Improving Lanthanide Nanocrystal Colloidal Stability in Competitive

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Improving Lanthanide Nanocrystal Colloidal Stability in Competitive Aqueous Buffer Solutions using Multivalent PEG-Phosphonate Ligands Pengpeng Cao, Lemuel Tong, Yi Hou, Guangyao Zhao, Gerald Guerin, Mitchell A. Winnik,* and Mark Nitz* Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario, Canada, M5S 3H6 S Supporting Information *

ABSTRACT: The range of properties available in the lanthanide series has inspired research into the use of lanthanide nanoparticles for numerous applications. We aim to use NaLnF4 nanoparticles for isotopic tags in mass cytometry. This application requires nanoparticles of narrow size distribution, diameters preferably less than 15 nm, and robust surface chemistry to avoid nonspecific interactions and to facilitate bioconjugation. Nanoparticles (NaHoF4, NaEuF4, NaGdF4, and NaTbF4) were synthesized with diameters from 9 to 11 nm with oleic acid surface stabilization. The surface ligands were replaced by a series of mono-, di-, and tetraphosphonate PEG ligands, whose synthesis is reported here. The colloidal stability of the resulting particles was monitored over a range of pH values and in phosphate containing solutions. All of the PEG-phosphonate ligands were found to produce non-aggregated colloidally stable suspensions of the nanoparticles in water as judged by DLS and TEM measurements. However, in more aggressive solutions, at high pH and in phosphate buffers, the mono- and diphosphonate PEG ligands did not stabilize the particles and aggregation as well as flocculation was observed. However, the tetraphosphonate ligand was able to stabilize the particles at high pH and in phosphate buffers for extended periods of time.

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INTRODUCTION

The current state-of-the-art labels for mass cytometry are metal chelating polymers (MCP) that can be readily conjugated to antibodies. These conjugates carry up to 250 copies of a lanthanide ion and are very effective for multiplexed mass cytometry experiments in which the biomarker is present at levels greater than 104 per cell.7−9 A shortcoming of this approach is that it does not have the sensitivity needed to detect biomarkers present at lower expression levels.10 In order to overcome this limitation, we have begun an investigation of methods to provide lanthanide nanoparticles (NPs) that are colloidally stable in biological media, have low nonspecific binding, and to which antibodies can be covalently attached for mass cytometry applications. Lanthanide NPs have the potential to dramatically improve the sensitivity of mass cytometry experiments. For example, a spherical NP such as NaTbF4 with a diameter (d) of 10 nm, assuming bulk density for the NP, would contain 8000 Tb atoms, and a 15-nmdiameter particle would have 15 000 Tb atoms. To proceed, we take advantage of the rapid progress in the synthesis of lanthanide (Ln)-based NPs for different

Biomarker profiling is used increasingly to differentiate normal cells from pathological cells with applications in early disease detection, accurate diagnosis, and to inform personalized health care decisions.1,2 Flow cytometry is used widely to obtain biomarker information on heterogeneous cell populations.3 However, flow cytometry is limited in the number of biomarkers that can be interrogated simultaneously due to the spectral overlap of available fluorophores.4 A new technology, mass cytometry, using element tags rather than fluorescent dyes, has been developed to dramatically increase multiplexed biomarker quantification.5,6 In mass cytometry, biomarkers are recognized by isotopically tagged antibodies. Antibody-labeled cells are introduced individually but stochastically into the plasma torch (ca. 7000 K) of an inductively coupled plasma mass spectrometer (ICP-MS), where they are vaporized, atomized, and ionized. The quantitative detection of a particular isotope tag indicates the level of expression of a specific biomarker. Lanthanide metals are ideal isotopic tags for mass cytometry. They comprise 15 elements with very similar chemistry, many stable isotopes, and low natural abundance. Using mass cytometry, the analysis of more than 30 biomarkers quantitatively in a multiplexed fashion has been achieved.4 © 2012 American Chemical Society

Received: July 4, 2012 Revised: August 1, 2012 Published: August 21, 2012 12861

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applications. Some research groups are interested in Gd3+-based NPs such as NaGdF4 and Gd2O3 as contrast enhancing agents for magnetic resonance imaging (MRI).11−14 Another class of applications takes advantage of Ln-ion doping in a variety of different nanoparticle matrices (Y2O3, NaYF4, NaGdF4, KGdF4, BaGdF5) for optical upconversion.15−22 For example, βNaYF4:Yb3+Er3+ and NaGdF4:Yb3+Er3+ NPs excited with a near-infrared laser (e.g., 978 nm) emit visible light in the wavelength range of 500−600 nm.23−29 These NPs can be used for optical imaging or as sensitizers for photodynamic therapy.30 These NPs, with the exception of the oxide-based materials, are commonly synthesized by a sequence of reactions involving high temperature (300 °C) in an organic medium containing oleic acid (OA) and 1-octadecene (ODE), and yield nanoparticle products that are coated with a surface layer of OA molecules. The OA molecules are surfactant-like surface ligands and provide colloidal stability in nonpolar solvents. A challenge for bioanalytical or therapeutic applications is to transform or encapsulate these particles so that they form stable colloidal solutions in aqueous media. The requirements for NPs designed for mass cytometry are different than those intended for the applications described above. Mass cytometry requires particles with robust colloidal stability to avoid nonspecific interactions with cellular components and uniform size, preferably less than 15 nm to allow for intracellular tagging applications. In comparison, there is no need for contrast enhancing MRI NPs to have a narrow size distribution. Particle aggregation is not an issue as long as the aggregates are colloidally stable. For optical upconversion (or “down conversion”), the low optical absorption cross section of the lanthanide ions means that the NPs require a certain minimum size (optical path length) for excitation to be efficient. These types of NPs normally have diameters of at least 60 nm and often d > 100 nm. Control of the crystal structure of the particle matrix is also important for efficient emission after excitation. There are two general approaches to prepare aqueous colloidally stable NP solutions from NPs prepared in organic media and coated with a stabilizing layer of OA molecules. One approach is to encapsulate the NPs, for example, with block copolymers that have both a hydrophobic block and a hydrophilic block such as PEG that will provide steric colloidal stability in water and serve as a protein repellent corona. The nanoprecipitation method developed by Prud’homme is a leading example of this methodology.23,31 Alternatively, one can carry out ligand exchange reactions, displacing OA molecules with other surfactant-like species that confer water solubility to the NPs. A number of different examples have been reported in the literature. For Ln2O3-type NPs, these examples include poly(acrylic acid) (PAA), PAA-PEG block copolymers, and PAA to which PEG-NH2 molecules have been grafted to the PAA chains in the surface of the NPs. Another approach, reported by van Veggel, used methoxy-PEG-phosphate to displace OA from NaYF4 NPs.24 Phosphates are known to bind to lanthanide metals with greater affinities than carboxylic acids.24,32,33 For many applications, the NPs have to remain colloidally stable in a wide range of buffer media, including phosphate buffered saline (PBS) and cell growth media containing serum. This appears not to be a problem with the NPs coated by nanoprecipitation as reported by Prud’homme,23 but is a more serious problem for NPs solubilized in aqueous media by ligand exchange. For example, van Veggel and co-workers24 reported

