Lanthanide-Dipicolinic Acid Coordination Driven Micelles with

Oct 19, 2015 - The hydrodynamic radii hardly change over time; we found the similar phenomena in the previous study with Zn-BP micelles. ..... Moughto...
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Lanthanide-Dipicolinic Acid Coordination Driven Micelles with Enhanced Stability and Tunable Function Junyou Wang,† R. H. Marleen de Kool,† and Aldrik H. Velders*,†,‡ †

Laboratory of BioNanoTechnology, Wageningen University, Dreijenplein 6, 6703 HB Wageningen, The Netherlands Interventional Molecular Imaging Laboratory, Department of Radiology, Leiden University Medical Centre, Leiden, The Netherlands

‡

S Supporting Information *

ABSTRACT: Lanthanide-incorporated polymer micelles have been prepared driven by the lanthanide-dipicolinic acid (LnDPA) coordination. The terdentate DPA ligand is grafted to the PVP block of a diblock copolymer poly(4-vinylpyridine)-bpoly(ethylene oxide) (P4VP48-b-PEO193). Upon addition of Eu(III) ions to a solution of the DPA16-g-P4VP48-b-PEO193 block copolymer, intermolecular cross-links form and the ligand-carrying blocks assemble, leading to the formation of micelles, stabilized by the hydrophilic PEO blocks. The DPA exhibits a dual function in this study. First, the chelate group strongly coordinates to Eu(III) in a three to one ratio, and leads to high stability of the formed micelles, as proven by light scattering and luminescence spectroscopy. Second, DPA acts as an antenna that transfers energy to the Eu(III) ion and dramatically enhances the luminescence emission. The Eu(III) emission is moreover most sensitive for local environment and allows to shine light on the internal structure of this class of self-assembled 36 nm size soft nanoparticles. With the same strategy gadolinium(III) can be incorporated providing micelles which show enhanced magnetic relaxation rates. Micelles capping a mixture of Eu(III) and Gd(III) show both enhanced luminescence emission and magnetic relaxation rates, and the functions can be tuned by regulating the mixing ratio of Eu(III) and Gd(III), showing great potential for developing multimodal imaging agents for diagnostic as well as therapeutic applications.

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INTRODUCTION The binding strength of metal−ligand bonds is intermediate between weak noncovalent interactions and covalent bonding, and can be tuned by selecting specific meal-ligand pairs, resulting in kinetically inert or labile bonds.1−3 Such versatile properties make the metal−ligand coordination motif very attractive in the field of directed self-assembly, where the interactions can be strong enough to initiate or drive assembly but weak enough to allow consecutive reorganization of the assembly upon physical-chemical stimuli. Indeed, ingenious strategies have been developed in recent years, to build up organic−inorganic hybrid supramolecular structures, such as polymers, micelles, vesicles, dendrimers, hydrogels, or selfassembled monolayers.4−11 The combination of metal ions and polymers is a popular methodology to explore not only new structures, but also to develop functional materials; metal ions can procure electrochemical, fluorescent, catalytic, or magnetic properties while the polymers act as building blocks for assembly into well-controlled structures.12−16 A particularly interesting class of polymers is constituted by those with pendant coordination groups on the polymer chains that can associate with metal ions to consequently cross-link polymer blocks. For example, mixing Fe(III) and poly(acrylic acid) (PAA) results in a hydrogel in aqueous solution;17 diblock copolymers with pyridine groups in one of the blocks form micelles with Pt(II) and Eu(III).18−20 However, pyridine © XXXX American Chemical Society

