Enhanced Cell Uptake of Superparamagnetic Iron Oxide

Bioconjugate Chem. 2008, 19, 2375–2384

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Enhanced Cell Uptake of Superparamagnetic Iron Oxide Nanoparticles Functionalized with Dendritic Guanidines Amanda L. Martin,† Lisa M. Bernas,|,§ Brian K. Rutt,|,§ Paula J. Foster,|,§ and Elizabeth R. Gillies*,†,‡ Department of Chemistry, Department of Chemical and Biochemical Engineering, Department of Medical Biophysics, and Imaging Research LaboratoriessRobarts Research Institute, The University of Western Ontario, 1151 Richmond Street, London, Ontario N6A 5B7, Canada. Received May 20, 2008; Revised Manuscript Received October 14, 2008

Magnetic resonance imaging (MRI) is a powerful tool for the diagnosis of disease and the study of biological processes such as cancer metastasis and inflammation. Superparamagnetic iron oxide (SPIO) nanoparticles have been shown to be effective contrast agents for labeling cells to provide high sensitivity in MRI, but this sensitivity depends on the ability to label cells with sufficient quantities of SPIO, which can be challenging for nonphagocytic cells such as cancer cells. To address this issue, a novel cell-penetrating polyester dendron with peripheral guanidines was developed and conjugated to the surface of SPIO. The functionalized nanoparticles were characterized by transmission electron microscopy, infrared spectroscopy, and dynamic light scattering, and it was found that the surface functionalization reaction proceeded to completion and did not have any adverse effects on the SPIO. In GL261 mouse glioma cells, the dendritic guanidine exhibited remarkably similar cell-penetrating capabilities to the HIV-Tat47-57 peptide for the transport of fluorescein, and when conjugated to SPIO, it provided significantly enhanced uptake in comparison with nanoparticles having no dendron or dendrons with hydroxyl or amine peripheries. This uptake led to substantial decreases in the transverse relaxation time (T2) of labeled cells relative to control cells. While the nanoparticles functionalized with dendritic guanidines exhibited somewhat greater toxicity than those functionalized with dendrons having hydroxyl or amine peripheries, they were still relatively nontoxic at the low concentrations required for labeling.

INTRODUCTION Magnetic resonance imaging (MRI) is an important noninvasive technique for the diagnosis of disease and the study of biological processes. Since the inception of MRI, several million contrast agent injections have been performed. These agents increase the signal contrast between normal and diseased tissues by altering the nuclear relaxation times of water molecules in their vicinity. In addition to low molecular weight complexes based on gadolinium (Gd3+) (1), a class of contrast agents of growing importance is superparamagnetic iron oxide (SPIO) nanoparticles (2). These nanoparticles induce local inhomogeneities in the magnetic field, resulting in regions of hypointensity in T2-weighted MR images. Several SPIO agents are clinically approved by the United States Food and Drug Administration for use in liver and spleen imaging, as well as gastrointestinal imaging (3). In vivo administration of SPIO has been investigated primarily by two routes. In some applications, SPIO is injected intravenously and reaches the target site either passively or by the conjugation of an active targeting group to the SPIO surface (4, 5). Alternatively, for the study of biological processes such as metastasis and stem cell transplantation, cells can be labeled with SPIO in vitro then tracked by MRI following in vivo injection (6-8). Recent work by Heyn and co-workers has demonstrated that even single SPIO labeled cells can be detected in vivo by MRI, thus illustrating the immense promise of this technique (9). The ability to detect labeled cells by MRI depends on the quantity of SPIO in the cells. Thus, the efficiency with which * Author to whom correspondence should be addressed. E-mail: [email protected]. Phone: 519-661-2111 ext 80223. Fax: 519-661-3022. † Department of Chemistry. ‡ Department of Chemical and Biochemical Engineering. § Department of Medical Biophysics. | Imaging Research LaboratoriessRobarts Research Institute.

cells can be loaded with SPIO is a major determinant for MR sensitivity at the single cell level. For biological applications, SPIO is typically coated with a water soluble polymer such as dextran (10) or poly(ethylene oxide) (11, 12) in order to provide water solubility and biocompatibility, but these polymers are not designed to significantly enhance the uptake of SPIO into cells. While cells such as macrophages can be readily labeled with adequate quantities of SPIO due to their inherent capability to phagocytose material in the extracellular medium, many other cell lines, including cancer cells do not readily phagocytose SPIO. This challenge can be overcome in some cases by the use of complexes formed between SPIO and transfection agents such as Superfect, Lipofectamine, and poly(L-lysine) (13); however, this is a multistep process that must be optimized on a case by case basis and care must be taken to control the size and polydispersity of the resulting complexes. An alternative approach that has been investigated involves the direct conjugation of cell-penetrating peptides such as HIV-Tat (14, 15) or protamine (7) to the SPIO surface. This method provides a welldefined agent that can be used to label cells in a single step, but a limitation is the availability of peptides on a large scale due to the expense of peptide or protein synthesis and concerns regarding immunogenicity when they are isolated from natural sources. In addition, due to the many reactive functional groups present in peptides, their conjugation to the nanoparticle surface is nontrivial, involving first activation and then conjugation via a multistep process. Thus, the goal of this work was the development of a simple and versatile process for modulating the cell uptake of SPIO via functionalization of the nanoparticle surface. Well-defined branched molecules called dendrimers were anticipated to be an ideal backbone for the covalent functionalization of the SPIO surface in order to modulate biological properties and cell uptake (16, 17). Dendrimers are prepared

10.1021/bc800209u CCC: $40.75  2008 American Chemical Society Published on Web 11/18/2008

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by a stepwise synthetic sequence providing molecules with welldefined molecular weight and composition. They can frequently be prepared on a multigram scale by exploiting very high yielding reactions and using a minimal number or even no chromatographic purification steps. Thus far, there have been a couple of reports of dendrimer coated SPIO for cell labeling, but in these cases, carboxyl terminated dendrimers were used as a stabilization agent during the nanoparticle synthesis and the mode of attachment of the dendrimer to the nanoparticle surface is still not well understood (6, 18, 19). For this work, it was proposed that by using wedges of dendrimers, commonly referred to as dendrons, the multiple functional groups at the dendron’s periphery could allow for the introduction of multiple biological ligands in a well-defined display to modulate properties such as cell uptake and targeting, while the dendron’s focal point would provide a site for attachment to the nanoparticle surface. Recent work by the groups of Goodman (20), Wender (21), and Harth (22) has demonstrated that dendrons functionalized at their peripheries with guanidines can significantly enhance the uptake of chromophores into cells in a manner similar to cell-penetrating peptides such as HIV-Tat (23) and protamine (24), which contain high levels of the amino acid arginine. However, the ability of these dendritic molecules to deliver large and challenging cargo such as nanoparticles into cells has not yet been demonstrated. With the aim of modulating the uptake of SPIO by cells for in vitro cell labeling, we describe here the preparation of a polyester dendron having multiple peripheral guanidine groups and its conjugation to SPIO. This new dendritic guanidine was found to have a cell penetrating capability very similar to that of the HIV-Tat47-47 peptide, and upon conjugation to SPIO, it was found to significantly enhance the cell uptake of the nanoparticles, leading to substantial changes in T2 in MR images. Its effect on the toxicity of the nanoparticles was also investigated.

EXPERIMENTAL PROCEDURES General Procedures and Materials. All chemicals were purchased from commercial sources and used without further purification unless otherwise noted. Anhydrous acetonitrile, dichloromethane, and N,N-dimethylformamide (DMF) were obtained from a solvent purification system. Triethylamine was distilled from CaH2. Column chromatography was performed using silica gel (0.063-0.200 mm particle size, 70-230 mesh). Dialysis was performed using a Spectra/Por regenerated cellulose membrane. Ultrafiltration was carried out using a 300 KDa molecular weight cutoff (MWCO) membrane of regenerated cellulose purchased from Amicon. Infrared spectra were obtained as thin films on NaCl plates or as KBr pellets. 1H NMR spectra were obtained at 400 or 600 MHz, and 13C NMR spectra were obtained at 100 or 150 MHz. Chemical shifts are reported in ppm and are calibrated against residual solvent signals of CDCl3 (δ 7.26, 77.2) or CD3OD (δ 3.31, 49.15). All coupling constants (J) are reported in Hertz. Transmission electron microscopy (TEM) was carried out using a carbon Formvar grid and a Phillips CM10 microscope operating at 80 kV with a 40 µm aperture. Dynamic light scattering (DLS) was performed at a concentration of 1 mg of iron/mL in water using a Malvern Zetasizer Nano-S instrument. Quantification of Accessible Amine Groups on the Nanoparticle Surface. Amine functionalized, cross-linked dextran coated Fe3O4 nanoparticles (1) were prepared as previously reported (7, 25). The nanoparticles (0.81 mg of Fe, as determined spectrophotometrically following treatment with HCl and H2O2 (7)) were then diluted into 0.5 mL of 25 mM pH 7.4 citrate buffer, and the rhodamine derivative 2 (26) (18 mg, 0.025 mmol) was added, followed by diisopropylethylamine (DIPEA) (20 µL). The reaction mixture was stirred overnight in the dark at

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Figure 1. TEM image of nanoparticle 1 (scale bar ) 20 nm).