that methoxy-PEG-phosphate stabilized NaYF4-based NPs were soluble in water, but unless an excess of ligand was present in the solution, the NPs precipitated in the presence of phosphate buffer as well as in solutions of pH 8 or higher. For mass cytometry applications, we would like nanoparticles that are uniform in size, with well-defined ligands on their surface. PEG-based ligands bearing multiple anchoring groups bind to NP surfaces with higher affinities and provide enhanced stabilization to NPs under competitive biological conditions.35−38 The best example of this approach is the multidentate thiol-containing ligands synthesized by Mattoussi et al. to stabilize semiconductor and metallic NPs in extreme conditions.39 To obtain particles that are colloidally stable and avoid aggregation, we designed and synthesized divalent and tetravalent PEG-phosphonate-based ligands, using lysine as a scaffold. To accommodate biofunctionalization of NPs stabilized with this ligand, we also synthesized a corresponding tetradentate ligand with a free −NH2 at the distal end of the PEG chain. The main conclusion of our work is that increasing the valence of the phosphonate-based ligand for the NaLnF4 NPs dramatically improved the stability of the particles, in competitive buffer solutions such as PBS. NPs stabilized with the tetradentate ligand remain colloidally stable and do not aggregate in PBS buffer, even over a time scale of weeks.

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EXPERIMENTAL SECTION

Instrumentation. 1H-, 13C-, and 31P-NMR spectra were recorded on a Varian 400 or Bruker 400 MHz spectrometers at T = 297 K. High-resolution mass spectra were obtained from an ABI/Sciex QStar mass spectrometer with an ESI source. Right-angle dynamic light scattering (DLS) measurements were performed using an (ALV/DLS/ SLS-5000) instrument equipped with an ALV-5000/EPP multiple digital time correlator and laser goniometer system ALV/CGS-8F S/N 025 with a helium−neon laser (Uniphase 1145 P, output power of 22 mW and wavelength of 632.8 nm) as light source. All nanoparticle solutions were measured after passage through a 0.2 μm cellulose filter. Transmission electron microscopy (TEM) images were obtained with a Hitachi H-7000 TEM. OA-coated NPs dispersed in cyclohexane were drop-cast onto hydrophobic Formvar-carbon coated copper TEM grids. PEG-coated NPs dispersed in DI water were drop-cast onto pretreated hydrophilic TEM grids. To prepare the hydrophilic grids, the grids were treated with a 1 wt % aqueous Triton X-305 solution, which was then dried. These TEM grids were then washed 4 times with DI water by dipping in water and gentle pad drying with filter paper between each washing step. The polydispersity indices of PEG starting materials were determined by gel permeation chromatography (GPC) using tetrahydrofuran (THF) as the eluent (flow rate: 1.0 mL/min) and a Viscotek TDA 302 triple detector array liquid chromatography instrument. The system is equipped with three linear mixed-bed polyanalytik columns with exclusion limits of 70k, 400k and 4000k. A 10-point calibration curve was prepared using polystyrene standards with molecular weights between 575 and 2.6 × 106 g/mol. The RI trace was analyzed for PDI. Materials. All organic and inorganic compounds (except PEG compounds) and solvents were purchased from Sigma Aldrich. All PEG-based compounds were purchased from JenKem. 6-Azidohexanoic acid succinimidyl ester and (S)-lysine methyl ester dihydrochloride were synthesized according to literature procedures.40 Monophosphate-PEG-OCH3 was prepared according to a previously published procedure.24 Silica chromatography was performed with SiliCycle Silica-P Flash Silica Gel. Nanoparticle Synthesis. NaLnF4 nanoparticles were synthesized as described by Qian and Zhang with some small modifications.41 For NaHoF4 nanoparticles, HoCl3·6H2O (0.38 g, 1 mmol), oleic acid (16 mL), and 1-octadecene (16 mL) was heated to 150 °C in a threenecked flask under vacuum for 1 h until a clear solution formed. After 12862

