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Assembly of double-hydrophilic block copolymers triggered by gadolinium ions: new colloidal MRI contrast agents Camille Frangville, Yichen Li, Claire Billotey, Daniel R. Talham, Jacqueline Taleb, Patrick Roux, Jean-Daniel Marty, and Christophe Mingotaud Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.6b00664 • Publication Date (Web): 25 May 2016 Downloaded from http://pubs.acs.org on May 30, 2016
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Assembly of double-hydrophilic block copolymers triggered by gadolinium ions: new colloidal MRI contrast agents Camille Frangville,† Yichen Li,‡ Claire Billotey,|| Daniel R. Talham,‡ Jacqueline Taleb,|| Patrick Roux,|| Jean-Daniel Marty*,†, Christophe Mingotaud*,† †
Laboratoire des IMRCP, Université de Toulouse, CNRS UMR 5623, Université Paul Sabatier, 118, route de Narbonne 31062 Toulouse Cedex 9, France ‡
Department of Chemistry, University of Florida, Gainesville, FL 32611-7200, USA
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EMR 3738 Ciblage Thérapeutique en Oncologie, Université de Lyon, Université Jean Monnet, Hospices Civils de Lyon, 42023 Saint-Etienne Cedex 2, France ABSTRACT: Mixing double-hydrophilic block copolymers containing a poly(acrylic acid) block with gadolinium ions in water leads to the spontaneous formation of polymeric nanoparticles. With an average radius near 20 nm, the nanoparticles are exceptionally stable, even after dilution and over a large range of pH and ionic strength. High magnetic relaxivities were measured in vitro for these biocompatible colloids, and in vivo magnetic resonance imaging on rats demonstrates the potential utility of such polymeric assemblies.
polyion complexes (HPICs) resulting from the addition of oppositely charged polyvalent metal ions (e.g. Cu2+, Zn2+, Ca2+, Al3+, La3+) are also known.13-16 Colloidal HPICs that strongly bind Gd3+ ions have potential to be effective relaxivity agents, however, to the best of our knowledge, no attempts to form HPICs with gadolinium ions have been reported.
KEYWORDS: Polymer, block copolymer, micelle, MRI, gadolinium, nanoparticle Magnetic Resonance Imaging (MRI) is a valuable and noninvasive medical imaging technique capable of probing the morphology and health of biological tissues.1 The technique relies on nuclear magnetic resonance of tissue water protons and longitudinal relaxation times (T1), transversal relaxation (T2) as well as proton density are all factors providing contrast between tissue types, including pathological states. Endogenous contrast agents (CAs) are often needed to enhance or optimize MRI definition.2 The two most common classes of contrast agents are: (a) T1 or positive contrast agents that shorten proton longitudinal relaxation time and (b) T2 or negative contrast agents that shorten proton transversal relaxation time. Whereas superparamagnetic iron oxide nanoparticles are most widely studied as T2 agents,3 molecular complexes and nanoparticles based on the lanthanide ions Gd3+ are most often employed as T1 agents.4 Molecular gadolinium chelates have been commercialized (e.g. DOTA: Dotarem®, DTPA: Magnevist®, ProHance®), are used clinically and are still the most widely studied class of contrast agent.4 However, molecular agents possess limitations, such as rapid elimination and complications from extravasation, and they are less efficient at the higher magnetic fields of modern MRI instruments, so there is still a need for concepts for more efficient CAs with enhanced relaxivity enhancement or extended circulation lifetime.5 Immobilization of Gd-complexes onto macromolecules is one way to slow down rotational motion, providing more efficient relaxation,6,7 although the synthetic pathways to such functionalized polymers can be complicated. An alternative to polymers functionalized with chelating groups is to use interactions between metal ions and ionic polymers. A class of colloidal ionic polymers receiving considerable attention are double hydrophilic block copolymers (DHBCs) with an ionizable complexing block and a neutral block.8-11 Although DHBCs are most often formed as polyion complexes with oppositely charged polymers,12 hybrid
Scheme 1. The addition of gadolinium ions to a double hydrophilic block copolymer forms polymeric nanoparticles (in yellow: ionizable block; in blue: neutral block).