groups, and in most cases also carboxylic groups, act as monodentate ligands and compromise the stability of the formed structures.21−23 Alternatively, in this respect, grafting multidentate coordination groups, for example, dipicolinic acid (DPA) or terpyridine groups, on polymer chains demonstrates a more attractive strategy.24−27 We have grafted DPA ligand on the PVP block of the diblock copolymer poly(4-vinylpyridine)-b-poly(ethylene oxide) (P4VP48-b-PEO193).25 The association of the obtained polymer, DPA16-g-P4VP48-b-PEO193 with Zn(II) was studied and the formed micelles were found to be highly stable, the metal binding constant of DPA being about a factor of 105 larger than those of pyridine or carboxylic acid groups.21,23 Although the formation and stability were firmly confirmed, the internal coordination structure of such micelles is missing. Moreover, developing new functions by applying different metal ions, for example, lanthanide, demonstrates more interests and potential for further applications. Herein, we present the assembly of the DPA16-g-P4VP48-b-PEO193 diblock copolymer with Eu(III) and Gd(III). DPA is a well-known chelate group in lanthanide coordination chemistry, providing not only the strong coordination bond, but also the effective Received: June 9, 2015 Revised: October 5, 2015

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Langmuir energy transfer to the coordinated lanthanide ions (“antenna effect”), particularly with a high quantum yield to europium and terbium.28,29 Based on the Eu-DPA pair, we expect the formed micelles showing high stability combined with excellent luminescence. Moreover, the luminescence steady-state and lifetime data reveal details about the coordination structure in the micellar core, which is instrumental for designing new functional self-assembling and responsive materials based on coordination polymers. The presented method allows to pack different lanthanide ions and tune the mixing ratio and corresponding functions, paving the way for further applications of hybrid micelles, for example, in biomedical imaging or material design.

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KR =

1 C = 1 + 2B2 S(q) M

Itoluene

× R toluene ×

KR C 1 C = + 2B2 2 Rθ M M

2 nsolvent 2 ntoluene

(1)

R h = kTq2 /6πη Γ

(2)

PDI = μ2 /Γ 2

(3)

(7)

By plotting KRC/Rθ versus C, molar mass of micelle is obtained from the intercept, allowing further calculating the aggregation number of the micelles. UV−vis and Luminescence Spectroscopy. DPA, DPA16-gP4VP48-b-PEO193 were dissolved directly in MOPS buffer, whereas P4VP48-b-PEO193 was dissolved in water at pH 2 and then diluted in MOPS buffer. The obtained solutions were put in 1.0 cm cells (quartz) for the UV−vis absorption spectra recorded with a Varian 50 double beam absorption spectrometer. Steady state luminescence spectra were recorded with a Edinburgh Fluorescence spectrometer (FLS900) and the lifetime measurements were performed on a Cary-4000 spectrometer. For the latter, the excitation was set at 280 nm and the intensity decay at 614 nm was recorded over time. The concentration of Eu(III) was fixed at 0.12 mM in all samples for the luminescence measurements. Transmission Electronic Microscopy (TEM). Copper grid (400 mesh) covered with a carbon film was first processed with a plasma cleaner (Harrick, PDC-32G). Then a drop of micellar solution was deposited onto the grid and excess solution was removed by filter paper after 1 min. Visualization was performed on a JEOL TEM 1100 transmission electron microscope and images were collected from different regions on the grid. Staining agent was not needed because micelles contain hundreds of metal ions in the core and showed sufficient contrast. Magnetic Relaxation Measurements. The longitudinal relaxation times T1 of micellar solutions were measured using a Maran Ultra spectrometer (Resonance Instruments, Abingdon, U.K.) operating at 30.9 MHz proton resonance frequency. A combined Inversion Recovery Carr−Purcell−Meiboom−Gill (IR-CPMG) pulse sequence was used to measure T1. Data were analyzed using a home-written IDL program (Research Systems Inc., Boulder, CO) and expressed as the relaxation rate R1, known as 1/T1.

where Isample is the scattering intensity of the micellar solution and Isolvent is the intensity of the solvent. Itoluene is the scattering intensity of toluene (the reference), Rtoluene is the known Rayleigh ratio of toluene (2.1 × 10−2 m−1) and n is the refractive index. The CUMULANT method30 was used to analyze the mean apparent hydrodynamic radius (Rh) and the polydispersity index (PDI). Rh is calculated from the average decay rate Γ and the PDI from the second moment μ2, according to the following formulas:

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RESULTS AND DISCUSSION The modification of the P4VP48-b-PEO193 diblock copolymer was reported in a previous study, and around 16 DPA groups (on average) are grafted on the PVP block, which make the obtained polymer, DPA16-g-P4VP48-b-PEO193 soluble in water at pH ≥ 6.25 Therefore, we selected MOPS buffer at pH 7 in this study, to be sure that the DPA16-g-P4VP48-b-PEO193 polymer itself is soluble and the micellization can occur upon mixing with metal ions. The DPA groups offering a strong driving force for micellization upon mixing with Eu(III) ions, and hydrophilic PEO block stabilizes the cross-links of the polymer blocks into the colloidal domain leading to micellar structures (Scheme 1). The formed micelles are called Eu-BP micelles with BP representing the DPA modified diblock copolymer DPA16-g-P4VP48-b-PEO193. As shown in Scheme 1 (bottom), the nine coordination sites of Eu(III) can be ideally

where q is the scattering vector, k is the Boltzmann constant, T is the absolute temperature, η is the viscosity of the solvent. The CONTIN method31,32 was used to analyze the distribution of particle radii. The absolute Γ values vary a lot from different samples, which makes it difficult to compare the results directly. Therefore, we normalized Γ with the highest value for each sample,33 and show it as G(Γ) in the profiles. The Rayleigh ratio can be linked to concentration and mass of the scattering objects:

KR C 1 1 1 = × × Rθ M P(qR ) S(q)

(6)

where B2 is the second virial coefficient. Substitution into eq 3, we get

Materials. The diblock copolymer poly(4-vinylpyridine)-b-poly(ethylene oxide), P4VP48-b-PEO193, (Mw/Mn = 1.10, Mn= 13.5 k) was purchased from Polymer Source. The modification with DPA is described in previous work and the obtained polymer is called DPA16g-P4VP48-b-PEO193 with g representing graft.25 The degree of grafted groups is around 34%, which means around 16 DPA ligands are present in one PVP block. Europium nitrate pentahydrate, Eu(NO3)3· 5H2O (analytical grade), Gadolinium chloride GdCl3·6H2O, Zn(NO3)2·4H2O and 3-(N-morpholino) propanesulfonic acid sodium salt (MOPS) were obtained from Aldrich and used without further purification. All stock solutions were prepared in MOPS buffer (20 mM, pH 7) with 0.1 M NaCl. The micelles were made by direct mixing the aqueous solutions of modified polymer DPA16-g-P4VP48-bPEO193 and metal ions, followed by sonication for 5 min and consecutive equilibration for about 24 h. Light Scattering. Light scattering at an angle of 90 deg was performed with an ALV light scattering-apparatus, equipped with a 400 mW argon ion laser operating at a wavelength of 532.0 nm. All the measurements were performed at room temperature. The light scattering intensity is expressed as the excess Rayleigh ratio Rθ, define as

Isample − Isolvent

(5)

where NAv is Avogadro’s number, λ0 is the wavelength of the incoming beam (532.0 nm), and dn/dc is the refractive index increment of the micelles, which is taken from the previous study on Zn(II) incorporated micelles with the same polymer. In our experiments, the scattering vector q = (4πn/λ0)sin(θ/2) is approximately 0.023 nm−1 (θ = 90°), so that qR is small for the micelles and we therefore assume that P(qR) = 1. At low concentrations, the structure factor can be approximated as

EXPERIMENTAL SECTION

Rθ =

2 4π 2n2 ⎛⎜ dn ⎞⎟ NAvλ 04 ⎝ dc ⎠

(4)

where C is the weight concentration of micelles, M is their molecular mass, and R is the radius of the object that contribute to scatter light. P(qR) and S(q) are the form factor and the structure factor, respectively. KR is an optical constant defined as B