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(4 H, m, CH2), and 1.40−1.38 (2 H, m, CH2); 31P NMR (400 MHz; CDCl3, δ) 26.0. 6-(4-((Bis(phosphonomethyl)amino)methyl)-1H-1,2,3-triazol-1yl)hexanyl-PEG-OCH3 (4). Compound 3 (0.25 g, 0.10 mmol) in dry DCM (2 mL) was cooled to 0 °C. Trimethylsilyl bromide (0.13 mL, 1.0 mmol) was added dropwise and the reaction mixture was allowed to warm to room temperature and stirred overnight. The solvent was removed under reduced pressure, and the residue was redissolved in MeOH (2 mL) and stirred at room temperature for 5 h. Compound 4 was isolated (0.21 g, 84%) after MeOH methanol was removed by evaporation. 1H NMR (400 MHz; CDCl3, δ): 4.43 (2H, br s, triazoleCH2), 3.79 (2 H, t, J = 4, CCH2N), 3.61−3.59 (184 H, m, -(CH2)44-), 3.30 (3 H, s, OCH3), 3.43 (4 H, t, J = 4 Hz, PCH2N), 2.18−2.14 (2 H, m, CH2CO), 1.53 (2 H, br s, CH2), and 1.29−1.05 (4 H, m, CH2); 31P NMR (162 MHz; D2O, δ) 7.40, 6.46. Methyl 2,6-Bis(6-azidohexanamido)hexanoate1 (5). Lysine (0.72 g, 3.12 mmol) and N,N-diisopropylethylamine (14.6 mL, 113.2 mmol) were dissolved in 30 mL dry DMF. 6-Azidohexanoic acid succinimidyl ester (1.44 g, 5.66 mmol) was added to the mixture and the solution was stirred overnight at room temperature. The solvent was removed under reduced pressure. The residue was redissolved in EtOAc (50 mL), washed with 0.5 M citric acid (3 × 50 mL), NaHCO3 (3 × 50 mL), and brine. The organic layer was dried over MgSO4, concentrated, and purified by silica column, eluting with 95:5 EtOAc−MeOH, to give product 5 (0.82 g, 67%) as a clear yellow oil. RF (95:5 EtOAc−MeOH) 0.47; 1H NMR (400 MHz; CDCl3, δ): 6.19 (1 H, d, J = 7.7 Hz, NH), 5.70 (1 H, s, NH), 4.57 (1 H, td, J = 8.0, 4.7 Hz, CH), 3.74 (3 H, s, CH3), 3.31−3.21 (6 H, m, NHCH2, CH2N3), 2.28−2.23 (2 H, m, COCH2), 2.18 (2 H, t, J = 7.5 Hz, COCH2), and 1.24−1.90 (18 H, m, CH2); 13C NMR (75 MHz; CDCl3, δ): 173.58, 173.55, 173.32, 53.01, 52.28, 51.84, 39.39, 37.00, 36.75, 32.59, 29.46, 29.21, 29.19, 26.98, 26.90, 25.80, 25.61, 22.92; HRMS m/z calculated for C19H35N8O4 [MH+] 439.27865, found 439.27813. 2,6-Bis(6-azidohexanamido)hexanoic Acid (6). Compound 5 (0.82 g, 1.86 mmol) and NaOH (5.5 mL of 1 M solution) were added into methanol (5 mL). The mixture was stirred at 0 °C for 3 h. The solvent was removed under reduced pressure. The residue was redissolved in EtOAc (30 mL) and washed with 0.5 M NaHSO4 (3 × 50 mL) and brine. The organic layer was dried over MgSO4 and purified by silica column, eluting with 95:5 EtOAc−MeOH, to give product 6 (0.72 g, 91%) as a clear yellow oil. RF (95:5 EtOAc− MeOH) 0.42; 1H NMR (400 MHz; CDCl3, δ): 6.78 (1 H, d, J = 7.2 Hz, NH), 5.97 (1 H, s, NH), 4.53 (1 H, dt, J = 7.5, 3.8 Hz, CH), 3.39− 3.17 (6 H, m, NHCH2, CH2N3), 2.29 (2 H, td, J = 7.3, 1.9 Hz, COCH2), 2.22 (2 H, t, J = 7.5 Hz, COCH2), and 1.97−1.30 (18 H, m, CH2); 13C NMR (75 MHz; CDCl3, δ): 174.59, 174.49, 173.32, 52.82, 51.86, 51.82, 39.28, 37.02, 36.58, 29.69, 29.21, 29.18, 29.16, 26.92, 26.89, 25.80, 25.66, 22.54; HRMS m/z calculated for C18H33N8O4 [MH+] 425.26271, found 425.26248. 2,5-Dioxopyrrolidin-1-yl-2,6-bis(6-azidohexanamido) Hexanoate (7). Compound 6 (910 mg, 2.14 mmol) was activated by N,N′dicyclohexylcarbodiimide (463.7 mg, 2.25 mmol) and N-hydroxysuccinimide (259.1 mg, 2.25 mmol) in dry DCM (8 mL), after stirring at room temperature overnight. The reaction mixture was filtered through a Celite plug and washed with EtOAc. This organic filtrate was washed with NaHCO3 (3 × 100 mL) and brine, and dried over MgSO4. Purification of the residue by silica column, eluting with 90:10 DCM−MeOH, gave the product as an opaque oil (0.8 g, 72%), RF (90:10 DCM−MeOH) 0.55; 1H NMR (400 MHz; CDCl3, δ): 6.37 (1 H, d, J = 7.5 Hz, NH), 5.90 (1 H, s, NH), 4.93 (1 H, td, J = 7.1, 5.4 Hz, CH), 3.37−3.15 (6 H, m, NHCH2, CH2N3), 2.85 (4 H, s, COCH2), 2.27 (2 H, t, J = 7.3 Hz, COCH2), 2.19 (2 H, t, J = 7.5 Hz, COCH2), and 1.74−1.28 (18 H, m, CH2); 13C NMR (75 MHz; CDCl3, δ): 173.79, 173.42, 172.79, 168.10, 51.26, 50.31, 38.29, 36.36, 35.85, 31.32, 28.77, 28.61, 28.58, 28.38, 28.25, 25.62, 25.23, 24.93, 21.58; HRMS m/z calculated for C22H35N9O6 [MH+] 522.27, found 522.2783. Bis(azido)-Lys-PEG-OCH3 (8). A solution of NH2−PEG2000−OCH3 (1.16 g, 0.58 mmol) and triethylamine (0.26 mL, 1.83 mmol) was