room temperature and then dialyzed against water for 24 h with several water changes. The UV-visible absorbance of the resulting solution was measured, and the concentration of conjugated dye 2 was calculated based on the extinction coefficient of the dye in water (ε ) 96100 M cm-1, λ ) 567nm). Synthesis of Water Soluble Linker 4. Under a nitrogen atmosphere 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)acetic acid 3 (27) (0.50 g, 2.2 mmol, 1.0 equiv), N-hydroxysuccinimide (0.25 g, 2.2 mmol, 1.0 equiv), and DCC (0.47 g, 2.3 mmol, 1.1 equiv) were dissolved in 20 mL of anhydrous acetonitrile. This solution was stirred at 65 °C for 6 h and then was cooled to room temperature, and the dicyclohexylurea byproduct was removed by filtration. The solvent was removed under reduced pressure to provide 0.69 g (96%) of the product 4 as a colorless oil. 1H NMR (400 MHz, CDCl3) δ: 4.53 (s, 2H), 3.81-3.79 (m, 2H), 3.73-3.65 (m, 8H), 3.39 (t, 2H, J ) 5.1), 2.85 (br s, 4H). 13C NMR (400 MHz, CDCl3) δ: 169.2, 166.3, 71.5, 70.8, 70.2, 66.7, 50.9, 25.8. IR (cm-1, thin film from CH2Cl2): 3330, 2930, 2870, 2110, 1820, 1790, 1740. MS calcd for [M + H]+ (C12H19O7N4): 331.1. Found: (ES+) 331.1. Conjugation of Linker 4 and Dye 2 to Nanoparticles (Synthesis of Nanoparticles 5). To a solution of nanoparticle 1 (10 mg of Fe) in 1.1 mL of 25 mM pH 7.4 citrate buffer was added linker 4 (8.2 mg, 25 µmol) in 0.25 mL of 1:1 DMSO/ H2O followed by the rhodamine derivative 2 (1.7 mg, 2.5 µmol) and DIPEA (50 µL). The solution was stirred overnight in the dark and then dialyzed against pure water for 24 h to provide nanoparticle 5. The presence of the azide group on the nanoparticle surface was verified by IR spectroscopy (KBr pellet) (Figure 3a), while the amount of conjugated dye was quantified by UV-visible spectroscopy to be 0.21 µmol/mg of Fe. Synthesis of N,N′-Bis(tert-butoxycarbonyl)-6-guanidinylcaproic Acid (8). 6-Aminocaproic acid (6) (0.51 g, 3.9 mmol, 1.0 equiv) was dissolved in 10:1 CH3CN/H2O (60 mL), and NEt3 (4.0 mL) was added. At 0 °C, N,N′-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine (7) (1.0 g, 3.2 mmol, 0.83 equiv) was added and the mixture was stirred overnight. The reaction was then diluted with EtOAc (100 mL) and washed with saturated NaHCO3 (2 × 50 mL), 1 M KHSO4 (2 × 50 mL), and finally brine (50 mL). The organic phase was dried with MgSO4 and filtered, and the solvent was removed under reduced pressure to yield 1.1 g (90%) of 8 as a white solid. 1H NMR (400 MHz, CDCl3) δ: 8.32 (t, 1H, J ) 4.6), 3.41-3.36 (m, 2H), 2.34 (t, 2H, J ) 7.4), 1.67-1.62 (m, 4H), 1.49 (s, 18H), 1.43-1.38 (m, 2H). 13C NMR (100 MHz, CDCl3) δ:

Superparamagnetic Iron Oxide Nanoparticles

Figure 2. Hydrodynamic diameters of the nanoparticles prior to and after surface functionalization via the “click” reaction, as determined by dynamic light scattering.

Figure 3. Infrared spectra of (a) nanoparticle 5, (b) dendron 13, and (c) functionalized nanoparticle 19.

163.71, 156.34, 153.49, 83.27, 79.48, 40.92, 28.92, 28.47, 28.26, 28.17, 26.55, 24.67. IR (cm-1, thin film from CH2Cl2): 3330, 3140, 2980, 2935, 1720, 1640, 1620. MS calcd for [M + H]+ (C17H31O6N3): 373.5. Found: (ES+) 373.2. Synthesis of Dendron 12. Dendron 11 (28) (50 mg, 21 µmol, 1.0 equiv) and the acid 8 (0.14 g, 0.38 mmol, 18 equiv) were dissolved in anhydrous N,N-dimethylformamide (DMF) (4.0 mL), and O-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) (0.14 g, 0.38 mmol, 18 equiv) was added, followed by 1-hydroxybenzotriazole (HOBt) (51 mg, 0.38 mmol, 18 equiv) and DIPEA (74 µL, 0.43 mmol, 20 equiv). The reaction mixture was stirred under nitrogen for 48 h. The product was purified by dialysis against DMF followed by silica gel chromatography using EtOAc and then 1:1 CH2Cl2/MeOH to provide 60 mg (65%) of 12 as a colorless oil. 1H NMR (400 MHz, CDCl3) δ: 11.48 (s, 8H), 8.30 (t, 8H, J ) 4.8), 6.57-6.54 (m, 6H), 4.73 (d, 2H, J ) 2.2), 4.31-4.16 (m, 28H), 3.51-3.47