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Langmuir Scheme 1. Top: Structure of DPA16-g-P4VP48-b-PEO193 Diblock Copolymers and Illustration of the Micellization Driven by Eu-DPA Coordination. Bottom: Coordination Structure of [Eu(DPA)3]3− and Charge Distribution in the Micellar Core

Figure 1. Variations of light scattering intensity, expressed by the excess Rayleigh ratio R90, and hydrodynamic radius of the formed micelles over time. Eu-BP micelles are prepared in 20 mM MOPS buffer at pH 7, [Eu(III)] = 0.34 mM.

to reach the final micellar state. In view of this observation, all the samples were prepared and equilibrated for about 24 h in the following studies. Figure 2a shows the light scattering intensity and hydrodynamic radius of Eu-BP micelles as a function of Eu/DPA mixing ratio. The intensity at the first point (pure polymer) is low, indicating only soluble BP coils present in solution. Upon mixing with Eu(III), the intensity increases with increasing Eu/ DPA ratio up until 0.35, corresponding to a Eu/DPA ratio of 1 /3, where Eu(III) is coordinated with three DPA ligands. No variation of intensity is found upon further increasing the Eu(III) amount. The hydrodynamic radius of the formed micelles is 18 ± 3 nm, and is independent of the Eu/DPA ratio. The constant light scattering intensity and radius at Eu/DPA ratios higher than 1/3 indicates that excess of Eu(III) ions either has no effect on the micellar structure, so the excess of Eu(III) stay free in solution, or only influences the internal coordination structure (core), vide infra, but does not break the micellar structure or change the macroscopic size of the micelles. The polydispersity, PDI (Figure 2b) and CONTIN analysis of the micellar size and size distribution (SI, Figure S1), confirms that there is only one predominant particle species in solution at all Eu/DPA ratios. Further light scattering studies provide additional information about micelles under charge stoichiometric conditions. Figure 3a shows the intensity decay upon diluting the micelle with buffer solution. The critical micelle concentration, CMC, is estimated by extrapolating the line to zero, and is found around Eu(III) concentration of 5 μM, suggesting the coordination between Eu(III) and DPA indeed to offer a strong driving force for micellization. Based on the molecular mass of micelles obtained from Figure 3b (see also experimental part, eq 7), we find aggregation numbers of around 80 for the BP per micelle, holding around 450 Eu(III) ions in the micellar core. The core radius is estimated from TEM image (SI, Figure S2), and is around 8 nm, so the PEOshell thickness is about 10 nm, considering the 18 nm hydrodynamic radius. These values fit well with our previous study on Zn-BP micelles.25 Having characterized the micelles with light scattering, we consecutively investigated the optical properties of the micelles. A most illustrative and dramatic enhancement of the

occupied by three DPA ligands at the Eu/DPA ratio of 1/3. The resulting coordination structure carries three net negative charges, [Eu(DPA)3]3−, which are compensated by the three positive charges on the quaternized pyridines on the PVP chain carrying the corresponding DPA ligand (Scheme 1). Hence, the designed Eu-BP micelles are expected to have a nearly neutral micellar core, viz. without counterions, at the stoichiometric coordination ratio. We studied the micelle formation by means of light scattering, characterized by the increase of scattered light. The light scattering intensity of Eu-BP micelles prepared at Eu/ DPA ratio of 1/3, increases over time and reaches a constant value after about 24 h (Figure 1). The hydrodynamic radii hardly change over time; we found the similar phenomena in the previous study with Zn-BP micelles.25 We hypothesize that the DPA ligands of DPA16-g-P4VP48-b-PEO193 can be involved in the formation of both intramolecular complexes, where Eu(III) binds with two or three DPAs from the same BP chain, and intermolecular complexes, in which the Eu(III) binds to DPA moieties of different BP chains, leading to micelles. A single DPA16-g-P4VP48-b-PEO193 polymer string with only intramolecular coordination structures does practically not contribute to the scattered light because of the low contrast compared to the surrounding solvent, and in fact we observe just low intensity shortly after mixing the components. The increase of the intensity with time indicates that besides intramolecular complexes micellar structure are eventually formed due to formation of intermolecular (interpolymer) coordination complexes. Apparently, this reorganization is related to an energy barrier, leading to a relatively long time C