the solution was cooled to room temperature, a solution of MeOH (10 mL) containing NH4F (0.15 g, 4 mmol) and NaOH (0.10 g, 2.5 mmol) was added dropwise at 50 °C and stirred for 30 min after the addition was completed. The mixture was heated to 100 °C slowly under vacuum to evaporate MeOH, and then it was heated to 300 °C at a heating rate of 20 °C/min and maintained for 1 h under nitrogen. After the solution was cooled to room temperature, the nanoparticles were precipitated with EtOH and centrifuged with a benchtop centrifuge at 14 000 rpm (11 000g) for 10 min. The as-prepared oleic acid coated NPs in the mixture of octadecene and oleic acid were precipitated with ethanol and sedimented by centrifugation. The liquid was decanted, and the particles were immediately redispersed in THF (2.5 mL). Then, ethanol (15 mL) was added, and the sample was centrifuged at 4000 rpm (7500g, 35 min) to sediment the particles. The supernatant was discarded, and the process was repeated a total of three times to obtain clean NaHoF4. The syntheses of three other types of NaLnF4 NPs (Ln: Eu, Gd, Tb) followed a similar synthetic route, except that the initial reaction mixture was heated to 125 °C instead of 150 °C under vacuum for 1 h. After the removal of the MeOH solution at 100 °C, the reaction was heated to 300 °C at a slower heating rate, 13 °C/min. The size of the nanoparticles was characterized by TEM and, in solution, by DLS. The TEM images were analyzed with a trial version of Fovea Pro 4.0 which is an image editing plug-in for Photoshop developed by John Russ.42 In order to determine the size of the NPs, the TEM image was first corrected for brightness, contrast, and nonuniform background intensity. The image was then reduced to a binary image via bilevel thresholding and segmentation. NPs that were not correctly binarized were removed before taking the measurements. Synthesis of PEG Ligands. Tetramethyl ((Prop-2-yn-1ylazanediyl)bis(methylene))bis(phosphonate) (1). Propargyl amine (0.5 g, 9.1 mmol) and formaldehyde (1.7 mL of a 37% by weight solution in water) in THF (40 mL) were stirred at room temperature for 5 min. Dimethyl phosphite (2 g, 18.2 mmol) was added to the mixture dropwise over 5 min. The mixture was refluxed at 70 °C overnight. The solvent was removed under reduced pressure. The residue was purified by silica column chromatography, eluting with 95:5 DCM−MeOH, to give product 1 (1.62 g, 60%) as a light yellow oil. RF (95:5 DCM−MeOH) 0.34; 1H NMR (400 MHz; CDCl3, δ): 3.80 (12 H, d, J = 10.7 Hz, CH3O), 3.76 (2 H, d, J = 2.2 Hz, CH2), 3.14 (4 H, dd, J = 11.4, 1.1 Hz, CH2), and 2.28 (1 H, t, J = 2.3, CH); 13 C NMR (75 MHz; CDCl3, δ): 74.58, 53.04, 50.28, 48.66, 46.22; 31P NMR (400 MHz; CDCl3, δ) 25.94; HRMS m/z calculated for C9H19NO6P2 [MNa+] 322.0576, found 322.0590. 6-Azidohexanyl-PEG-OCH3 (2). 6-Azidohexanoic acid succinimidyl ester (0.41 g, 0.19 mmol) dissolved in 1 mL of dry DMF was added slowly to 1 mL dry DMF containing NH2−PEG−OCH3 (0.37 g, 0.18 mmol) and triethylamine (0.1 mL, 0.72 mmol). The reaction mixture was allowed to stir at room temperature overnight. The solvent was removed under reduced pressure, and the crude product was precipitated from THF (5 mL) at −20 °C to afford a light yellow solid 2 (0.32 g, 84%). 1H NMR (400 MHz; CDCl3, δ): 3.80−3.51 (m), 3.35 (3 H, s, OCH3), 3.24 (2 H, t, J = 6 Hz, CH2N3), 2.16 (2 H, t, J = 6 Hz, CH2CO), 1.66−1.57 (4 H, m, CH2), and 1.40−1.38 (2 H, m, CH2). 6-(4-((Bis((dimethoxyphosphoryl)methyl)amino)methyl)-1H1,2,3-triazol-1-yl)hexanyl-PEG-OCH3 (3). Compound 2 (0.31 g, 0.14 mmol) and compound 1 (54.2 mg, 0.18 mmol) were dissolved in a solvent mixture of t-butanol/MeOH/H2O (1.5 mL/3 mL/1.5 mL). Sodium ascorbate (5.7 mg, 0.029 mmol) and catalytic CuSO4 (1.8 mg, 0.0072 mmol) were added to the solution and it was stirred at room temperature overnight before the solvent was evaporated under vacuum. The crude product was redissolved in THF (3 mL) and the insoluble impurities were removed by a syringe filter (0.2 μm, PTFE Acrodisc). The product was further purified by precipitation in THF at −20 °C to afford a light yellow compound 3 (0.28 g, 80%).1H NMR (400 MHz; CDCl3, δ): 4.38 (2 H, br s, triazole-CH2), 3.81−3.78 (14 H, m, CH3OP, CCH2N), 3.65−3.63 (m), 3.37 (3 H, s, OCH3), 3.13 (4 H, dd, J = 4 Hz, PCH2N), 2.28 (2 H, t, J = 4 Hz, CH2CO), 1.66−1.57 12863

