ARTICLE pubs.acs.org/Langmuir
Surface Modification of Gadolinium Oxide Thin Films and Nanoparticles using Poly(ethylene glycol)-Phosphate Andree-Anne Guay-Begin,†,‡ Pascale Chevallier,† Luc Faucher,†,‡ Stephane Turgeon,† and Marc-Andre Fortin*,†,‡ †
Axe metabolisme, sante vasculaire et renale, Centre hospitalier universitaire de Quebec (CRCHUQ-MSVR), Laboratoire de biomateriaux pour l0 imagerie medicale (BIM), Centre de recherche sur les materiaux avances (CERMA) and Centre quebecois sur les materiaux fonctionnels (CQMF) ‡ Department of Mining, Metallurgical and Materials Engineering, Universite Laval, Quebec, Canada, G1V 0A6
bS Supporting Information ABSTRACT: The performance of nanomaterials for biomedical applications is highly dependent on the nature and the quality of surface coatings. In particular, the development of functionalized nanoparticles for magnetic resonance imaging (MRI) requires the grafting of hydrophilic, nonimmunogenic, and biocompatible polymers such as poly(ethylene glycol) (PEG). Attached at the surface of nanoparticles, this polymer enhances the steric repulsion and therefore the stability of the colloids. In this study, phosphate molecules were used as an alternative to silanes or carboxylic acids, to graft PEG at the surface of ultrasmall gadolinium oxide nanoparticles (US-Gd2O3, 23 nm diameter). This emerging, high-sensitivity “positive” contrast agent is used for signal enhancement in T1-weighted molecular and cellular MRI. Comparative grafting assays were performed on Gd2O3 thin films, which demonstrated the strong reaction of phosphate with Gd2O3 compared to silane and carboxyl groups. Therefore, PEG-phosphate was preferentially used to coat US-Gd2O3 nanoparticles. The grafting of this polymer on the particles was confirmed by XPS and FTIR. These analyses also demonstrated the strong attachment of PEG-phosphate at the surface of Gd2O3, forming a protective layer on the nanoparticles. The stability in aqueous solution, the relaxometric properties, and the MRI signal of PEG-phosphate-covered Gd2O3 particles were also better than those from non-PEGylated nanoparticles. As a result, reacting PEG-phosphate with Gd2O3 particles is a promising, rapid, one-step procedure to PEGylate US-Gd2O3 nanoparticles, an emerging “positive” contrast agent for preclinical molecular and cellular applications.
’ INTRODUCTION With seven unpaired electrons in its 4f orbitals, paramagnetic gadolinium is used to increase the longitudinal relaxation rate of hydrogen protons in aqueous solutions and in biological tissues, which translates into MR signal increase in T1-weighted images.1,2 Therefore, chelated Gd is used in a large fraction of MRI clinical scans nowadays.3 However, most single Gd chelates are not appropriate for molecular and cellular contrast agent (CA) applications (e.g., cell tracking), mainly because they are not detected very sensitively (only one Gd per CA unit), and because they do not internalize well into cells.4,5 Metal oxide nanoparticles are now being used in a broad spectrum of biomedical applications. In particular, coupling metal oxide nanoparticles with magnetic resonance imaging (MRI) has allowed the development of a new class of superparamagnetic and paramagnetic CAs that can be used in molecular and cellular imaging. Recently, ultrasmall gadolinium oxide particles (US-Gd2O3, 23 nm diameter) have been developed, expressing high longitudinal relaxivities (r1) and low r2/r1 ratios, which are ideal for “positively contrasted” T1-weighted imaging r 2011 American Chemical Society
applications.610 Compared to Gd chelates, they concentrate hundreds of paramagnetic atoms into very small nanoparticulate volumes, therefore providing higher relaxivities per unit of Gd. This new class of contrast agents has been applied to cell labeling in vitro and to intravenous injections in rodents.7,1017 Nanoparticles administrated in vivo and accumulating at specific sites could induce very strong and specific contrast enhancement effects compared to conventional Gd chelates. One of the most critical aspects in the design of optimal nanoparticulate CAs is colloidal stability. In fact, this parameter is of utmost importance for preserving the relaxometric properties of the paramagnetic suspension.18 The nonspecific adsorption of proteins from the surrounding environment is also a critical aspect that could cause nanoparticle aggregation and limit the performance of CAs. Therefore, molecules such as poly(ethylene glycol) (PEG) are used to stabilize US-Gd2O3 particles.6,7,9 Received: July 19, 2011 Revised: September 27, 2011 Published: October 04, 2011 774
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Langmuir Indeed, PEG is a water-soluble and biocompatible polymer, widely used to reduce cell and protein adsorption, as well as the opsonization of systemically administrated particles.19 This resistance is attributed to the generation of a steric barrier by the PEG chains, which are highly hydrated and mobile.20,21 In order to strongly attach the polymer on metal oxide surfaces, it is necessary to use PEG molecules containing at least one functional group expressing a high affinity for the surface to treat. In addition to attachment, this functional group should ideally add a protective effect to the nanoparticle system. The adsorption of carboxylic acids is probably the most widely used approach to coat and stabilize ultrasmall metal oxide nanoparticles. However, the bonding between COOH and metal oxides is often pH sensitive and its stability is variable depending on the type of bonding.22,23 In order to limit nanoparticle aggregation, to prevent the lixiviation of metal ions, and to improve the biocompatibility, silanization has been used as the main strategy to graft PEG and to protect the oxide surface of magnetic particles.6,9,22,24,25 Although silanes allow the fabrication of relatively well-controlled thin coatings on metal oxides, they can form multilayers through uncontrolled polymerization. The procedure of silanization is also more sensitive to solvent, temperature, and trace amounts of water than by using other functional groups. Finally, silica shells at the surface of "positive" CAs could impede the T1-relaxation enhancement performance of these products, since they introduce an important distance between the paramagnetic atoms and the surrounding water protons. In order to provide an alternative to silanes, phosphate-based molecules could be used as metal oxide ligands. Phosphonic and phosphoric acids, as well as organophosphates, have provided chemically well-controlled, structurally ordered, and relatively stable self-assembled monolayers (SAM) on oxide surfaces of reactive metals. Indeed, long-chain phosphonic and phosphoric acids lead to dense, well-ordered SAM on ZrO2 and TiO2.2628 Using a similar approach, alkyl phosphate solutions have been used on a variety of metal oxide substrates (Al2O3, Ta2O5, Nb2O5, ZrO2, and TiO2).2932 For instance, octadecyl phosphate has been shown to spontaneously form adlayers from heptane/2-propanol solutions on tantalum oxide (Ta2O5) surfaces.33,34 More recently, alkane phosphates conjugated to PEG have been successfully applied to the design of SAM, with the aim of developing optimal biomedical surfaces preventing the nonspecific adsorption of proteins, bacteria, and cells.35,36 The thickness of such layers was evaluated to about 1.3 nm, for a molecular grafting density of 2 molecules/nm2.36 Therefore, the use of this family of molecules has been considered as a promising approach to stabilize metal oxide nanoparticles. Indeed, alkyl phosphonates and phosphate surfactants, as well as phosphoric and phosphonic acids, have been used to coat magnetic nanoparticles, with evidence of high binding energies.3740 In fact, the study of alkanephosphonic acid adsorption on the surface of amorphous ferric oxide particles has led to the establishment of two possible bonding schemes for the phosphonate ions on Fe3+, with either one or two oxygen atoms of the phosphonate group binding to the surface.41 So far, the use of phosphates as ligands to graft PEG at the surface of rare-earth oxide thin films and nanoparticles such as Gd2O3 has never been reported. In the present article, we report on the use of PEG-phosphate molecules as a promising alternative to carboxylation and silanization of metal oxide particles. This approach could provide a one-step procedure to PEGylate ultrafine Gd2O3 nanoparticles. In the first step of this study, we used Gd2O3 thin films to
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demonstrate the reactivity of PEG-phosphate with the rare-earth oxide. This system provided a comparative model to investigate the PEG coupling efficiency using phosphate instead of carboxyl and silane functionalities. Moreover, these Gd2O3 thin films can be useful to study the interaction of many biochemical molecules with rare-earth ions. In fact, the study of Gd3+ catalytical properties in biochemical and biological environments and processes is a very active field, in direct link with MRI contrast agent applications. Then, we applied this one-step PEG-phosphate grafting methodology to coat US-Gd2O3 nanoparticles. The particles were characterized by XPS and FTIR. The hydrodynamic size of the Gd2O3 particles, their stability in aqueous solutions, and their relaxometric properties were also measured and compared with those from non-PEGylated Gd2O3 nanoparticles.
’ MATERIALS AND METHODS Materials. α-Methoxy-ω-phosphate poly(ethylene glycol) (PEGphosphate, MW 5000 g/mol, Chemicell, Germany) was used to treat both Gd2O3 thin films and nanoparticles. Poly(ethylene glycol) silane (mPEG-silane, MW 5000 g/mol, Laysan, AL, USA) and poly(ethylene glycol) bis(carboxymethyl) ether (PEG diacid, Mn ∼ 600 g/mol, Aldrich, ON, Canada) were used for PEG-grafting comparative assessment on Gd2O3 thin films (Supporting Information). Nanoparticle Synthesis. Ultrasmall gadolinium oxide (US-Gd2O3) nanoparticles showing a fine and narrow particle size distribution were prepared by the polyol method. 23,42 The synthesis was reported elsewhere.12,18 Briefly, Gd(NO3)3 3 6H2O (99.99%, Aldrich, ON, Canada) was dissolved in diethylene glycol (DEG, g99.0%, Fluka, ON, Canada). Then, a small amount of hydrolysis agent (NaOH, Sigma-Aldrich, ON, Canada) was added. After different temperature steps,18 the final product was cooled, centrifuge-filtered (Sartorius Stedim Biotech, Germany, pore size 0.2 μm, 1500 G for 30 min), and dialyzed in ultrapure water (18 MΩ 3 cm) or in ethanol (Commercial Alcohols, ON, Canada) for 24 h to eliminate free Gd3+ ions and excess DEG. A membrane pore size of 1000 MW (Spectra/Por no. 6, Rancho Dominguez, CA, USA) was used, the solvent was changed five times, and the sample-to-volume ratio was kept to about 1:1000. After dialysis, the samples were transferred in 15 mL tubes and the volume was adjusted to 6 mL ([Gd] ≈ 10 mM). The products were then sonicated for several hours to disperse the particles, followed by centrifugation (1000 G for 15 min). Gadolinium Oxide Thin Film Preparation. To demonstrate the reactivity of phosphate molecules with the rare-earth oxide, Gd2O3 was deposited on flat silicon wafers. In brief, double-side polished silicon (100) wafers (University Wafer, MA, USA, thickness: 500550 μm) were first washed in TL2 solution, a mixture of ultrapure water, 30% hydrogen peroxide (H2O2, Fluka, ON, Canada) and concentrated hydrochloric acid (Fisher, Canada) (6:1:1), at 80 °C for 10 min. The wafers were removed from the solution, thoroughly rinsed with ultrapure water and anhydrous ethanol, and dried under a stream of medical grade air. Then, the substrates were cleaned in TL1 solution, a mixture of ultrapure water, 30% H2O2 and 25% ammonia (Fisher, IL, USA) (5:1:1), at 80 °C for 10 min. The wafers were removed from the solution, thoroughly rinsed with ultrapure water and anhydrous ethanol, quickly dried under medical air, and stored in argon. The cleanliness of the substrates was confirmed by X-ray photoelectron spectroscopy (XPS). