Luminescent Nanoparticles with Lanthanide-Containing Poly(ethylene

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Luminescent Nanoparticles with Lanthanide-Containing Poly(ethylene glycol)−Poly(ε-caprolactone) Block Copolymers David C. Thévenaz, Christophe A. Monnier, Sandor Balog, and Gina L. Fiore* Adolphe Merkle Institute, University of Fribourg, Chemin des Verdiers 4, CH-1700, Fribourg, Switzerland S Supporting Information *

ABSTRACT: Lanthanide-containing nanoparticles have attracted much attention due to their unique optical properties and potential in nanotechnological applications. An amphiphilic block copolymer of poly(ethylene glycol)-b-poly(ε-caprolactone) methyl ether (mPEGPCL) was functionalized with a dipicolinic acid (dpa) moiety and coordinated to lanthanide ions to afford [Ln(dpa-PCL-PEG-OCH3)3](HNEt3)3 (Ln = Eu3+, Tb3+). Micelle-like nanoparticles of dpa-PCLPEG-OCH3 macroligand and metal-centered polymers were prepared by solvent displacement methods. Dynamic light scattering analysis (DLS) and cryogenic transmission electron microscopy images confirmed the presence of solid sphere (47 nm in diameter) morphologies. The viability and stability of the lanthanide complexes in micelle-like nanoparticles was explored by DLS and luminescence spectroscopy, and found to be stable for several weeks.

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INTRODUCTION Amphiphilic block copolymers based on poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL) are of broad interest in biomedical applications.1−5 These materials can form various self-assembled nanostructures in solution (e.g., spheres,6−8 wormlike micelles,6−9 vesicles8,10), films,11,12 and hydrogels,3,13−16 and have been used in applications for tissue engineering4,13,17,18 and as nanocarriers for the delivery of drugs.13,18−21 Control over the molecular architecture and morphologies can be readily achieved with polymerization techniques and preparation conditions of the self-assembly process. For instance, PEG-b-PCL wormlike micelles can be formed with a PEG weight fraction of 0.42; whereas spheres are favored for weight fractions of ca. 0.5.2,9,19 Additionally, solution-assembled morphologies can be tuned in a controlled fashion by varying processing conditions, such as aging, salt concentration, and temperature, to result in platelets, cylinders, spheres, and intermediate structures.6,22−25 The incorporation of a metal center into polymeric materials offers another kind of architectural tunability. In particular, in inorganic−organic hybrid materials that feature site-isolated metal centers within well-defined polymer architectures, the metal can serve as an architectural motif, responsive cross-link, chromophore, and in some cases as a protecting group26−28 and catalyst28 in the material.29−35 Metal-centered polymers are highly modular and have many tunable features, and by varying the metal, ligand, and polymer composition these materials can be tailored for specific applications.31,34,36−40 Furthermore, the incorporation of a luminescent metal-center offers the advantage of both a built-in optical tag and a chromophore which can give information about the local environment. Lanthanide complexes are one such class of materials as they © XXXX American Chemical Society

offer several unique optical properties including long excitedstate lifetimes, a large Stokes shift between excitation and emission wavelengths, sharp emission lines and are also insensitive to oxygen quenching and resistant to photobleaching.41 The interesting optical behaviors are attributed to the f−f transitions and the shielding of the 4f electrons from lower-lying 5s and 5p orbitals. Lanthanide ions are also known to have very low molar absorptivities; however, coordination to certain ligands allows for the absorbance of UV light and efficient energy transfer to the lanthanide ion, from which light is emitted.42,43 The combination of polymers with built-in optical centers can be interesting as a new class of nanocarriers and as alternatives to organic dyes or quantum dots. An early example of europium-centered polymers was reported by Bender et al., where β-diketonate-functionalized polymers were coordinated to Eu3+ ions and the resulting materials selfassembled into a lamellar morphology in polymer films.44 Recently, Wang and co-workers demonstrated that lanthanide complexes and other metal complexes can serve as imaging agents, cross-linkers, and also the driving force for the formation of micelle-like structures when ligand-functionalized block copolymers are combined with lanthanide ions (i.e., Eu3+, Gd3+) in solution.45−49 Here, we report on the investigation of amphiphilic block copolymers that feature site-isolated luminescent lanthanide metal centers. Diblock copolymers based on PEG and PCL segments were prepared via controlled polymerization techniques and then coupled with dipicolinic acid (dpa) Received: July 20, 2014 Revised: September 17, 2014

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columns along with Wyatt Technology Corp. (Optilab REX interferometric refractometer, miniDawn TREOS laser photometer) and Agilent Technologies instrumentation (series 1200 HPLC) and Wyatt Technology software (ASTRA) were used for GPC analysis. The incremental refractive index (dn/dc) was estimated by a singleinjection method assuming 100% mass recovery from the columns. Excitation and emission spectra were recorded on a PTI C720 fluorescence spectrometer using right angle illumination. For excitation, a XeArc lamp was used along with a PETI 814 photomultiplier detection system for all luminescence experiments. Thermogravimetric analysis (TGA) was conducted using a MettlerToledo STAR thermogravimetric analyzer from 25 to 500 °C at a heating rate of 10 °C/min under N2. Differential scanning calorimetry (DSC) measurements were performed using a Mettler-Toledo modulated DSC. Analyses were carried out in modulated mode under a nitrogen atmosphere (amplitude ± 1 °C; period = 60 s; heating/cooling rate = 10 °C/min; range −80 to 100 °C). Reported values of thermal events are from the second heating cycle and the reverse heat flow curve (Td = decomposition temperature of 5% weight loss; Tm reported as the peak maximum). Light scattering measurements were performed with a commercial goniometer instrument (3D LS Spectrometer, LS Instruments AG, Switzerland). The primary beam was formed by a linearly polarized and collimated laser beam (HeNe, 632.8 nm, 21 mW), and the scattered light was collected by single-mode optical fibers equipped with integrated collimation optics. The collected light was coupled into two highsensitivity APD detectors (PerkinElmer, Single Photon Counting Module), and their outputs were fed into a two-channel multiple-tau correlator (Correlator.com). The signal-to-noise ratio was improved by cross-correlating these two channels. Cryogenic transmission electron microscopy (cryo-TEM) images were recorded using a FEI Tecnai F20 cryogenic transmission electron microscope with an operating tension of 200 kV. Images were taken under low-dose conditions with an UltraScan 1000 CCD sensor (Gatan, Inc.) with an image resolution of 2048 × 2048 pixels. 4-Hydroxypyridine-2,6-dicarboxylic Acid Dibenzyl Ester, 2. The product was prepared by the method of Velasco et al.51 with the following modifications for the purification. The following reagent loadings were used for the reaction conditions: oxalyl chloride (8.20 mL, 97.7 mmol), chelidamic acid (5.04 g, 27.5 mmol), DMF (1.1 mL, 14.2 mmol) in anhydrous THF (220 mL) and benzyl alcohol (6.00 mL, 57.7 mmol), and diisopropylamine (19.20 mL, 109.9 mmol) in anhydrous THF (26 mL). The crude product was recrystallized from CH3OH (2×), and the off-white solid was collected via vacuum filtration. The crude product was dissolved in CH2Cl2 (75 mL) and washed once with aqueous solution of NaOH (10−6 M, 40 mL). The aqueous layer was then extracted with additional CH2Cl2 (2 × 75 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo and dried at 40 °C for 3 days to afford a white solid: 2.1 g; 5.78 mmol; 21%. 1H NMR (300 MHz, DMSO-d6): δ 11.56 (s, 1H, PyrOH), 7.79−7.08 (m, 12H, ArH, PyrH), 5.38 (s, 4H, CH2Ph). 13C NMR (75 MHz, DMSO-d6): δ 165.90, 163.99, 149.40, 135.75, 128.48, 128.19, 115.42, 66.76. ESI-MS (pos. mode): calcd for [C21H17NO5 + H]+, 364.11; found, 364.1. mPEG-PCL, 3. The block copolymer was prepared as previously reported by Fiore and Fraser.38 A flame-dried Kontes flask was charged with poly(ethylene glycol) methyl ether (mPEG, Mn = 2000 g/mol) (1.01 g, 0.505 mmol) and ε-caprolactone (8.1 mL, 73.1 mmol) under a nitrogen atmosphere in a glovebox. The Kontes flask was sealed, and the reaction mixture placed in an oil bath at 110 °C to create a homogeneous melt. Under a positive flow of nitrogen, a 207.4 mM solution of Sn(oct)2 in hexanes (48 μL, 10.0 μmol) was added to the reaction mixture. The reaction vessel was resealed and heated at 110 °C for 7 h. The resulting viscous reaction mixture was then cooled to room temperature, dissolved in a minimal amount of CH2Cl2, and precipitated (3×) into cold stirring hexanes (−70 °C). The product was collected by vacuum filtration and dried in vacuo to afford mPEGPCL as a white solid: 4.43 g, 88%. 1H NMR (300 MHz, CDCl3): δ 4.05 (t, J = 6.7 Hz, −CH2CH2O), 3.64 (s, −CH2CH2−), 3.37 (s, PEG CH3OCH2), 2.30 (t, J = 7.5 Hz −CO2CH2CH2−), 1.73−1.54(m,

ligands. In a single step, the resulting dpa-PCL-PEG-OCH3 macroligands were coordinated to a lanthanide salt (Eu3+, Tb3+) and then formulated as micelle-like nanoparticles with a PCL core and PEG corona (Figure 1). The color of emission

Figure 1. Schematic representation for nanoparticle preparation of [Ln(dpa-PCL-PEG-OCH3)3](HNEt3)3 (Ln = Eu, Tb) materials.

and morphology of the luminescent nanoparticles were tuned in a relatively straightforward approach by either changing the lanthanide ion or tuning the solution-assembly conditions, respectively. The synthesis, thermal analysis, luminescent properties, and solution-assemblies and morphologies of [Eu(dpa-PCL-PEG-OCH3)3](HNEt3)3 and [Tb(dpa-PCLPEG-OCH3)3](HNEt3)3 are described.

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EXPERIMENTAL SECTION

Materials. Tetrahydrofuran was dried by passage through alumina columns.50 Poly(ethylene glycol) methyl ether (mPEG; Mn = 2000 g/ mol) (Aldrich) was dissolved in toluene, dried by azeotropic distillation using a Dean−Stark trap, and concentrated in vacuo; the residual solvent was removed in vacuo and the dried mPEG and then stored in the glovebox under a nitrogen atmosphere. ε-Caprolactone (Aldrich) was dried over CaH2 and distilled under reduced pressure prior to use. Chloroform-d was passed through a short plug of dry, activated (Brockman I) basic alumina prior to use. Chelidamic acid (Intratrade Chemicals), triethylamine (Aldrich), europium(III) chloride hexahydrate (EuCl3·6H2O) (Aldrich), terbium(III) chloride hexahydrate (TbCl3·6H2O) (Aldrich), Gibco PBS pH = 7.4 (Life Technologies), and all other reagents were used as received. Methods. 1H NMR (300 or 500 MHz), 13C NMR (75 or 126 MHz), and 19F NMR (282 MHz) spectra were recorded on a Bruker Advance III in CDCl3 or DMSO-d6. 1H NMR coupling constants are given in hertz. 1H NMR spectra were referenced to the signal for residual CH3Cl or DMSO at 7.26 or 2.50 ppm, respectively. Experiments for end-group analysis were performed using a relaxation delay time of 10 s to allow for the relaxation of all protons in the polymer chain and ensure maximum signal acquisition. 13C NMR spectra were referenced to the signal of CDCl3 or DMSO-d6 at 77.16 or 39.52 ppm. Number-weighted molecular weights (Mn), massweighted molecular weights (Mw) and polydispersity index (PDI) were determined by gel permeation chromatography (GPC) (THF, 40 °C, 1.0 mL/min) using multiangle laser light scattering (MALLS) (λ = 658 nm, 25 °C) and a refractive index (λ = 658 nm, 40 °C) detection. A Polymer Laboratories 5 μm mixed-C guard column and two GPC B