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dissolved in dry DMF (3 mL) and stirred at room temperature. Compound 7 (0.32 g, 0.61 mmol) dispersed in dry DMF (3 mL) was added dropwise. The mixture was stirred overnight at room temperature. DMF was removed under reduced pressure and the crude product was precipitated from THF (5 mL) at −20 °C to afford a white solid product 8 (1.13 g, 81%). 1H NMR (400 MHz; CDCl3, δ): 6.92 (1 H, d, J = 7.3 Hz, NH), 6.44 (1 H, s, NH), 4.46 (1 H, td, J = 7.8, 4.6 Hz, CH), 3.88−3.59 (172 H, m, -(CH2)44-), 3.34 (3 H, s, OCH3), 3.29−3.12 (6 H, m, NHCH2, CH2N3), 2.28 (2 H, t, J = 7.5 Hz COCH2), 2.20 (2 H, t, J = 7.5 Hz COCH2), and 1.92−1.04 (18 H, m, CH2). Bis(dimethyl phosphonate)-Lys-PEG-OCH3 (9). Compound 8 (1.0 g, 0.43 mmol) and compound 1 (0.31 g, 1.04 mmol) were dissolved in a solvent mixture of t-butanol/MeOH/H2O (5 mL/10 mL/5 mL). Sodium ascorbate (33.3 mg, 0.4 mmol) and catalytic CuSO4 (10.5 mg, 0.042 mmol) were added to the solution and it was stirred at room temperature overnight before the solvent was evaporated under vacuum. The crude product was redissolved in THF (5 mL) and insoluble impurities were removed by a syringe filter (0.2 μm, PTFE Acrodisc). The product was further purified by precipitation in THF at −20 °C to afford a brown compound 9 (1.62 g, 63%). 1H NMR (400 MHz; CDCl3, δ): 6.79 (1 H, s, NH), 6.40 (1 H, s, NH), 4.35−4.24 (5 H, m, CH, CH2-triazole), 3.73 (28 H, m, CH3OP, CCH2N), 3.59− 3.55 (187 H, m, -(CH2)44-), 3.31 (3 H, s, CH3O), 3.17−3.02 (10 H, m, NCH2P, NHCH2), 2.24−2.07 (4 H, m, COCH2), and 1.90−0.93 (18 H, m, CH2); 31P NMR (162 MHz; CDCl3, δ) 26.52, 25.98. Bis(phosphonate)-Lys-PEG-OCH3 10. Compound 9 (1.6 g, 0.53 mmol) in dry DCM (10 mL) was cooled to 0 °C. Trimethylsilyl bromide (1.62 g, 10.6 mmol) was added dropwise and the reaction mixture was allowed to warm to room temperature and stirred overnight. The solvent was removed under reduced pressure and the residue was redissolved in MeOH (10 mL) and stirred at room temperature for 5 h. Compound 10 was isolated (1.22 g, 79%) after the solvent was removed by evaporation. 1H NMR (400 MHz; CDCl3, δ): 4.48 (4 H, t, J = 6.7 Hz, CH2-triazole), 4.16−4.19 (1 H, m, CH), 3.72−3.69 (184 H, m, -(CH2)44-), 3.39 (3 H, s, CH3O), 3.30−3.04 (10 H, m, NCH2P, NHCH2), 2.26 (4 H, d, J = 31 Hz, COCH2), and 1.85− 0.93 (18 H, m, CH2); 31P NMR (162 MHz; D2O, δ) 7.40, 6.46. Ligand Exchange Experiments. The as-prepared oleic acidcoated NPs in the mixture of octadecene and oleic acid were precipitated with ethanol and sedimented by centrifugation at 4000 rpm (7500g, 35 min). The supernatant was decanted, and the particles were immediately redispersed in THF (2.5 mL). Then, ethanol (15 mL) was added, and the sample was centrifuged at 4000 rpm (7500g, 35 min) to sediment the particles. The supernatant was discarded, and the process was repeated a total of three times. Nanoparticles (∼20 mg) washed as described above were redispersed in dry THF (5 mL) to give a clear solution. Then, a PEG ligand sample (30 mg) in THF (2 mL) was added, and the mixture was stirred at room temperature overnight. At this point, the NPs were precipitated with hexanes (50 mL), centrifuged at 4000 rpm (7500g, 40 min) to form a pellet, and the supernatant was removed. The pellet was redispersed in 5 mL THF by sonication, and the hexane precipitation step was repeated twice. After the final wash, a gentle stream of air was applied to the NPs in the tube to remove traces of solvent left in the sample. The dried pellet was readily dispersed in distilled water (15 mL) and the homogeneous suspension was transferred to a spin filter with a ∼10 000 MWCO. The sample was concentrated to 1.5 mL by centrifugation at 3000 rpm (5600g, 30 min). This washing step was repeated twice with distilled water (15 mL) to remove excess PEG ligands. As an additional washing step, mono- and diphosphonate-methoxy-PEG-coated NPs were dissolved in 15 mL MES buffer (25 mM, pH 5.5) and concentrated to 1.5 mL using a spin filter. The tetra-phosphonate-methoxy-PEG coated NPs were redispersed in PBS buffer and similarly concentrated to 1.5 mL using a spin filter. To remove the salts in the buffers, all methoxy-PEGcoated NPs were redispersed in 15 mL of water and concentrated to 1.5 mL using a spin filter in the final washing step. The PEG ligandcoated NPs were stored in water at a concentration of 10 mg/mL at

room temperature. The original oleic acid-coated NPs and PEGcoated NPs were characterized by TEM and DLS measurements. Stability Assessments of PEG-Coated Nanoparticles. Aqueous solutions at particular pH values were prepared by adding diluted NaOH and HCl to distilled water or to buffer and monitoring with a standard pH electrode. The stability test of methoxy-PEG-coated NPs was carried out in water at pH 3, 7, and 9, standard PBS solution (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4·H2O, 2 mM KH2PO4, pH 7.4) and 200 mM sodium phosphate buffer (pH 7.4). The final concentration of PEG-coated NPs was adjusted to 1 mg/mL for DLS measurements.

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RESULTS AND DISCUSSION This paper focuses on the synthesis of novel bidentate and tetradentate PEG-phosphonate ligands and their application as stabilizing surfactants for lanthanide NPs in water and in competitive buffer solutions. The structures of the four PEG ligands examined are shown in Figure 1. Monophosphate-PEG-

Figure 1. Structure of the PEG (MW∼2000) ligands for nanoparticle surface modification.