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Figure 2. Light scattering intensity (a), hydrodynamic radius and polydispersity, PDI (b) of the formed micelles as a function of Eu/DPA mixing ratio. Note that micelles are prepared separately under the designed ratios.

Figure 3. a: light scattering intensity of micelles (Eu/DPA at 1/3) versus the concentration of Eu(III). b: KRC/R90 plotted as a function of C, the sum concentration of polymer and Eu(III) subtracted by the CMC.

ligands. The broad band in the range of 320−370 nm is attributed to absorption of the free pyridine groups on the polymer chain which have not been functionalized. Even though the intensity of the broad peak is low, it suggests that the pyridine is in close proximity to the metal ion and possibly even involved in the Eu(III) coordination; in fact, the strain in the polymer backbone might either facilitate or hamper the coordination of the pyridine and DPA groups to the metal ions. Finally, the absorption of Eu(III) ions gives a weak peak at 395 nm, corresponding to the 7F0-5L6 transition. Such a distribution implies that the brightest emission should be obtained from exciting the DPA ligand at 280 nm and, indeed, Figure 5c shows the emission curves upon exciting the micellar solution at 280, 360, and 395 nm. The typical transitions of Eu(III) from 5 D0 to the 7FJ ground states are evident, with emission peaks located at 580 (J = 0), 593 (J = 1), 614 (J = 2), 649 (J = 3), and 688, 693, 703 nm (J = 4).28 The emission upon excitation at 280 nm is much stronger than when exciting at 360 or 395 nm, confirming that the DPA ligand functions as a good antenna for the Eu(III) ion. In Figure 5d, the intensity of luminescence at 614 nm is plotted as a function of Eu/DPA mixing ratio. The excitation, λex, was set at the absorption maximum of DPA, 280 nm, and the ligand concentration being fixed at 1 mM for all the samples. This concentration is high enough to get a significant inner-filter effect,36 however, this systematically affects all the samples, so the obtained curve still reveals the real relation between the emission intensity and Eu/DPA mixing ratio. We find the maximum emission is at Eu/DPA ratio of 1/3, nicely

luminescence is found upon mixing the Eu(III) and BP (Figure 4). Figure 5a displays the UV absorption spectra of DPA and of

Figure 4. Exciting Eu(III) (left), polymer (middle) and micelle (right) solutions with a UV-lamp (254 nm). Eu(III) and DPA are kept at the same concentrations in all solutions, ∼ 0.05 and 0.15 mM, respectively.

the diblock copolymer before (P4VP48-b-PEO193) and after (DPA16-g-P4VP48-b-PEO193) modification. P4VP48-b-PEO193 shows two absorption features: one peak at 254 nm and one broad band in the range of 320−370 nm, corresponding to the π−π* and n-π* transitions of pyridine groups, respectively.34 The DPA ligand gives one absorption band at 280 nm,35 and the polymer after modification shows a combined absorption of pyridine and DPA. Based on the absorption curve, we can assign the peaks in the excitation curve of the Eu-BP micelles obtained by monitoring the emission at 614 nm (Figure 5b). The profile displays a maximum at 280 nm, corresponding to the absorption of DPA D

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Figure 5. a: UV−vis spectra of DPA, P4VP48-b-PEO193, and DPA16-g-P4VP48-b-PEO193 copolymers. b, c: excitation spectra, b (blue line, λem = 614 nm) and emission spectrum, c (λex = 280, 360, and 395 nm, corresponding to red, green, and black lines) of Eu-BP micelles (Eu/DPA at 1/3). d: normalized emission intensity at 614 nm (λex = 280 nm) of the formed micelles as a function of Eu/DPA ratio.