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(m, 16H), 3.39-3.35 (m, 16H), 2.60-2.64 (m, 1H), 2.55 (t, 16H, J ) 6.1), 2.18 (t, 16H, J ) 7.4), 1.66-1.54 (m, 24H), 1.49 (s, 144H), 1.37-1.33 (m, 16H), 1.27 (s, 3H), 1.25 (s, 6H), 1.23 (s, 12H). 13C NMR (150 MHz, CDCl3): δ 173.1, 171.9, 171.8, 171.7, 156.1, 153.2, 82.9, 80.0, 77.3, 75.8, 65.2, 65.0, 64.8, 53.0, 46.7, 46.5, 46.4, 40.7, 36.2, 34.9, 34.1, 28.6, 28.3, 28.0, 26.5, 26.0, 25.2, 17.8, 17.5, 17. 4. IR (cm-1, thin film from CH2Cl2): 3330, 2980, 2930, 2120, 1740, 1735, 1725, 1640, 1615. MS: calcd for [M + H]+ (C198H341N32O70): 4287.3. Found: (ES+) 4282.2. Synthesis of Dendron 13. Dendron 12 (77 mg, 18 µmol) was dissolved in 2 mL of 1:1 trifluoroacetic acid/CH2Cl2, and the solution was stirred at room temperature for 2 h. The solvent was removed under reduced pressure to yield 63 mg (98%) of 13 as a colorless oil in the form of its TFA salt. 1H NMR (600 MHz, CD3OD): δ 4.77 (d, 2H, J ) 2.2), 4.22 (m, 28H), 3.43 (t, 16H, J ) 6.7), 3.16 (t, 16H, J ) 7.1), 2.93 (t, 1H, J ) 2.3), 2.56 (t, 16H, J ) 6.7), 2.20 (t, 16H, J ) 7.4), 1.64-1.55 (m, 24H), 1.41-1.37 (m, 16H), 1.35 (s, 3H), 1.29 (s, 6H), 1.26 (s, 12H). 13C NMR (600 MHz, CD3OD): δ 174.6, 174.1, 173.1, 173.0, 172.3, 77.0, 75.5, 65.0, 52.5, 46.2, 46.1, 46.0, 39.9, 35.8, 35.2, 34.9, 33.2, 28.5, 25.8, 24.9, 16.8. IR (cm-1, thin film from THF/MeOH): 3400, 2930, 2120, 1650, 1460. MS: calcd for [M + H]+ (C118H205N32O38): 2677.5. Found: (MALDI-TOF) 2678.4. Synthesis of Dendron 14. Dendron 12 (25 mg, 5.8 µmol, 1.0 equiv) was dissolved in 0.5 mL of 1:1 THF/H2O. To this was added 3-amino-1-azidopropane (29) (5.9 mg, 58 µmol, 10 equiv), CuSO4 (15 µL of a 0.5 M solution in H2O, 7.5 µmol, 1.3 equiv), sodium ascorbate (200 µL of a 0.5 M solution in H2O, 100 µmol, 17 equiv), and finally bathophenanthrolinedisulfonic acid disodium salt (5.0 mg, 8.5 µmol, 1.5 equiv). The reaction mixture was stirred for 24 h at room temperature. The product was purified by dialysis against DMF to provide 15 mg (60%) of 14 as a viscous colorless oil. 1H NMR (400 MHz, CDCl3) δ: 11.49 (s, 8H), 8.32 (s, 8H), 6.61 (br s, 8H), 5.26 (br s, 2H), 4.25-4.16 (m, 28H), 3.65 (s, 2H), 3.50-3.46 (m, 16H), 3.40-3.36 (m, 16H), 2.57-2.54 (m, 16H), 2.43-2.40 (m, 1H), 2.21-2.17 (m, 16H), 1.66-1.54 (m, 24H), 1.49 (s, 144H), 1.37-1.33 (m, 16H), 1.25-1.21 (m, 24H). 13C NMR (600 MHz, CDCl3): δ 180.0, 173.2, 171.9, 171.8, 171.6, 156.1, 138.1, 83.0, 79.2, 70.5, 64.9, 64.8, 58.0, 46.4, 46.2, 40.7, 36.4, 36.2, 34.9, 34.1, 31.4, 28.8, 28.2, 28.0, 26.5, 25.3, 17.9, 17.8, 17.7. IR (cm-1, thin film from CH2Cl2): 3330, 2980, 2935, 2240, 2100, 1740, 1735, 1645, 1615, 1575, 1455. MS: calcd for [M + H+]+ (C201H341N36O70): 4379.8. Found: (ES+) 4379.2. Synthesis of Fluorescein Functionalized Dendron 16. Dendron 14 (10 mg, 2.3 µmol, 1.0 equiv) and fluorescein isothiocyanate isomer 1 (FITC) (3.5 mg, 9.2 µmol, 4.0 equiv) were dissolved in 2 mL of dry DMF. The resulting solution was cooled to 0 °C, and then 1 mL of dry triethylamine was added and the reaction mixture was stirred in the dark for 24 h. The product was purified by dialysis against DMF to provide 10 mg (95%) of the protected product as a viscous yellow oil. The protected derivative (10 mg, 2.2 µmol) was then dissolved in 2 mL of 1:1 trifluoroacetic acid/CH2Cl2, and the solution was stirred at room temperature for 2 h. The solvent was removed under reduced pressure to yield 8 mg (90%) of 16 as a viscous yellow oil. 1H NMR (600 MHz, CD3OD/CDCl3 1:1): δ 8.26 (d, 2H, J ) 9.4), 8.09 (d, 2H, J ) 9.4), 7.46 (d, 1H, J ) 8.8), 7.39 (d, 1H, J ) 8.8), 7.20-7.14 (m, 1H), 6.85 (d, 2H, J ) 9.4), 5.24 (br s, 2H), 4.22-4.17 (m, 28H), 3.80 (br s, 2H), 3.43-3.40 (m, 16H), 3.23 (br s, 2H), 3.13-3.10 (m, 16H), 3.03 (br s, 2H), 2.70 (br s, 1H), 2.55-2.53 (m, 16H), 2.19-2.16 (m, 16H), 1.59-1.55 (m, 24H), 1.37-1.33 (m, 16H), 1.24-1.21 (m, 24H). 13 C NMR (600 MHz, CDCl3): δ 174.5, 171.5, 170.9, 165.8, 162.0, 157.1, 156.0, 145.6, 129.7, 125.9, 125.1, 121.9, 121.8, 115.3, 70.1, 64.9, 64.8, 64.6, 48.9, 46.5, 40.9, 35.5, 34.9, 33.6,