Figure 1. Chemical structure of the diblock copolymer used in this work. Poly(acrylic acid) and a poly(ethylene oxide) block have average molecular weights of 6000 g.mol-1 and 3000 g.mol-1 respectively. The polymer is noted PEO6k-b-PAA3k. In this paper, we describe gadolinium-containing HPICs, Scheme 1, prepared from a commercially available diblock copolymer made of poly(acrylic acid), PAA, and poly(ethylene oxide) PEO blocks, Figure 1, that exhibit exceptionally high stability upon dilution, as well as toward pH changes or competing ions. In typical experiments, a concentrated solution of gadolinium ions is added to an aqueous solution of the diblock PEO6k-bPAA3k copolymer at a concentration of 0.1%wt. The mixture can be defined by the ratio, R, between the positive charge arising from the trivalent Gd3+ ions and the potentially available negative
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Nano Letters
The electroneutrality between gadolinium ions and fully ionized polymers should then correspond to R equal unity. At constant polymer concentration, Figure 2A shows modifications in particle size determined by dynamic light scattering as the ratio, R, is changed.
charges from the ionized or ionizable acrylic acid monomer units (AA) of the polymers: 3
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Figure 2. (A) Scattered light intensity (full squares) and Z-average size (full circles, in term of diameter given in nm) measured for mixed Gd3+/polymer solutions as the ratio, R, is varied (see supporting information Figure S1 for the corresponding correlograms). The Z-average size data for low values of R are poorly defined (presumably because of self-association of the copolymers in pure water) and are differentiated with open circles. The PEO6k-b-PAA3k polymer concentration was 0.1% wt with pH adjusted to 7 at room temperature. (B) ICP-AES determination of free Gd3+ concentration plotted versus R=3.[Gd3+]total /[AA] for 0.1%wt PEO6k-b-PAA3k. (C) Scattered light intensity by a PEO6k-b-PAA3k (0.1%wt)/Gd3+ HPIC (R=1) solution as a function of pH. The light scattering intensity increases linearly up to the value of R = 1, above which the scattered light intensity remains constant. The result suggests the formation of colloids that are larger than a single macromolecule, triggered by the added ions and completed upon achieving electroneutrality. Dynamic light scattering of the mixtures also shows a strong modification of the correlogram with the addition of gadolinium ions before stabilizing when the critical value of unity is reached (Supporting Information, Figure S1). Analysis of these correlograms in term of Zaverage size (Figure 2A) also shows two regimes, before or after R = 1. Moreover, for R larger than 1, the constant value of the Zaverage size, 33 ± 6 nm, strongly suggests the presence of large polymeric nanoparticles with a constant diameter. Titration by inductively coupled plasma atomic emission spectrometry (ICP-AES) of free gadolinium ions after filtering the polymer mixtures (see Figure 2B) reveals the quantity of free Gd3+ is negligible when the ratio R < 1 (about three orders of magnitude below the total concentration of gadolinium added to the solution). This result demonstrates that virtually all gadolinium ions are trapped within the polymeric nanoparticles (NPs), a pre-requisite for biological applications. Indeed, Gd3+ is known to interfere with Ca2+ ions and forms mineral deposits in capillary beds, having an LD50 close to 0.4 mmol.kg-1 in rats.18,19 In the gadolinium/polymer solutions, the concentration of free Gd3+ increases significantly and linearly only when the ratio, R, is greater than 1. This efficient trapping of the gadolinium ions within the NP structure suggests that leakage of gadolinium can occur mainly with disaggregation of the NPs. Similar behavior is achieved with europium ions, allowing the interactions between the metal ions and polymer to be followed with fluorescence, as the complexation chemistry of Eu3+ and Gd3+ is often very similar. Water coordination quenches lanthanide fluorescence, which can be regained upon displacing water with other coordinating ligands.17 Figure 3 plots the significant increase of Eu3+ fluorescence as the diblock copolymer is added to the metal ion (see Figure S2 for the fluorescence spectra). The