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Scheme 1

−CH2CH2CH2CH2CH2−), 1.47, 1.30 (m, −CH2CH2CH2−). GPC (MALLS): Mw = 10 000 g/mol; PDI = 1.12; dn/dc = 0.071. Bn2dpa-PCL-PEG-OCH3, 4. A solution of 3 (1.26 g, 0.126 mmol), 2 (0.18 g, 0.495 mmol) and triphenylphosphine (0.2 g, 0.763 mmol) in anhydrous THF (40 mL) was stirred at room temperature under a N2 atmosphere. The reaction mixture was cooled to −10 °C and diisopropyl azodicarboxylate (DIAD) (0.15 mL, 0.771 mmol) was then added dropwise. The reaction mixture was stirred for 3 days as it slowly warmed to room temperature. The reaction mixture was then precipitated into cold CH3OH (−70 °C), and the crude product collected by vacuum filtration and dried in vacuo (40 °C, 12 h). The crude polymer product was dissolved in a minimal amount of CH2Cl2, precipitated into cold stirring CH3OH (−70 °C), and collected by vacuum filtration and in vacuo (40 °C, 12 h) to afford a white powder: 1.04 g, 80%. 1H NMR (500 MHz, CDCl3): δ 7.73 (s, PyrH), 7.54− 7.29 (m, ArH), 5.43 (s, CH2Ph), 4.05 (t, J = 6.7 Hz, −CH2CH2O), 3.64 (s, PEG −CH2CH2−), 3.37 (s, PEG CH3OCH2), 2.31 (dt, J = 15.1, 7.6 Hz, −CO2CH2CH2−), 1.70−1.56 (m, −CH2CH2CH2CH2CH2−), 1.46−1.31 (m, −CH2CH2CH2−). 13C NMR (126 MHz, CDCl3) δ 173.65, 166.98, 164.65, 150.07, 135.57, 128.70, 128.63, 128.50, 114.58, 70.67, 67.83, 64.27, 59.17, 34.26, 28.46, 25.64, 24.69. GPC (MALLS): Mw = 10 700 g/mol; PDI = 1.10; dn/dc = 0.073; Tm = 28 °C (PEG), 53 °C (PCL); Td = 345 °C. dpa-PCL-PEG-OCH3, 5. The functionalized block copolymer 4 (0.91 g, 0.089 mmol), 10% Pd/C (128 mg), and THF (25 mL) were combined and stirred under a H2 atmosphere (1 atm) at room temperature for 18 h. The reaction mixture was then filtered to remove the catalyst and the THF was then removed in vacuo. The crude product was dissolved in a minimal amount of CH2Cl2 and precipitated into cold stirring hexane (−70 °C). The product was collected by vacuum filtration, dried in vacuo (40 °C for 18 h) to afford a white powder: 0.71 g, 80%. 1H NMR (500 MHz, CDCl3): δ 7.86 (s, PyrH), 4.05 (t, J = 6.7 Hz, −CH2CH2O), 3.64 (s, PEG −CH2CH2−), 3.37 (s, PEG CH3OCH2), 2.30 (t, −CO2CH2CH2−), 1.69−1.58 (m, −CH 2 CH 2 CH 2 CH 2 CH 2 −), 1.42−1.33 (m, −CH2CH2CH2−). 13C NMR (126 MHz, CDCl3) δ 173.68, 168.59, 164.03, 147.87, 114.19, 70.68, 64.27, 59.15, 34.24, 28.46, 25.64, 24.69; Tm = 28 °C (PEG), 54 °C (PCL); Td = 325 °C. Titration of 2,6-Pyridinedicarboxylic Acid (dpa) and dpaPCL-PEG-OCH3, 5, with EuCl3. A representative procedure is provided: 2,6-Pyridinedicarboxylic acid (dpa, 1.6 mg, 9.6 μmol) was dissolved in DMF (10 mL). A portion of this solution (2.5 mL) was added to a quartz cuvette, and a 10% Et3N/DMF solution (0.340 mL,

48.8 μmol Et3N) was then added. Aliquots of a EuCl3·6H2O solution (0.13 mL, 3.14 mM, 0.4 μmol) in DMF were added, and emission spectra were taken after stirring the solution for 5 min. The solution was excited at 270 nm, and the emission spectrum was monitored over the range of 400−800 nm. The maximum intensity at 615 nm was plotted as a function of equivalents of EuCl3. Self-Assembly and Nanoparticle Fabrication of Ln[(dpa-PCLPEG-OCH3)3](HNEt3)3. A representative procedure is provided. Self-Assembly. The macroligand, 5 (0.010 g, 0.98 μmol) was dissolved in DMF (1 mL). Portions of a 10% Et3N/DMF solution (0.134 mL, 19.28 μmol Et3N) and EuCl3·6H2O solution (0.11 mL, 2.89 mM, 0.32 μmol) in DMF were added dropwise to the macroligand and the solution was stirred at room temperature for 5 min. Nanoparticle Fabrication. Method A: The resulting polymeric metal complex solution in DMF was added dropwise at a constant rate into a vial of stirring Milli-Q water (10 mL). The suspension was stirred at room temperature for 5 min. DMF was then removed by extensive dialysis against Milli-Q water. The volume of the suspension was then adjusted to 20 mL to obtain a concentration of 0.5 mg of macroligand per milliliter. Method B: The reagent loadings from method A were used; however, water was slowly added to a polymeric metal complex solution in DMF. Characterization of Ln[(dpa-PCL-PEG-OCH3)3](HNEt3)3 SelfAssembly. Dynamic Light Scattering (DLS). Aliquots of the samples were filtered through 0.45 μm PVDF filters into cylindrical glass cells of 10 mm diameter. Measurements were performed at a scattering angle of 90° for 180 s at 25 °C in water. A viscosity of 0.893 mPa·s and a refractive index of 1.33 were used to calculate hydrodynamic radii. CONTIN method,52,53 cumulant,54 and Schulz−Zimm55,56 distributions were fitted to correlation function from DLS to obtain a mean diffusion coefficient that can be transferred into a hydrodynamic radius using the Stokes−Einstein relation.57 Sample Vitrification for Cryogenic Transmission Electron Microscopy (cryo-TEM). Aliquots of the samples were filtered through 0.45 μm PVDF filters, and 5 μL of sample solution was first deposited on a carbon-coated copper grid (Lacey carbon film grids, Plano GmbH). Excess of liquid was then carefully blotted away with filter paper (Whatman qualitative filter paper, grade 1) before plunging the sample into a liquid ethane bath cooled by liquid nitrogen. The vitrified specimens were then kept for storage in liquid nitrogen and analyzed the following day. C