OCH3, synthesized as described by Boyer et al.,24 serves as a reference for the other three polymeric ligands whose synthesis is described below. Diphosphonate-PEG-OCH3 (4) and tetraphosphonate-PEG-OCH3 (10) are useful for testing the stability of lanthanide nanoparticles in various buffers, but lack a functional group for further biological modifications. To meet this need, we also synthesized a functional ligand, tetraphosphonate-PEG-NH2 (13), with an amino group at the distal end of the PEG chain. For all four ligands, we employed PEG chains with a molecular weight of 2000 (ca. 44 EG units). Ligand Synthesis and Characterization. Three PEGs (HO-PEG-OCH3, NH2-PEG-OCH3, NH2-PEG-NHBoc) used in our synthesis to make mono-, di-, and tetravalent PEG ligands were carefully characterized by 1H NMR. The 1H NMR T1 and T2 relaxation constants of all PEG polymer samples were measured by a Varian 400 MHz spectrometer (Table S1, Supporting Information). The molecule NH2-PEG-OCH3 has the longest T1 decay time (3.24 ± 0.08 s). We assumed that all PEG derivatives synthesized would have T1 and T2 relaxation 12864

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Scheme 1. Synthesis of Di-phosphonate-PEG-OCH3 and tetra-Phosphonate-PEG-OCH3 Ligands

PEG end group to determine the efficiency of the transformations. The synthesis of diphosphonate-PEG-OCH3 (4) began by attaching 6-azido-hexanoic acid to H2N-PEG-OCH3 via DCC/ NHS coupling (Scheme 1). In parallel, a propargyl amine was functionalized with two protected phosphonate groups via condensation with formaldehyde and dimethyl phosphite in a Mannich-like reaction to give compound 1.43 Coupling the azido-functionalized PEG and compound 1 via a coppercatalyzed azide−alkyne (“click”) reaction gave the protected diphosphonate PEG (3). The dimethyl phosphonates were hydrolyzed in the final step with trimethylsilyl bromide (TMSBr) to release free phosphonate anchoring groups and give compound 4.43 To synthesize the tetradentate ligand, 6-azidohexanoic acid was attached to both amino groups of lysine methyl ester

constants similar to their PEG precursors. To obtain fully relaxed spectra with accurate proton integrations of all PEGcontaining molecules, a delay time of 16 s was used to obtain all 1 H NMR spectra. The degree of polymerization for all 3 PEG precursors determined by NMR was approximately 44 which corresponds to PEG with a molecular weight of 2000 g/mol. Gel permeation chromatography (GPC) using THF as the eluent and polystyrene standards was used to determine polydispersity index values (PDI = Mw/Mn) of HO-PEG-OCH3 (1.04), NH2-PEG-OCH3 (1.06), and NH2-PEG-NHBoc (1.02). These values confirm the narrow length distribution of the polymers employed. For the synthesized PEG ligands, 1H and 31 P NMR analysis was performed at each synthetic step to demonstrate the efficiency of the reactions. The integrated intensity of signals from protons on the anchoring group was compared to the signals from the PEG backbone as well as the 12865

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mL) for several months in cyclohexane often led to irreversible aggregation. TEM images of the NaLnF4 particles (Ln: Ho, Eu, Gd, Tb) prepared in this way are presented in Figure 2. The

(Scheme 1). After ester hydrolysis, the product 6 was converted to its N-hydroxysuccinimide ester 7 and reacted with 1 equiv of NH2-PEG-OCH3 in dry DMF to obtain the diazido-PEG derivative 8. This species was coupled to the diphosphonate functionalized propargylamine (1) via the copper-catalyzed click reaction. After the deprotection with TMS-Br, each PEG chain carries four metal-binding phosphonate anchoring groups at the end of the chain. Crude products from each synthetic step were purified by sequential precipitations in dry THF at −20 °C and collected by filtration. The yield of the amide conjugate was determined by 1H NMR where the integrations of protons from the lysine anchoring group were compared to the signals of protons from the PEG polymer backbone and its end groups. For example, in compound 8, the signal from the α carbon on the lysine at 4.46 ppm integrates to 1.2 proton (expected 1 proton), while the signal of the methoxy end group on the polymer chain at 3.34 ppm integrates to 3 protons (expected 3 protons) and the polymer backbone signal at 3.61 ppm integrates to 172 protons (expected 176 protons for 44 methylene repeating units). The close-to-expected integration values of 1H NMR signals demonstrated the effective amide bond formation. In compound 9, the signals from the 8 methoxy groups on the phosphonates (24 protons) and 4 protons on the branched propargylamine at 3.78 ppm integrate to 28.8 protons (expected 28 protons), while the signal of the methoxy end group on the polymer chain at 3.31 ppm integrates to 3 protons (expected 3 protons) and the polymer backbone signal at 3.55−3.59 ppm integrates to 187 protons (expected 176 protons for 44 methylene repeating units). The approximate 1:1 ratio between proton signals of the lysine anchoring group and the PEG polymer backbone indicates the high efficiency of the click-reaction. The appearance of a phosphorus signal in the 31P NMR spectrum after the coupling step confirmed the success of the reaction with the propargyl phosphonates. The disappearance of the proton signals from the methoxy groups on the phosphonates at 3.78 ppm and the significant signal shift in the 31P NMR spectrum after the deprotection step by TMS-Br indicate the formation of a new phosphorus species, the free phosphonate-containing PEG molecules. The same strategy was utilized to synthesize an amine functionalized tetradentate PEG ligand. The synthesis of tetra-phosphonate-PEG-NH2 ligand started with NH2-PEG-NHBoc. As with the bidentate ligand both 1H NMR and 31P NMR analysis were used to monitor the coupling efficiency between the lysine anchoring group and the PEG chain. In both coupling steps, high efficiency was observed. In the final deprotection step of the synthesis, TMS-Br not only removed the methyl groups of the phosphonate methyl esters, but also removed the Boc- protecting group. The free amine group on the PEG ligand can be used for future bioconjugation studies or other desired modifications. Nanoparticle Synthesis and Characterization. The nanoparticle synthesis followed the protocol reported by Qian and Zhang,28 in which the chloride salt of the lanthanide was first converted to the corresponding oleate in a mixture of oleic acid (OA) and 1-octadecene (ODE). After treatment with NH4F and NaOH, the mixture was heated to 300 °C to promote particle formation. The particles were purified by precipitation with excess ethanol followed by resuspension in THF. After three cycles of purification, the NPs were dispersed in cyclohexane. These particles also give stable colloidal solutions in organic solvents such as hexane and toluene. However, storing these NPs at high concentration (>40 mg/