change in fluorescence proves that the lanthanide ions interact strongly with the polymer. When the experiments were performed with simple PEO, the fluorescence intensity remained weak and constant for all values of R, confirming the lanthanide ions are only complexed by the carboxylate functions of the PAA block (Figure 3). Interestingly, when PAA homopolymer was used, the maximum fluorescence intensity was weaker than with the diblock system indicating more complete complexation of the lanthanide ion with the PAA of the diblock polymer. Relative intensity at 618nm
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Figure 3. Fluorescence intensity, in arbitrary units, at 618 nm attributed to the 5D0 → 7F2 transition of europium ions versus [Polymer Units]/(3[Eu3+]) (i.e. [AA]/(3[Eu3+]) for the diblock copolymer or and the PAA homopolymer, or EO]/(3[Eu3+]) for the PEO homopolymer). Open circles: homopolymer PEO (2k); full circles: homopolymer PAA (1k); full squares: diblock PEO6kb-PAA3k copolymer. For each series, polymer is added to a constant concentration,10-3 M, of Eu3+ ions at room temperature. Excitation wavelength: 252 nm. The size distribution of the NPs is monomodal for R close to unity or higher, as determined with DLS correlograms analyzed by a non-negative least squares (NNLS) method, with an average diameter of 32 ± 9 nm, determined by intensity analysis, or 23 ± 5 nm, by number analysis (Figure S3). Such values are larger than
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the ca 9 nm found for HPICs formed by La3+ and poly(acrylic acid)3k-b-poly(acrylamide)10k copolymers.15 As seen in Figure 4, transmission electronic microscopy of dried solutions shows small domains of high contrast with average diameter of 8 ± 2 nm.
As shown in Figure 5, the proton relaxation time of an aqueous solution of gadolinium ions is strongly shortened by the addition of copolymer. 120
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Figure 4. TEM images of a dried solution PEO6k-b-PAA3k (0.1%wt) + Gd3+ HPICs (R=1) and distribution of sizes of the high-contrast domains (assumed as disks of diameter d). Statistics are on ca 70 domains. The objects presumably arise from the gadolinium ions at the polymer complex core, which will be surrounded by polymer corona. Together, the structural analyses indicate association of the block copolymers with the gadolinium or europium ions to form hybrid inorganic/polymeric nanoparticles with diameter on the order of 20 nm. The formation of NPs is complete when the ratio R is close to unity. Sanson et al. 15 have shown (by SAXS and SANS) that the NPs formed by DHBC mixed with metal ions have a dense core and a diffuse corona (presumably made of the neutral blocks). Such a structure is also expected in the present NPs with a corona of PEO confirmed by zetametry. Indeed, the zeta potential of the pristine copolymers is negative due to the ionized carboxylate groups, but became null during the formation of the NPs (Figure S4). The NPs are therefore stabilized by steric hindrance and are poorly affected by the ionic strength of the solution even after a few months in 1 M NaCl (Figure S5). The integrity of the NPs is also maintained over a large range of pH. As shown in Figure 2C, the scattered light intensity from a 0.1%wt suspension of PEO6k-bPAA3k HPICs with Gd3+ (R=1) strongly decreases only below pH 4. ICP-AES titration suggests also a massive release of gadolinium ions for such low pHs (see Figure S6). Therefore, neutralization of the carboxylate anionic charges of the copolymers, which dissociates the NP complexes, is shifted toward lower pH by at least one unit compared to the free carboxylic function. The entanglement of PAA blocks and the inter-block bridging induced by the gadolinium ions appears to impart higher and somewhat unexpected stability of the NPs in solution. Such effects could compensate at least partially the individual weaker interactions between gadolinium ions and carboxylate functions compared to carefully designed ligand (such as DOTA) with Gd3+. The integrity of the Gd3+/PEO6k-b-PAA3k NPs is also maintained with high dilution. The scattered light intensity, or the fluorescence intensity in the case of Eu3+, of diluted solutions of the NPs is linear within the concentration range 0.1-10-4 % wt of the polymers, with the lower limit at the sensitivity limit of our instruments (Figure S7). Stability over this range of dilution demonstrates that the NPs are not in equilibrium with free copolymer and Gd3+ ions. This is confirmed by the recorded very slow disaggregation of the NPs when NPs solution was dialyzed against pure water. The striking stability of the HPICs leads to possible applications of the NPs in MRI, and magnetic relaxivity measurements were performed to assess the properties of the hybrid colloids.