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RESULTS AND DISCUSSION Macroligand Synthesis. A ligand-functionalized amphiphilic block copolymer was first prepared to explore the solution assemblies of metal-centered polymers (MCP). A dipicolinic acid derivative (1) was chosen as the ligand due to its ability to coordinate to lanthanide ions, and the spectroscopy of the respective complexes has been well characterized in the literature.58−60 The carboxylic acid groups of 1 were selectively protected with benzyl alcohol in order to circumvent side reactions and provide 2 with a pyridinic−OH that can be further functionalized (Scheme 1).51 A block copolymer of mPEG-PCL, 3, was also prepared by the ring-opening polymerization of ε-caprolactone using tin(II) 2-ethylhexanoate (Sn(oct)2) as a catalyst and poly(ethylene glycol) methyl ether (mPEG, Mn = 2000 g/mol) as the initiator (Scheme 1).38 The protected macroligand, 4, then was prepared by coupling 2 with 3 via Mitsunobu reaction and subsequently the benzyl protecting groups were removed by hydrogenation over Pd/ C to afford the dipicolinic acid-PCL-PEG-OCH3 macroligand, 5 (Scheme 1).

The thermal properties of the polymer products 4 and 5 were analyzed by TGA and DSC (Supporting Information Figures S10 and S11). Two melting peaks were observed at 28 and 54 °C for the PEG and PCL block, respectively. These values match well to those found in the literature.62,63 TGA measurements revealed that the polymer products, 4 and 5, are thermally stable until 345 and 325 °C, respectively. Metal-Centered Polymers and Solution Assemblies. The coordination of ligands with lanthanide ions is largely determined by electrostatic interactions and the directionality by ligand−ligand steric interactions and repulsions. Water molecules are known to be strong ligands to lanthanide ions (Ln3+) and complexes prepared under anhydrous conditions often undergo partial hydrolysis even in the presence of trace amounts of water resulting in the quenching of luminescence through O−H vibrations.41,64,65 However, multidentate ligands containing one or more negatively charged oxygen donor groups, such as dipicolinic acid, are known to form luminescent tris-complexes with Ln3+ ions even in the presence of water.58−60 Amphiphilic block copolymers of mPEG-PCL were previously reported to readily form spherical micelle-like nanoparticles in water.66−69 Based on the assumption that solution assembled nanostructures do not alter the coordination of dipicolinic acid with Ln3+ ions, the preparation of MCPs and solution assemblies were prepared in a single step (Figure 1). The coordination and structural properties among the lanthanide ions are quite similar, and it is often assumed that lanthanide complexes formed with a particular ligand under a common set of conditions are isostructural.41,70 The spectral properties of Eu3+ are also well-known and several of the emissive transitions (5D0 → 7FJ) exhibit splitting patterns and intensity variations which are relatively simple for making detailed spectra-structure correlations.71 For instance, the emission at 615 nm is of the 5D0 → 7F2 transition, and a peak that is uniform and without splitting is indicative of sample homogeneity.72 Therefore, we have chosen to perform a systematic study using Eu3+ which can easily be substituted with any lanthanide ion of choice to tune the emissive color. The MCP of [Eu(5)3](HNEt3)3 was prepared in situ by combining the amphiphilic macroligand of 5 with triethylamine and EuCl3 in DMF. The formation of [Eu(5)3](HNEt3)3 was confirmed by luminescence spectroscopy, the 5D0 → 7F2 emission peak (λ = 615 nm) did not display any splitting (Supporting Information Figure S17), and all emissive peaks corresponded well to a nonpolymeric [Eu(dpa)3](HNEt3)3 complex in DMF (Supporting Information Figure S20). In addition, a titration of 5 with EuCl3 was performed in DMF, and the intensity of the 5 D0 → 7F2 emission peak (λ = 615 nm) was monitored. A maximum emission intensity was found when 1 equiv of Eu3+ per 3 equiv of macroligand was added, corresponding to a triscomplex (Supporting Information Figure S25). As a control, a similar titration was performed using EuCl3:dpa and a slightly lower maximum intensity was observed in comparison to [Eu(5)3](HNEt3)3 (Supporting Information Figure S25). These results suggest that the polymer does not affect the coordination of 5 to the metal center, and an increase of intensity is consistent with previous reports where the polymer shell can have a protective nature and diminish luminescence quenching due to metal−metal interactions and the coordination of water and other donors to the Eu3+ center.73−75 Micelle-like nanoparticles of amphiphilic block copolymers, such as PEG-b-PCL, are often prepared by solvent-displace-