Figure 2. TEM images of oleic acid-coated NaLnF4 NPs. The space bars in each image are 100 nm.

particles are nearly spherical in shape. Image analysis gave mean diameters of 16 ± 1.2 nm for NaHoF4, 8.8 ± 0.5 nm for NaEuF4, 8.9 ± 0.4 nm for NaGdF4, and 11 ± 0.4 nm for NaTbF4. Ligand Exchange. Ligand exchange was carried out by stirring an excess of PEG ligands dissolved in THF with a THF solution containing oleic acid-coated NaLnF4 NPs. We typically employed about a 50 wt % excess of ligand compared to nanoparticles, and the mixture was stirred at room temperature overnight. At this point, the PEG-coated NPs and excess ligand were precipitated with hexane, while the oleic acid released from the NPs was removed with the organic solvent. The NPs obtained in this way readily dispersed in water to yield transparent solutions. To remove excess ligand, the PEG-coated NPs were washed with water and then with buffer using spin filters with a pore size small enough to retain the NPs but large enough (MWCO ∼10 000) to allow the PEG ligands (MW ∼2000) to pass. We used more vigorous buffer washes with the multidentate ligands (see the Experimental section for details), to avoid nonspecific PEG adsorption to the NPs. Brigger and co-workers have observed that free PEGs can loosely associate with nanoparticle surfaces and form a PEG layer.44 The ligand exchange reaction was equally effective for the series of different NP compositions and sizes described above. One indication of the colloidal stability and absence of aggregation of the PEGcoated particles prepared in this way is that they gave wellseparated particles when drop-cast onto hydrophilically coated TEM grids. Three examples are presented in Figure 3 for NaHoF4 NPs following ligand exchange with methoxy-PEGmonophosphate, methoxy-PEG-diphosphonate, and methoxyPEG-tetraphosphonate. Aqueous solutions of the particles were also examined by right-angle dynamic light scattering (DLS). In Figure 4, we 12866

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Figure 4. DLS CONTIN plots for NaLnF4 NPs comparing the hydrodynamic radii for the OA-NPs in THF with the methoxy-PEGtetraphosphonate coated particles in water.

pH and Buffer Effects on the Colloidal Stability of PEG-Coated NaHoF4 NPs. To examine the influence of pH and of phosphate as a competitive buffer on NP colloidal stability, we focused on NaHoF4 NPs. Some experiments relied on visual observations to assess sample flocculation, whereas transparent solutions were examined by DLS to assess changes in particle size or the onset of particle aggregation. For these experiments, we compare NaHoF4 NPs after exchange with methoxy-PEG-monophosphate, methoxy-PEG-diphosphonate, and methoxy-PEG-tetraphosphonate ligands. The results of the DLS measurements are presented in Figure 5. At pH 7, PEG-coated NaHoF4 NPs with the mono-, di-, and tetradentate ligands gave monomodal distributions. For methoxy-PEG-phosphate, this result is consistent with that reported by the van Veggel group.24 The mean apparent hydrodynamic radii (Rh) for these samples are in the range of 18 to 21 nm, larger than the Rh of the OA-capped NaHoF4 NPs in THF (12 nm) (Figure 3). The CONTIN plots confirm the colloidal stability and the absence of aggregation of the particles in water. An interesting question is whether the different ligands lead to different values of Rh, since the footprint of the ligand on the particle surface would be expected to increase from the monophosphate to the di- and then the tetraphosphonate. There is some indication that the monophosphate leads to a slightly larger value of Rh, perhaps associated with closer packing on the particle surface and, as a consequence, a slightly more extended brush into the aqueous medium. Detailed values of Rh and PDI for these samples are presented in SI Table S3. Solutions of the three different methoxy-PEG-coated NaHoF4 NPs were rendered acidic (to pH 3) by addition of HCl to the solution at pH 7 and basic by addition of NaOH. The final concentrations of PEG-NPs were kept constant (1 mg/mL) and the samples were evaluated by DLS (Figure 5A,C, respectively). At pH 3, all three types of PEG-NPs showed

Figure 3. TEM images of NaHoF4 NPs following ligand exchange, respectively, with methoxy-PEG-monophosphate, methoxy-PEG-diphosphonate, and methoxy-PEG-tetraphosphonate. The space bars in each image are 100 nm.

compare CONTIN plots of OA-stabilized NaEuF4, NaGdF4, and NaTbF4, NPs in THF with corresponding plots obtained for these NPs in water after ligand exchange with methoxyPEG-tetraphosphonate. These plots show that particles are monomodal with a very narrow size distribution. Values of the hydrodynamic radii (Rh) and polydispersity (PDI) values of these particles calculated from the DLS autocorrelation functions are presented in SI Table S2. These Rh values are approximately 2 to 3 nm larger than the radii determined by TEM, due to the presence of the oleates on the surface of the NPs. Following ligand exchange, one sees an increase in Rh, accompanied by a broadening of the distribution, and the presence, in the case of NaEuF4 NPs, of a tiny amount of aggregates. Values of Rh and PDI for these NPs are also presented in SI Table S2. The hydrodynamic radius of all of the PEG-coated NPs increased by about 10 nm, due to the size of PEG2000 ligands anchored at the surface, compared to the diameter of the dry particles determined by TEM. This formation of a 10-nm-thick corona around an inorganic NP core after coating with PEG chains with MW of ∼2000 Da is consistent with a report by Budijono et al. for block copolymer coated NPs.45 12867

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Figure 5. DLS CONTIN plots of PEG-coated NaHoF4 NPs in water at different pH values and in PBS buffer. The colloidal stability of the monophosphate-PEG-, diphosphonate-PEG-, and tetra-phosphonate-PEG-coated NPs were monitored at (A) pH 3; (B) pH 7; (C) pH 9; and (D) in PBS buffer at pH 7.4. All samples were prepared at 1 mg/mL PEG-NP concentration.

