth factor is fenestrated. Cancer Res. 57, 765–72. (42) Haag, R. (2004) Supramolecular drug-delivery systems based on polymeric core-shell architectures. Angew. Chem., Int. Ed. 43, 278–82.

Bioconjugate Chem., Vol. 20, No. 11, 2009 2031 (43) Zheng, G., Chen, J., Li, H., and Glickson, J. D. (2005) Rerouting lipoprotein nanoparticles to selected alternate receptors for the targeted delivery of cancer diagnostic and therapeutic agents. Proc. Natl. Acad. Sci. U.S.A. 102, 17757– 62. (44) Chen, J., Corbin, I. R., Li, H., Cao, W., Glickson, J. D., and Zheng, G. (2007) Ligand conjugated low-density lipoprotein nanoparticles for enhanced optical cancer imaging in vivo. J. Am. Chem. Soc. 129, 5798–9. (45) von Maltzahn, G., Ren, Y., Park, J. H., Min, D. H., Kotamraju, V. R., Jayakumar, J., Fogal, V., Sailor, M. J., Ruoslahti, E., and Bhatia, S. N. (2008) In vivo tumor cell targeting with “click” nanoparticles. Bioconjug. Chem. 19, 1570–8. (46) Lupold, S. E., Hicke, B. J., Lin, Y., and Coffey, D. S. (2002) Identification and characterization of nuclease-stabilized RNA molecules that bind human prostate cancer cells via the prostate-specific membrane antigen. Cancer Res. 62, 4029–33.

BC900404Y