Table 1. Molecular Weight Data for Amphiphilic Block Copolymers polymer 3 4 5

Mn (GPC)a b

8900 9700c −d

Mw (GPC)a b

10 000 10 700c −d

Mn (NMR)e

PDI

10 300 10 700 10 500

1.12 1.10 −d

a

Molecular weights determined by GPC (MALLS, THF). bdn/dc = 0.071 mL/g. cdn/dc = 0 .073 mL/g. dMeasurements could not be performed due to strong interactions of 5 with the GPC columns (Supporting Information Figure S9). eMolecular weights determined by 1H NMR end-group analysis.

The resulting polymer products, 3 and 4, were analyzed by GPC and NMR. GPC analysis revealed that the low polydispersity index of 3 (PDI = 1.12) was retained after the Mitsunobu coupling (Table 1, Supporting Information Figure S8) which suggests that the reaction conditions did not affect the polymer backbone. Additionally, the degree of chain-end functionalization of 4 was evaluated by 1H NMR end-group analysis by comparing the relative integrations of the benzylic peaks and pyridinic protons corresponding to the protected ligand (ArH and PyrH) versus the chemical shifts of −CH2CH2− from the PEG backbone and the −OCH3 endgroup of mPEG-PCL (Supporting Information Figure S3). Using this approach, the degree of functionalization was determined to be 95%. This was further confirmed by using the method of Keizer et al.61 to determine the amount of terminal −OH groups remaining after the Mitsunobu coupling, and a 99% conversion was determined (Supporting Information Figure S4). Upon deprotection of 4 via hydrogenation conditions, the resulting macroligand, 5, was analyzed by 1H NMR and 13C NMR. A disappearance of the chemical shifts corresponding to the benzyl protecting groups (ArH) and a shift of the PyrH of the dipicolinate moiety from 7.73 to 7.86 ppm indicates that complete deprotection was achieved (Supporting Information Figures S3 and S6). Additionally, a disappearance of the chemical shifts corresponding to the benzylic groups (135.57, 128.70, 128.63, 128.5, and 67.83 ppm) was also observed in 13C NMR analysis (Supporting Information Figures S5 and S7), further verifying the removal of the benzyl protecting groups. D

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ment methods with THF or DMF, which are eventually removed by dialysis with water. The morphology, average size, and polydispersity of particles can be influenced by several parameters, such as the polarity index of the organic solvent, the rate of addition of polymer solution, and the order of addition during the solvent displacement process.1,76,77 For instance, larger spherical particles and vesicle structures are often obtained when water is slowly added to an organic polymer solution. However, spherical particles with small hydrodynamic radii often result when the reverse occurs and an organic polymer solution is slowly added to water.76 Moreover, particles prepared by the addition of a polymer DMF solution to water tend to be more uniform and smaller in size in comparison to acetone or THF due to the high polarity index of DMF, resulting in efficient solvent diffusion and polymer dispersion.76 However, a large number of parameters are often involved in solvent-displacement methods and to control all of them is not trivial; therefore, small variations in size and dispersity can be observed between batches.7,76,77 On the basis of these findings, the parameters for nanoparticle formation of MCPs were systematically tuned to determine the optimal conditions for control over the particle morophology, average size, and polydispersity. Using two different solvent displacement (also known as nanoprecipitation) approaches, micelle-like nanoparticles of MCPs were prepared either by the slow addition of water to an organic polymer solution or vice versa. First, water was slowly added dropwise to a stirring solution of [Eu(5)3](HNEt3)3 in DMF. Samples were then dialyzed against water (2 days) to remove the organic solvent, and the resulting suspensions were colorless in visible light and emitted red when exposed to UV excitation (λ = 254 nm) (Figure 2), indicating the presence of

Table 2. Summary of Hydrodynamic Diameters and Polydispersities of dpa-PCL-PEG-OCH3 Block Copolymer Nanoparticles water added to organica

polymer

organic added to watera × ×c

[Eu(5)3](HNEt3)3 × ×c [Tb(5)3](HNEt3)3 (5)(HNEt3)2 (5)(HNEt3)2

× × ×

Dh [nm]b 24 bimodal 66 15 20 16 18

PD (std/mean)b 0.39 0.30 0.28 0.41 0.37 0.71

a

Nanoparticles were prepared using DMF as the organic solvent, unless indicated otherwise. bSchulz−Zimm distribution55,56 was used to fit correlation functions and converted into a hydrodynamic radius using the Stokes−Einstein relation.57 The standard deviation was determined for each batch where three aliquots of a sample were measured three times each. The standard deviation for all measurements was less than 1%. cNanoparticles were prepared using THF as the organic solvent.