photographic images in Figure 6. On the left are three solutions in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM

monomodal CONTIN plots. NaHoF4 NPs coated with both diand tetra-phosphonate-PEG ligands maintain a narrow size distribution with PDI values of 0.25 and 0.19, respectively, which are not very different. The NPs bearing PEGmonophosphate ligands show substantial broadening, characteristic of some particle aggregation. We did not monitor changes in the DLS profile of this sample over extended times. At pH 9, aggregation was much more pronounced for the NPs stabilized by the PEG-monophosphate and PEG-diphosphonate ligands with a prominent secondary peak seen for aggregates with Rh values >100 nm. The tetra-phosphonatePEG stabilized NPs remain stable at pH 9 for extended periods of time (weeks). The most important results are those in PBS buffer. Inorganic phosphate acts as competitive NP ligand for polymeric phosphate ligands. Boyer et al.24 have shown that, while PEG-monophosphate ligands will provide aqueous colloidal stability to upconverting rare earth NPs in the presence of phosphate buffer if an excess of PEG-monophosphate is present in solution, the NPs flocculate when that excess is removed. Similarly, we found that NaHoF4 NPs stabilized by methoxy-PEG-monophosphate in water flocculated when transferred to PBS buffer. Corresponding NPs stabilized by PEG-diphosphonate ligands gave transparent solutions in PBS buffer. As one can see from the DLS data in Figure 5D, the most prominent peak is due to aggregates of these NPs. Only in the case of the PEG-tetraphosphonate ligands did the particles maintain their colloidal stability without detectable aggregation. Here, the tetra-phosphonatePEG coated NP sample has Rh of 24 nm with the same narrow PDI (0.18) found in water at pH 7. Monitoring by DLS showed that, on a time scale of weeks, no sign of aggregation could be detected. A limited number of experiments were carried out at much higher (200 mM, pH 7.4) phosphate buffer concentration. Under these much harsher conditions, the particles tended to aggregate and flocculate. NaHoF4 NP samples at 10 mg/mL made it easier to visualize precipitation. This can be seen in the

Figure 6. Stability of NaHoF4 NPs stabilized with methoxy-PEGphosphate-, methoxy-PEG diphosphonate-, and methoxy-PEG tetraphosphonate surface ligands in different buffer solutions. Left: PEGNPs in PBS buffer solution. Right: PEG-NPs in 200 mM sodium phosphate buffer. The NP concentration in all solutions was 10 mg/ mL.

Na2HPO4·H2O, 2 mM KH2PO4, pH 7.4) that mimics biological conditions. For these experiments, a precipitate can be seen in the NP solution for the methoxy-PEG-monophosphate-coated NPs. The NPs coated with the PEG-diphosphonate and PEGtetraphosphonate ligands remain transparent. At 200 mM phosphate buffer, a copious precipitate can be seen in the samples containing the monophosphate- and diphosphonatecoated NPs. Here, the NPs precipitated out of solution within minutes. Some turbidity, but no precipitate, appears in the solution of NPs coated with the PEG-tetraphosphonate. This 12868

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Langmuir turbidity took a few hours to become apparent and then remained stable. While the slight cloudiness indicates that some NP aggregation occurred under these conditions, the most important conclusion is that the tetra-phosphonate ligand provides exceptionally stable chelation to the NP surface.

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REFERENCES

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CONCLUSIONS We synthesized three different methoxy-PEG derivatives and an end-functional amino-PEG derivative based upon PEG precursors with Mn = 2000 (44 EG units) to serve as monodentate, bidentate, and tetradentate ligands for NaLnF4 nanoparticles (NPs) in aqueous media. Methoxy-PEG-monophosphate was compared with a methoxy-PEG-diphosphonate and a methoxy-PEG-tetraphosphonate for their ability to impart colloidal stability to these nanoparticles, particularly in phosphate buffer. These ligands were able to displace oleate from the as-synthesized NPs. Following ligand exchange, the NPs formed stable colloidal solutions in water at neutral pH. The exchange worked equally well for a series of NaLnF4 NPs (Ln = Ho, Eu, Gd, Tb). Detailed examination of colloidal stability as a function of pH and in the presence of phosphate buffer focused on 14-nm-diameter NaHoF4 NPs. Monophosphate-coated particles showed indication of aggregation in water at pH 3 and at pH 9, where a second peak at Rh > 100 nm could be seen in a CONTIN plot of dynamic light scattering (DLS) measurements. The NPs coated with methoxy-PEG-diphosphonate were stable at pH 3 but showed an aggregate peak by DLS at pH 9. The most important results were obtained from experiments in phosphate buffer. The PEG-monophosphate particles precipitated in both PBS buffer and at higher concentration (200 mM, pH 7.4) phosphate buffer. The methoxy-PEG-diphosphonate coated NPs gave transparent solutions in PBS buffer but showed signs of partial aggregation by DLS. These NPs precipitated in 200 mM phosphate buffer. The methoxy-PEG-tetraphosphonate coating provided excellent and robust colloidal stability. In PBS buffer at 1 mg/mL, there was no sign of aggregation by DLS. The particle size and size distribution was identical to that in water at neutral pH. At higher concentration (10 mg/mL) in 200 mM phosphate buffer, the solutions acquired a slight but noticeable turbidity, but no precipitate formed. These results demonstrate that the multidentate ligands bind to NaLnF4 NPs with higher avidity, thereby providing enhanced colloidal stability to the NPs. ASSOCIATED CONTENT

S Supporting Information *

Tables describing the 1H NMR T1 and T2 Relaxation Constants of PEG2000 samples and the hydrodynamic radii of NPs before and after ligand exchange measured by DLS. 1H NMR spectra of compounds 4, 10, and 13. This material is available free of charge via the Internet at http://pubs.acs.org.

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ACKNOWLEDGMENTS

The authors thank NSERC Canada and DVS Sciences for their support of this research.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; [email protected]. ca. Notes

The authors declare no competing financial interest. 12869

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