28.1, 25.8, 24.9, 17.3, 17.1. IR (cm-1, thin film from CH2Cl2/ MeOH): 3369, 2980, 2930, 2102, 1739, 1681, 1674, 1652, 1594, 1526, 1456, 1436. MS: calcd for [M + Na]+ (C142H234NaN37O41S): 3168.7. Found: (ES+) 3167.7. Conjugation of Dendrons 9, 11, and 13 to Nanoparticle 5 (Synthesis of Nanoparticles 17, 18, and 19). The desired dendron (9, 11, or 13) (10 µmol, 2 equiv per azide) was dissolved in a minimum volume of water and was added to a solution of nanoparticle 5 (2 mg of Fe in 1 mL of water). To this was added sodium ascorbate (4 mg, 20 µmol) followed by CuSO4 (1.2 mg, 5 µmol), and the reaction mixture was stirred overnight under a nitrogen atmosphere. The resulting solution was dialyzed against 10 mM ethylenediaminetetraacetic acid (EDTA), followed by pure water. IR spectroscopy (KBr pellet) on a lyophilized sample showed disappearance of the peak corresponding to the azides on the nanoparticles, confirming completion of the reaction (Figure 3c and the Supporting Information). Cell Culture. GL261 mouse glioma cells obtained from the NCI-Frederick Cancer Research National Tumor Repository were maintained at 37 °C and 5% CO2 in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco). Evaluation of the Cell Uptake of Dendron 16. Cells were plated at 8 × 105 cells/well in 6-well plates and allowed to adhere for 24 h. At this point, the medium was aspirated to remove dead cells and replaced with 1 mL of fresh medium to which dendron 16 or FITC-Tat47-57 (Ana Spec) was added at concentrations of 0.1, 1.0, or 10 µM. The cells were incubated at 37 °C for either 10 min or 1 h. At this point, the cells were washed twice in the plates with Hank’s Buffered Salt Solution (HBSS, Gibco), detached using trypsin-EDTA for 3 min, quenched with RPMI/10% FBS, and then resuspended in 0.5 mL of HBSS and kept on ice until analysis. The cells were analyzed for fluorescein fluorescence by flow cytometry on a BD FACSCalibur flow cytometer (BD Biosciences, USA). Basal fluorescence was given by GL261 cells that were not incubated with the agents. Evaluation of Cell Uptake of Nanoparticles 5, 17, 18, and 19. In order to assess cellular uptake of the nanoparticle agents, the experiment was carried out as described above, except that the agents were added at a concentration of 25 µg Fe/mL, and the cells were incubated for 30 min or 2 h and then analyzed by flow cytometry for rhodamine fluorescence. Confocal Microscopy. Cells were plated on collagen-coated coverslips (BD Biosciences) in 6-well plates at a density of 5 × 105 cells/well and allowed to adhere overnight. Agents were then added at 25 µg of Fe/mL in 1 mL of fresh media, and the cells were incubated at 37 °C for 2 h. The cells were then washed with HBSS, fixed with 10% formalin for 10 min, washed again, and turned onto glass slides for confocal microscopy. Images were obtained using a confocal laser scanning microscope (LSM 510, Carl Zeiss Inc.) using a 63× (N.A. ) 1.4) objective and an excitation wavelength of 543 nm (He-Ne laser). Evaluation of Agent Toxicity. The cytotoxicity of the agents was evaluated using a colorimetric MTT assay (Sigma). Cells were plated at 5 × 104 cells/well in 50 µL in a 96-well plate and allowed to adhere overnight. Iron oxide agents were then added at varying concentrations up to ∼200 µg Fe/mL, giving a total volume of 100 µL/well. After 24 h, the MTT reagent (10 µL) was added to the cells and the cells were returned to 37 °C for 3 h, during which time the MTT reagent was metabolized by viable cells, resulting in the formation of insoluble purple formazan crystals. The crystals were then solubilized by the addition of a detergent, and the plate was incubated for 4 h in the dark at room temperature. The

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absorbance read on a plate reader at 590 nm and normalized to the background at 650 nm is proportional to the number of viable cells. Magnetic Resonance Imaging of Labeled Cells. GL261 cells were plated in T75 flasks and allowed to reach 80% confluency. Nanoparticle 19 was incubated with the cells at a concentration of 12 µg of iron per mL for 2 h. Control cells were incubated in the absence of nanoparticles. The cells were then washed in the flasks four times with HBSS, detached, and washed once more by resuspension in 5 mL of HBSS and centrifugation. The cells were transferred to mini NMR tubes (New Era Enterprises, Inc.) and centrifuged at 10000g for 5 min to form dense pellets. T2 measurements of unlabeled and labeled cell pellets were made using a spin echo sequence (TE ) 12-300 ms, TR ) 2500 ms, 2 mm slice thickness) on a 3T MRI scanner (GE).

RESULTS AND DISCUSSION Preparation of Nanoparticles for Functionalization. A number of synthetic protocols ranging from aqueous to high temperature thermal decomposition methods have been reported for the synthesis of SPIO (30). For this work, the procedure involving the decomposition of FeCl2 and FeCl3 in the presence of ammonium hydroxide and dextran was used, as it readily provided water soluble nanoparticles for further functionalization (25). Treatment with epichlorohydrin followed by ammonium hydroxide, as described by Weissleder and co-workers, provided nanoparticle 1, with a cross-linked dextran shell and surface amine groups (7). These nanoparticles were characterized by transmission electron microscopy (TEM) (Figure 1), and the size of the iron oxide core was found to be approximately 5 nm, as previously reported (31). Dynamic light scattering was used to determine that the total hydrodynamic diameter of the nanoparticles, including the dextran coating, was 16 nm (Figure 2). The polydispersity index for the nanoparticles was relatively low at 0.26. The concentrations of iron in nanoparticle solutions were determined spectrophotometrically following nanoparticle degradation with hydrochloric acid and hydrogen peroxide (7). In addition, the dextran was quantified using the phenol sulfuric acid assay (32), and it was found that the mass ratio of dextran/ iron in the nanoparticles was approximately 1.9:1, similar to what was expected based on previous reports (31). As the surface amine groups are critical for further functionalization of 1, it was important to quantify the number of amine groups that were available for potential derivatization. By treatment of the nanoparticles with a large excess of the N-hydroxysuccinimidyl carbonate activated rhodamine derivative 2 (26), followed by removal of excess dye by dialysis, it was found that there were 23 µmol of amine available for functionalization per milligram of iron based on quantification of the dye by UV-visible spectroscopy. Although the concentration of reactive amines on the nanoparticle surface has not been explicitly quantified in this way before, conjugations to SPIO have generally been carried out at ratios on the order of micromoles of agent per milligrams of iron, thus indicating that nanoparticle 1 has more than enough amines available (7, 33, 34). Amines provide good functional handles for bioconjugation reactions with a number of functional groups including carboxylic acids, N-hydroxysuccinimidyl esters, and isothiocyanates in water. However, when the biological ligands of interest have internal amines or other nucleophilic functional groups, the preparation and conjugation of the above derivatives can be problematic. Therefore, in order to develop a highly general technique to introduce dendrons with a wide variety of functional groups on their peripheries, surface amines on 1 were converted to azides using a water soluble linker (Scheme 1). Azides allow



Scheme 1

Scheme 2

for highly chemoselective and functional group tolerant Cu(I) catalyzed “click” cycloaddition reactions with alkynes to be carried out on the nanoparticle surface under aqueous conditions (35). These cycloaddition reactions have been found to be very high yielding, even in the presence of significant steric hindrance (36), and are therefore ideal for the conjugation of macromolecules such as dendrons. This reaction has previously been demonstrated to be effective for conjugating molecules to the surface of SPIO (37, 38). As shown in Scheme 1, the linker 4 was prepared by the reaction of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)acetic acid (27) (3) with N-hydroxysuccinimide using N,N′-dicyclohexylcarbodiimide in acetonitrile. This linker was then reacted with nanoparticle 1 in pH 7.4 citrate buffer at a ratio of 2.5 µmol of linker per milligram of iron to provide 5. Due to the large excess of amines on the nanoparticle surface, the number of surface azides was limited by the quantity of linker added. The presence of the azide groups on the nanoparticle surface was verified using infrared (IR) spectroscopy by the presence of the distinctive azide stretch at 2100 cm-1 (Figure 3a). In addition to the azide functional handles for conjugation of the dendrons, a fluorescent probe was required in order to characterize the biological properties of the nanoparticles, such as their cellular uptake and intracellular trafficking. This was accomplished in the same step using the rhodamine derivative 2 at a ratio of 0.25 µmol/mg of iron. After the reaction, all residual small molecules and salts were removed by dialysis. The quantity of conjugated dye on 5 was determined by UV-visible spectroscopy to be 0.21 µmol/mg of iron, thus verifying the high yield of the nanoparticle amine derivatization using N-hydroxysuccinimidyl derivatives. In addition, by comparison of the fluorescence with a sample having a normalized absorbance at 567 nm, it was determined that fluorescence quenching of the rhodamine by the nanoparticles was negligible. Synthesis of a Dendritic Guanidine. For preparation of the dendritic guanidine transporter, a guanidine derivative with a carboxylic acid functional handle was prepared. 6-Aminocaproic acid (6) was reacted with N,N′-bis(BOC)-1-amidinopyrazole (7) to provide N,N′-bis(BOC)-6-guanidinylcaproic acid (8) as shown in Scheme 2. Selection of this linker length was based on previous reports demonstrating that guanidine functionalized

dendrons with more flexible spacers exhibited increased cellpenetrating capabilities relative to those that were less flexible (21). A polyester dendron based on 2,2-bis(hydroxymethyl)propionic acid was selected as the dendritic scaffold due to its ease of synthesis, biodegradability, and biocompatibility (39, 40). The alkyne functionalized dendron 9 was prepared as previously reported (41), and then the peripheral hydroxyl groups were converted to amines by reaction with anhydride 10 followed by deprotection to provide 11, with more nucleophilic functional handles for further derivatization (Scheme 3) (28). The guanidine derivative 8 was then coupled to the peripheral amines using O-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) and hydroxybenzotriazole (HOBt). Excess 8 and other byproducts were removed by dialysis and standard column chromatography to provide dendron 12. Removal of the BOC protecting groups using 1:1 trifluoroacetic acid/dichloromethane provided dendron 13 with eight peripheral guanidine groups in the form of their trifluoroacetate salts. Overall, this synthetic route is simple and amenable to scale up, as no HPLC and only one standard column chromatography purification step is required throughout the entire synthesis. In order to investigate the efficacy of the dendritic guanidine relative to the well established molecular transporter HIVtat47-57, a fluorescein labeled dendron was prepared. As shown in Scheme 4, an amine was introduced to the focal point of dendron 12 by reaction of the focal point alkyne with 3-amino1-azidopropane (29) to provide 14. This amine was then reacted with fluorescein isothiocyanate (FITC) (15). Excess FITC was removed by dialysis, and then dendron 16 was obtained upon removal of the BOC protecting groups. Conjugation of Dendrons to Nanoparticles. With dendrons having peripheral hydroxyls (9), amines (11), or guanidine groups (13) in hand, each was conjugated to the surface of nanoparticle 5 to provide functionalized nanoparticles 17, 18, and 19, respectively. As shown in Scheme 5, each coupling was carried out using click reaction conditions consisting of 5, 5 mM CuSO4, 20 mM sodium ascorbate, and 2 equiv of the dendron per azide group on the nanoparticle. After 24 h, excess reagents were removed by dialysis against 10 mM EDTA followed by water. The EDTA dialysis was critical in removing residual copper salts. In the absence of this step, high levels of toxicity were observed in preliminary work. Characterization of the resulting nanoparticles by TEM verified that the iron oxide core was not degraded during the conjugation and that the size remained approximately 5 nm. DLS showed that only a small increase in the hydrodynamic diameter of the nanoparticles was observed following conjugation, accompanied by a small increase in the polydisperity index to 0.28, which is consistent


Martin et al.

Scheme 3

Scheme 4

with the small size of the dendron relative to the initial nanoparticles and very minimal aggregation (Figure 2). IR spectroscopy on dried samples of the nanoparticles showed that, in the case of all three dendrons, the click reaction proceeded to completion with complete disappearance of the peak at 2100 cm-1 corresponding to the azide group. In addition, changes in the region of the spectrum corresponding to CdO stretching and NsH bending (∼1500-1700 cm-1) were observed, which is consistent with the introduction of the dendrons bearing multiple ester and amide groups (Figure 3b and c). In contrast, subjecting nanoparticle 5 to the click conditions in the absence of dendron did not lead to any change in the IR spectrum. Thus, the click reaction proved to be highly effective for the conjugation of dendrons having a variety of functional groups on their peripheries. Biological Evaluation. GL261 mouse glioma cells were selected for biological studies because they are nonphagocytic cells, which are generally challenging to load with SPIO in the absence of a transfection agent. It is of significant interest to be able to track cancer cells using MRI in order to reach a better understanding of the cell invasion process, so the development

of nanoparticles that are capable of more effectively labeling such cells would contribute significantly to the imaging studies. First, the efficacy of the fluorescein labeled dendritic guanidine transporter 16 was compared to that of a fluorescein labeled HIV-Tat47-57 oligopeptide (FITC-LC-YGRKKRRQRRR-NH2) using flow cytometry. The effects of both incubation time and concentration were investigated. As shown in Figure 4, it was found that, at a concentration of 1.0 µM, dendron 16 and Tat47-57 exhibited very similar levels of uptake at both 10 min and 1 h time points. At a concentration of 10 µM, very similar uptake was observed after 10 min, while the uptake of Tat47-57 was approximately 2-fold higher after 1 h. Overall, both cellpenetrating agents exhibited time- and concentration-dependent uptake, and the similarity in the uptake of the new dendritic guanidine compared to Tat47-57 is remarkable, given that it does not contain a specific amino acid sequence and is an unoptimized structure. Next, the cellular uptake of the functionalized SPIO nanoparticles 5, 17, 18, and 19 was investigated. The cells were incubated for 30 min or 2 h with the nanoparticles at a concentration of 25 µg of iron per mL, and then cell uptake



Scheme 5

was assessed by flow cytometry based on the fluorescence of the rhodamine dye that was conjugated at the same density on each set of nanoparticles. After 30 min, only nanoparticle 19 gave rise to a new population of cells with increased fluorescence (Figure 5a) while the fluorescence of all other cells remained identical to that of control cells that were not exposed to any nanoparticles. After 2 h, cells incubated with nanoparticle 19 exhibited significantly increased fluorescence, as shown in Figure 5b, while cells incubated with all other nanoparticles remained at background levels. This result indicates that the dendritic guanidines specifically enhance the uptake of SPIO into GL261 cells and that the effect is not simply due to a cationic charge on the nanoparticles.

To verify the flow cytometry results, confocal microscopy was performed on cells following 2 h incubations with nanoparticles 5, 17, 18, and 19 at a concentration of 25 µg of iron per mL. As shown in Figure 6, cells incubated with nanoparticle 19 were strongly fluorescent, while control cells and cells incubated with nanoparticles 5, 17, and 18 did not show any fluorescence using the same microscope settings. Based on the confocal images, it is difficult to determine the exact intracellular location of the nanoparticles or whether it is even a single compartment. Thus, further detailed microscopy studies will need to be carried out to determine this, as well as the effects of cell type, incubation time, and fixation (42). Dendritic guanidines have been demonstrated to provide delivery to different intracellular compartments depending on subtle differences in the dendritic structure (22), but it is not yet clear how a nanoparticle-based cargo may affect the intracellular trafficking. For this work, the location of the SPIO within cells is not a critical aspect of its ability to provide contrast, unless it causes undesirable alterations in the behavior of the labeled cells. No signs of nanoparticle toxicity were observed in the cells during this study. To show that the enhanced uptake observed using flow cytometry and confocal microscopy results in an enhanced MRI response, GL261 cells were incubated with nanoparticle 19 at a concentration of 12 µg of iron per mL for 2 h, washed, and pelleted. T2 measurements of unlabeled and labeled cell pellets were made using a spin echo sequence on a 3T MRI scanner. T2 values were 65 and 6.1 ms for unlabeled and labeled cell

Figure 4. Flow cytometry analyses of GL261 cells following incubation with dendron 16 or HIV-Tat47-57: (a) 1.0 µM for 10 min; (b) 1.0 µM for 1 h; (c) 10 µM for 10 min; (d) 10 µM for 1 h.

Figure 5. Flow cytometry analyses of GL261 cells following incubation with nanoparticles 5, 17, 18, or 19 at a concentration of 25 µg Fe/mL for (a) 30 min or (b) 2 h.


Martin et al.

Figure 8. Viability of GL261 cells after a 24 h incubation with nanoparticles 5, 17, 18, and 19, as measured by the MTT assay. Error bars represent standard deviations on three measurements.

Figure 6. Confocal laser scanning microscopy image of GL261 cells following a 2 h incubation with nanoparticle 19 at a concentration of 25 µg of Fe/mL.

polycationic nanoparticles and transporters (47). It is likely that the toxicity of the nanoparticles can be modulated in the future by tuning the ratio of dendron to nanoparticles as well as the length and hydrophobicity of the linker used to conjugate the guanidine groups to the dendron periphery.

CONCLUSIONS

Figure 7. MRI signal intensity of unlabeled cells and cells labeled with nanoparticle 19, measured at 3T using a spin echo sequence with varying echo times (TE).

pellets, respectively (Figure 7). This significant difference in T2 values illustrates the utility of these nanoparticles for labeling cells for detection by MRI. Finally, to investigate the toxicities of the nanoparticles as a function of their surface coating, the GL261 cells were incubated with varying concentrations of nanoparticles 5, 17, 18, and 19 for a 24 h period, and then an MTT assay was performed to quantitatively assess cell viability. The results of this assay are shown in Figure 8. Consistent with previous experiments demonstrating the high biocompatibilities of dendrimers based on 2,2-bis(hydroxymethyl)propionic acid (39, 43), nanoparticle 17 exhibited a relatively similar toxicity profile to the underivatized nanoparticle 5 which is similar to those that have been widely used in a number of cell labeling applications (44-46). Nanoparticle 18, prepared from the dendron 11 with peripheral amines, also had a similar toxicity profile. Guanidine functionalized nanoparticle 19 did exhibit somewhat increased toxicity, but it remained relatively nontoxic at the low concentrations used for cell labeling. It is possible that its toxicity at higher concentrations arises from their ability to penetrate and create defects in cell membranes, as has been observed for various

In conclusion, SPIO with surface azide groups was prepared and used to conjugate dendrons having various peripheral functional groups and focal point alkynes. In particular, a new dendritic guanidine was prepared and was found to have a cell penetrating capability remarkably similar to that of the HIVTat47-57 oligopeptide. Upon conjugation to the nanoparticle surface, this dendron provided significantly enhanced uptake of SPIO into GL261 mouse glioma cells in comparison to dendrons with hydroxyl or amine peripheries. This level of uptake was demonstrated to provide a decrease in the T2 of labeled cells relative to control cells by an order of magnitude. At the concentrations used for cell labeling, the functionalized nanoparticles were found to be relatively nontoxic. It is also noteworthy that the nanoparticles at all stages of synthesis were found to be stable over periods of months when stored at 4 °C in neutral solutions. This represents the first successful example of the transport of a biologically relevant nanoparticle cargo by a dendritic guanidine transporter and promises to provide improved labeling of cells with SPIO for enhanced sensitivity in cellular MRI. In the future, this dendron will also be investigated for its ability to transport other challenging cargo such as quantum dots and polymer assemblies. In addition, as this work demonstrates that dendrons are an effective means of presenting biologically relevant functionality on the nanoparticle surface, it is anticipated that this surface functionalization approach can be expanded to include other biological targeting ligands such as carbohydrates that can also benefit from a multivalent dendritic display.

ACKNOWLEDGMENT We thank NSERC, the Canada Research Chairs Program, and the Ontario Institute for Cancer Research One Millimetre Cancer Challenge Program for funding this work. A.L.M. is supported by an Ontario Graduate Scholarship in Science and Technology. B.K.R. receives salary support from the Barnett-Ivey Heart and Stroke Foundation of Ontario Endowed Chair award. L.M.B. is supported by CIHR Grant MGC-14973. Jessica Watson is acknowledged for assistance in the preparation of compound 4 and Michael Maris for compound 8.


Supporting Information Available: NMR spectra of all new molecules; IR characterization of dendrons 9 and 11 and nanoparticles 17 and 18. This information is available free of charge via the Internet at http://pubs.acs.org.

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Enhanced Cell Uptake of Superparamagnetic Iron Oxide

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