approximately 47 nm, the nanoparticles exhibit a hollow inner structure, suggesting the formation of vesicles, whereas particles of a smaller diameter (i.e., less than ca. 47 nm) are solid spheres (Figure 3, Supporting Information Figure S16). This divergence in structure becomes more apparent in the intensity profiles of the suggested particles. The overall wall thickness was measured to be 17.5 nm and remained constant regardless of the particle size (Supporting Information Figure S16), further supporting the presence of vesicles. However, when nanoparticles were also prepared using THF as the organic solvent, and a Dh of 15 nm and PD of 0.28 were obtained (Table 2). Solution assemblies of [Eu(5)3](HNEt3)3 were also prepared by the dropwise addition of a MCP DMF solution into stirring water. The organic solvent was then removed by extensive dialysis against water (2 days), and the resulting suspensions were analyzed by DLS and cryo-TEM. Using this method, DLS analysis showed that smaller nanoparticles with a Dh of 24 nm and PD of 0.39 were obtained (Table 2), and only spherical micelle-like structures were observed by cryo-TEM (Figure 3 and Supporting Information Figure S14). Additionally, nanoparticles were also prepared using THF as the organic solvent, however, a bimodal distribution was observed in DLS analysis (Table 2). Moreover, the particles prepared from [Eu(5)3](HNEt3)3 in DMF were dialyzed against a PBS buffer solution (pH = 7.4) and the size/distribution was explored using DLS. Here, the particles increased in size (Dh = 42 nm), however the polydispersity remained constant. The increase in size is likely due to a change in the salt concentration which can influence the solvation of the PEG corona.24 The [Eu(5)3](HNEt3)3 nanoparticle suspensions were analyzed by luminescence spectroscopy to test the material stability and viability of the lanthanide complexes from the solution assembly conditions. The emission spectra of [Eu(5)3](HNEt3)3 nanoparticles displayed the characteristic 5D0 → 7FJ emissive transitions (Figure 4). Moreover, the 5D0 → 7F2 emission peak (λmax = 615 nm) corresponded well to the starting solutions and the morphology of the peaks did not display any splitting, suggesting a homogeneous environment. Thus, the [Eu(5)3](HNEt3)3 complex was not hydrolyzed during the solvent displacement procedure (Figure 4). Based

Figure 2. Images of [Eu(5)3](HNEt3)3 and [Tb(5)3](HNEt3)3 nanoparticles in water (0.5 mg/mL) under visible light (left) and UV excitation (λ = 254 nm) (right).

tris-europium dipicolinate complexes. The solution assemblies were characterized by DLS to determine both the hydrodynamic diameter (Dh) and polydispersity (PD). A Schulz− Zimm model was used for data analysis and compared with CONTIN and cumulant methods, and similar results were obtained for both methods; therefore, only data obtained by fittings with Schulz−Zimm distribution are reported in the present work. Nanoparticles displayed a Dh of 66 nm and PD of 0.30 (Table 2). The morphology of the nanoparticles was further investigated by cryo-TEM, which allows for direct visualization of the solution-assembled nanostructures formed in water. As can be seen in Figure 3, a broad distribution of spherical particle sizes is visible, and the overall diameters are consistent with DLS data (Supporting Information Figure S12). Upon a closer investigation, a variation in morphology is observed with an increase in particle size (Supporting Information Figure S15). Above an overall diameter of E

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Figure 3. Representative images of [Eu(5)3](HNEt3)3 micelle-like nanoparticles obtained by cryo-TEM. Solution-assemblies were prepared by a solvent displacement method from a [Eu(5)3](HNEt3)3 DMF solution and the slow addition of water (A) or the slow addition of [Eu(5)3](HNEt3)3 DMF solution to water (B).