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Figure 5. Magnetic relaxation time (T1 and T2) measured at 1.4 T and 25°C of a solution of Gd3+ (10-3 mol.L-1) with increasing amount of added PEO6k-b-PAA3k expressed as 1/R = [AA]/(3.[Gd3+]). At polymer concentration sufficiently high to trap all Gd3+ ions within the NPs, the relaxation properties level off and become constant. The behaviour indicates that the NPs induce faster relaxation than free aqueous Gd3+ ions. The result was confirmed by measurements of the specific relaxivities of the Gd3+/PEO6k-bPAA3k NPs (Figure S8). The relaxivity values of 48 ± 2 mM-1.s-1 for r1 and 67 ± 2 mM-1.s-1 for r2 (B0 = 1.4 T) are remarkably high, much higher than standard molecular complexes, or even polymers modified by gadolinium complexes,5 but close to values found for large doped silica NPs.20 The spin-lattice (r1) relaxivity is only modestly decreased at elevated magnetic fields. Measurements performed at 7 T yield r1 = 15.4 mM-1.s-1 (Figure S9), larger than for the molecular complex, GdDOTA (r1 = 4.1 mM-1.s-1), or hybrid gadolinium oxide nanoparticles (r1 = 8.8 mM-1.s-1)21, resulting in very high T1 contrast (Figure S10). To explain such a high relaxivity, further studies are needed. However, following literature data,5 two main hypothesis can be proposed. Like for gadolinium complexes attached to dendrimers, the huge increase in the molecular weight of the gadolinium species due to the NPs formation should induce a much slower tumbling compared to the pristine gadolinium. Slowing tumbling means higher relaxivity. Furthermore, the gadolinium ions should be located in the center part of the NP, i.e. near the barycenter of the gadolinium species. Gadolinium will rotate at the same (and very low) rate than the whole NPs, increasing the observed relaxivity. The highly hydrophilic structure of the NPs made of ions and double hydrophilic block copolymers should also be a clear advantage compared to architecture made of amphiphilic copolymers or full inorganic nanoparticles. Indeed, water molecules should be able to diffuse somewhat easily within the NPs. This favors interaction between water molecules and all the gadolinium ions. The cytotoxicity of the polymeric NPs was found to be insignificant up to 1.5 mM Gd (Figure S11), allowing in vivo experiments on rats, performed to obtain preliminary determinations of MR contrast efficacy, pharmacokinetic properties, and tolerance. The in vivo MR contrast was assessed by performing cerebral angiography (Figure S12) after intravenous (IV) bolus injection of Gd3+/PEO6k-b-PAA3k NPs at 15 µmol/kg and comparing to the use of GdDOTA. The Gd3+/PEO6k-b-PAA3k NPs provide both higher intensity and more persistent enhancement (Figure S13) of the vascular signal, even at 1/3 the total Gd concentration of GdDOTA. The imaging confirms the in vitro results, demonstrating that
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Nano Letters Gd3+/PEO6k-b-PAA3k NPs provide easily visible contrast enhancement at lower Gd concentration. Tissue uptake and elimination properties were assessed using a T1-weighted dynamic sequence of coronal images centered on the abdominal cavity, acquired before and 1 h after IV injection of Gd3+/PEO6k-b-PAA3k NPs at 11.5 µmol/kg equivalent Gd concentration. Regions of interest (ROI) were drawn corresponding to one adrenal gland (AG), the pelvic cavities (PC) and the renal cortex (RC) of both kidneys, the liver (LI), and the bladder (BL) (see Figure 6 and Figure S14) to generate time activity curves. Maximum enhanced contrast of about +25% is achieved in T1weighted images of the adrenal gland after 50s of Gd3+/PEO6k-bPAA3k injection, followed by a slow decrease until reaching the initial value after 1 h, suggesting a slow decrease of the contrast agent in blood flow. Higher values of enhanced contrast of approximately +40% and +30% were measured for the right and left renal cortex regions, which also slowly decreased over about 1 h, consistent with the elimination function of kidneys. The signal in the pelvic cavities sharply increased, reaching maximum values of enhanced contrast of 64% after 1 min 40 s and 88% after 4 min 20 s. The signal then slowly decreased, taking longer than one hour to return to initial values.
60%. This result can be related to inhibited miction due to the gaseous anesthesia, which can also explain the lack of draining of contrast agents from the pelvic cavities. The bladder signal meanwhile was found to progressively increase, corresponding to progressive hepatobiliary elimination. These in vivo analyses reveal that Gd3+/PEO6k-b-PAA3k HPICs significantly increase contrast in all the organs studied. The blood circulation life-time is relatively long, with signal enhancement of ~ +20% in the adrenal gland even after 40 min, and urinary and hepatobiliary elimination processes were confirmed. Finally, in vitro toxicity tests show the intravenous dosage, up to the maximum of 15 µmol/kg Gd equivalent concentration used, were well tolerated by the rats. Gd quantification based on ICM-MS was performed within main organs, blood sample and urine, one hour after the NPs injection (see Table S1). These results confirmed quantitatively the high long time blood remanence, the high fast urine elimination, the low uptake in reticulo-endothelial system (RES) tissue, and the absence of consistent lung uptake. These preliminary studies of a relatively simple HPIC show surprisingly good stability, fast urinary elimination, low RES uptake and superior magnetic relaxivity properties in vitro and in vivo, even at high magnetic field. With long blood remanence and high induced contrast, this new type of Gd probe could be used for MRI at lower concentrations than currently used contrast agents, leading to enhanced safety. Complete long term toxicity analysis should be carefully performed in vivo to estimate the leakage rate of the gadolinium. However, the HPIC platform allows simple variation of the ions as well as the copolymers, affording easy-to-obtain improvement of stability of the Gd probe.22 Compared to polymeric MRI contrast agents previously described,23 the HPIC architecture is clearly obtained through a much simpler way, leading to well defined systems with biodegradability and toxicity easily modulated through the choice of the used copolymers. Furthermore, mixing of ions or polymers within this architecture opens many opportunities to develop new families of HPICs for future biological applications.
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Supporting Information Experimental procedures, complementary characterizations, MRI data. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION 40
Corresponding Author Dr C. Mingotaud,
[email protected] 20
Author Contributions These authors contributed equally.
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Figure 6. Top. Coronal T1-weighted images centered on the kidneys, before, during the IV injection, at the peak signal (1 min 40 s) and 55 min after injection of Gd3+/PEO6k-b-PAA3k HPICs. Bottom. Enhanced contrast (EHC %) relative to the background (BKG) vs. time measured for the adrenal gland (AG), left and right pelvic cavities (PC), left and right renal cortex (RC). Lines are guides to the eye. Elimination processes for the Gd3+/PEO6k-b-PAA3k HPICs were elucidated by monitoring signal enhancement vs time in the liver and bladder (Figures S14 and S15). For the liver, contrast increased over few minutes and then remained constant at about
FUNDING The authors declare no competing interest. They acknowledge the financial support of the EU (FEDER-35477 "Nano-objects pour la biotechnologie"), the ANR agency (ANR-DFG n° ANR-11INTB-1004, "Dendrion-Bio"), the Région Midi-Pyrénées (Chaire d'excellence Pierre de Fermat n° 12012781) and the CNRS.
ACKNOWLEDGMENT The authors wish to thank J-B. Langlois for technical support (CERMEP-Imagerie du vivant, Bron, France), B. Amouroux and K. Hicquet for the dialysis experiments.
REFERENCES
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