Figure 6. Luminescence decay (λex = 280 nm, λem = 614 nm) of Eu-BP micelles prepared at Eu/DPA ratio of 1/3 (a) and 2/3 (b) in H2O (green) and D2O (red). The black curve is the fitting curve and the obtained numbers are inserted in the picture. The intensities are normalized to the same initial values in order to highlight the decay difference. τ: lifetime, q: number of inner sphere water molecules.

Apparently, the formed micelles with the cross-linked core are highly stable and excess of Eu(III) cannot break the micelles even though they do disrupt the Eu(DPA)3 structures. Most likely, in case of Eu/DPA ratios higher than the optimal 1/3, also the non-DPA-functionalized pyridines are involved in Eu(III)-coordination, intramolecularly but most likely also intermolecularly, and as such further stabilizing the micelle structures, particularly at high Eu/DPA ratio. To better understand the coordination structure in the micellar core, we performed lifetime measurement of Eu-BP micelles (Eu/DPA at 1/3), both in H2O and D2O. An average lifetime of Eu(III) incorporated in the micellar core is obtained

corresponding to the results obtained by the light scattering. Interestingly, the emission intensity goes down upon further increasing the Eu(III) amount after the 1/3 stoichiometry. This suggests that excess of Eu(III) ions may go into the micellar core and break the fully coordinated Eu(DPA)3 structure, leading to more coordination sites of Eu(III) occupied by either pyridine or water molecules with a consequential net decrease of emission intensity. However, despite the Eu(DPA)3 structures might be converted into Eu(DPA)2 or even Eu(DPA) moieties, the micelle structure remains intact, as proven by light scattering results where the intensity and radius stay constant also at Eu/DPA ratios higher than 1/3. E

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Figure 7. Stability tests of Eu-BP micelles. a, b: Light scattering intensity, hydrodynamic radius (a) and luminescence intensity (b, λem = 614 nm)) at different salt concentration.

Figure 8. Light scattering intensity (a) and luminescence emission (b, λex = 280 nm) of Eu-BP and Zn-BP micelles before and after adding metal ions. A: Eu-BP micelles; A′: Eu-BP micelles with added Zn(II); B: Zn-BP micelles; B′: Zn-BP micelles with added Eu(III).

by fitting the decay curve and the numbers are shown in Figure 6a. First of all, the life times of Eu(III) micelles in H2O and D2O are around 1.0 and 1.5 ms, respectively, and are smaller than the reported values of [Eu(DPA)3]3− structures in H2O (∼1.6 ms) and D2O (∼3.0 ms).37,38 The faster decay in the micelles indicates that, although a Eu(III) ion in the micellar core stays roughly coordinated to three DPA ligands on average, the full coordination, and/or the optimal geometry of an isolated Eu(DPA)3 moiety is not reached. Part of the PVPblock’s pyridines that were not functionalized with a DPA moiety is probably also involved in the coordination, which is in correspondence with the result from the excitation spectrum (Figure 5b). As the DPA ligands are grafted on the polymer chain covalently, the Eu-DPA coordination may require an adaptive twist or deformation on the polymer backbone, which cannot be achieved completely. In other words, the failed adaptation of the polymer chain hampers the full and optimal coordination between Eu(III) and DPA, eventually leads to a variety of different Eu-DPA coordination structures in the micellar core. Nevertheless, the number of inner sphere water molecules, q, calculated by q = 1.2(1/τH2O − 1/τD2O − 0.25), is around 0.13, corresponding to a high coordination degree of the Eu(III) ions and (DPA) ligand.39,40 We also measured the life times of Eu-BP micelles prepared at Eu/DPA ratio of 2/3, corresponding to the double amount of the Eu(III) with respect to the stoichiometry.(Figure 6b) We find the q is much higher and around 0.72, confirming our light scattering and luminescence data that excess of Eu(III) indeed breaks the full