verified the formation of nanoparticles with a Dh of 20 nm and low polydispersity (PD = 0.41) (Table 2). Additionally, the [Tb(5)3](HNEt3)3 nanoparticle suspensions display the signature emissive 5D4 → 7FJ peaks of a Tb3+−dipicolinate complex (Figure 4).41 Thus, by changing the metal center, the emission color of the luminescent particles can be tuned while the size of the particles remains nearly constant for a given selfassembly condition (Figure 2, Table 2). A control experiment was also performed where 5 was deprotonated by triethylamine in DMF in the absence of any lanthanide ion. The dropwise addition of 5 into stirring water, followed by dialysis, resulted in nanoparticles with similar sizes (Dh = 16 nm, PD = 0.37) as the terbium- and europiumcontaining particles obtained under similar conditions (Table 2). This can be attributed that upon addition of an organic to water, the organic solvent diffuses quickly into the aqueous media and the polymer chains are rapidly frozen in the selective solvent. Therefore, similar sizes were obtained for the selfassembly of 5 in the presence and absence of a metal ion irrespective of the molecular architecture (linear block copolymer vs 3-arm metal-centered polymer).77 Additionally, water was slowly added to a solution of 5 in DMF/ triethylamine, and particles with a Dh of 18 nm and PD of 0.72 were observed, which are smaller in comparison to [Eu(5)3](HNEt3)3 (Dh = 66 nm; PD = 0.30) obtained under similar conditions (Table 2). This difference is likely due to a slower change in the polarity of the solvent system when water is added to the organic, also allowing for an increased amount of time for the polymer chains to diffuse and reorganize.77 Therefore, the variation in size is likely due to the difference in molecular architecture (linear block copolymer vs 3-arm metalcentered polymer). Finally, a solution of EuCl3 was added to a nanoparticle suspension of 5, and a red emissive color and characteristic emissive transitions of a [Eu(dpa)3](HNEt3)3 complex (Supporting Information Figure S24) were immediately observed, and both the size and polydispersity of the nanoparticles remained constant. These results demonstrate that nanoparticles can be first prepared and the metal can be added afterward. Moreover, nanoparticle suspensions of [Eu(5)3](HNEt3)3, [Tb(5)3](HNEt3)3, and the control (5)(HNEt3)2 were shown to be stable as a change in the DLS size and distribution was not observed over a several week time period. These results suggest that the self-assembly of this class

Figure 4. Excitation and emission spectra of (A) [Eu(5)3](HNEt3)3 and (B) [Tb(5)3](HNEt3)3 nanoparticles in water (0.5 mg/mL). The excitation spectrum (- - -) was recorded with a fixed emission at 615 nm. The emission spectrum () was recorded with a fixed excitation at 270 nm.

on these observations, the europium metal center was replaced with terbium to tune the color of emission from red to green (Figure 2). Using a similar procedure, [Tb(5)3](HNEt3)3 was prepared by combining 5 with triethylamine and TbCl3 in DMF. The emission spectra of [Tb(5)3](HNEt3)3 corresponded well to a nonpolymeric [Tb(dpa)3](HNEt3)3 complex (Supporting Information Figures S18 and S21). The resulting MCP DMF solution was then added to stirring water followed by extensive dialysis against water (2 days). DLS analysis F

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of PCL-b-PEG nanoparticles is mainly driven by the amphiphilic nature of the block copolymers and molecular architecture and not due to the presence of a metal complex core.

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CONCLUSION In summary, amphiphilic block copolymers of PCL-b-PEG were prepared using living polymerization techniques and functionalized with a dipicolinic acid (dpa) moiety. The dpaPCL-PEG-OCH3 macroligand was combined with lanthanide ions to produce luminescent [Eu(dpa-PCL-PEG-OCH3)3](HNEt3)3 and [Tb(dpa-PCL-PEG-OCH3)3](HNEt3)3 complexes with red and green color emission, respectively. Micelle-like nanoparticles were obtained using solvent-displacement methods, and both the size and morphology (47 nm diameter = vesicles) of the nanostructures was tuned by simply varying the self-assembly conditions. Additionally, the emissive color of the nanoparticles was readily varied by the lanthanide ion of choice (Eu3+ = red; Tb3+ = green), while the size of the nanostructure remain unchanged for a given self-assembly condition. This single-component system serves as a model for a new class of luminescent drug delivery vectors and alternative to existing bioprobes. The amphiphilic block copolymers could entrap hydrophobic drugs, while the unique optical properties of the lanthanide complexes can serve as optical tags to allow visualization in vitro.42,65,78−81 Finally, these materials are highly tunable in nature, and by changing the metal ion, polymer backbone, and self-assembly conditions, a myriad of applications can be targeted.

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ASSOCIATED CONTENT

S Supporting Information *

1 H NMR and 13C NMR spectra, gel permeation chromatography data, thermogravimetric analysis, dynamic scanning calorimetry, DLS analyses, excitation and emission spectra, and cryo-TEM images. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: gina.fi[email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Swiss National Science Foundation (200021_140723/1) and Adolphe Merkle Foundation for support of this research. The authors acknowledge the Dr. Alfred Bretscher Fund and the Microscopy Imaging Center (University of Bern) for the cryo-TEM facilities, and Prof. T. Jenny and F. Fehr for assistance with NMR experiments. Prof. Frederick S. Richardson is gratefully acknowledged for fruitful discussions and invaluable advice.

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