Eu(DPA)3 coordination into differently coordinated structures. We note that the data fitting and analysis is simplified and rather rude for such a heterogeneous coordination system, still, the observations are consistent with previous discussions, and provide a good complementation to the results from light scattering and the steady-state luminescence data. From the application point of view, stability of the micelle is another crucial factor to be concerned. In our previous studies,41−44 metal-incorporated complex coacervate core micelles (M-C3Ms) based on a three-building-blocks concept were prepared by the electrostatic interaction between an anionic coordination polymer formed from a bis(DPA) ligand (L2EO4) and a polycationic-neutral diblock copolymer. We have found that, first, both lanthanide and first row transition metal ions based C3Ms show strong dependence on ionic strength and dissociate completely upon the salt concentration exceeding a critical value, which is around 0.8 M (CNaCl) for Eu−C3Ms and 0.2 M for Zn−C3Ms. Second, the salt stability of Eu−C3Ms is higher than that of Zn−C3Ms, possibly due to the different structures and charge density of Eu-L2EO4 and ZnL2EO4. Under Eu/DPA ratio of 1/3, Eu-L2EO4 form net-work structures with three negative charges per coordination site, while Zn(II) demands 2 DPA groups to reach full coordination state. Hence, Zn-L2EO4 form linear structures with only two negative charge per coordination site. Here, with the designed DPA grafted polymers, we expect that the form micelles are stable against ionic strength. The light scattering intensity and size of Zn-BP micelles, in our previous study, stayed constant at F

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Figure 9. a: Light scattering intensity of Eu-BP, Eu−Gd−BP, and Gd-BP micelles. b: luminescence emission (λex = 280 nm, red bar) and magnetic relaxation rate (green bar) of Eu-BP, Eu−Gd−BP and Gd-BP micelles. [Ln(III)] = 0.34 mM for Ln-BP micelles, [Eu(III)] = [Gd(III)] = 0.17 mM for Eu−Gd−BP micelles.

CNaCl ≤ 0.4 M and increased with further increasing salt concentration.25 For Eu-BP micelles in the present work, we find that the light scattering intensity, hydrodynamic radius (Figure 7a) and normalized luminescence emission at 614 emission (Figure 7b) remain constant upon increasing the NaCl concentration to even 1 M. These findings indicate that Eu-BP micelles are more stable than the three component system Eu−C3Ms with bis(DPA) ligands, and likely also more stable than Zn-BP micelles. To evaluate whether the Eu-BP micelle is indeed more stable than Zn-BP micelle, we added Zn(II) into Eu-BP micellar solution and, oppositely, added Eu(III) to Zn-BP micellar solution, and recorded the variations on light scattering intensity and luminescence emission. Note that the polymer concentration (DPA group) was fixed here, and the added amount of Zn(II) or Eu(III) corresponds to the full coordination of DPA groups. Upon adding Zn(II) into EuBP micellar solution, the light scattering intensity increases a little, luminescence emission decreases about 30% and the hydrodynamic radius stays constant at 18 ± 3 nm. (Figure 8) On the other hand, upon adding Eu(III) into Zn-BP solution, the hydrodynamic radius stays at 20 ± 3 nm, but both light scattering intensity and luminescence emission increase intensely. The final mixtures, Zn(II) to Eu-BP and Eu(III) to Zn-BP, get the same combination of the different component, but different light scattering intensity and luminescence emission, confirming the M-BP micelles are kinetically trapped structures. These results suggest, again, excess of metal ions get into the formed micelles and affect the internal coordination structure without breaking the micelles, and Eu-BP micelles display a stronger ability against excess metal ions and salt compared to Zn-BP micelles and M-C3Ms. Finally, we demonstrated the general applicability of our micelle system by incorporating different functional Ln(III) ions. We prepared the gadolinium incorporated micelles (GdBP) and the micelles packing both Eu(III) and Gd(III) (Eu− Gd−BP) under the mixing ratio of 1/1. As shown in Figure 9a, Eu-BP, Eu−Gd−BP, and Gd-BP micelles show similar light scattering intensity and size (Rh: 18 ± 3 nm), indicating the micellar structure is independent of the packing metals. However, the corresponding functions are strongly relying on the incorporated Ln(III) ions. Gd(III) complexes are wellknown MRI (magnetic resonance imaging) contrast agents due to their seven unpaired electron spins which dramatically decrease the spin−lattice relaxation time of proximate water

molecules.45 With Gd-BP micelles, we find an enhanced longitudinal relaxation rate due to the increase of the rotational correlation time by forming micelles. The relaxation rate, R1 is around 5.7 s−1, (Figure 9b, green bar) corresponding to a reflexivity about 17.1 mMGd−1 s−1, which is comparable to the Gd−C3Ms in our previous report.43 Replacing half of the Gd(III) with Eu(III), the obtained Eu−Gd−BP micelles show enhancement not only of the magnetic relaxation rate, but also on the luminescence emission due to the presence of the Eu(III). The strength is about half (0.5 ± 0.05) of the full luminescent Eu-BP micelles or Gd-BP micelles on the magnetic relaxation. (Figure 9b) Such findings suggest that our method is easily adapted for preparing multimodal micellar structures with different functions based on the incorporated Ln(III) ions, and more interestingly, allows for combining different Ln(III) ions and tuning the functions simply by regulating the Ln(III) mixing ratio.

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CONCLUSION We have shown single block copolymers to form stable micelles incorporating Eu(III) ions in the core, resulting in a strong luminescence. The DPA ligand is grafted on the PVP block of a P4VP48-b-PEO193 diblock copolymer, and micellization occurs upon mixing with Eu(III) in aqueous solution. The obtained Eu-BP micelles get a core radius around 8 nm, in which several hundreds of Eu(III) ions are encapsulated and the core is surrounded by a PEO shell with a thickness of about 10 nm. Light scattering and luminescence measurements reveal the optimal micellization and emission are obtained under the Eu/ DPA ratio at 1/3, corresponding to a charge neutral, that is, counterion free, micellar core. While the excitation curve and lifetime tests suggest the Eu(III) in the micellar core is not fully coordinated by three DPA ligands, but partly coordinated by pyridine as well, attributable to the constrains of the polymer chain. Such an effect from the polymer backbone should be addressed with special attention when designing new functional structures based on similar systems and mechanisms. Because of the strong Eu-DPA coordination and the strongly crosslinked core structure, the Eu-BP micelles are highly stable. The method reported here demonstrates a general and robust way for preparing micelles incorporating different lanthanide ions. As proof of principle, the Gd-BP micelles with enhanced magnetic relaxation rate were prepared following the same strategy. Moreover, the Eu−Gd−BP micelles incorporating a mixture of Eu(III) and Gd(III) ions show enhancement on G

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both the luminescence emission and magnetic relaxation rate. More interestingly, the corresponding functions could be tuned by controlling the mixing ratio of Ln(III) ions, showing great potential for preparing novel multifunctional materials, for example, as multimodal diagnostic imaging and/or therapeutic agents.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b03226. CONTIN analysis of micellar size and TEM images of micelles, Figures S1−S2 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Daan W. de Kort (Laboratory of Biophysics, Wageningen University) for help with the magnetic relaxation measurements and Jan Bart ten Hove (Laboratory of BioNanoTechnology, Wageningen University) for help with TEM tests.

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REFERENCES

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DOI: 10.1021/acs.langmuir.5b03226 Langmuir XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.langmuir.5b03226 Langmuir XXXX, XXX, XXX−XXX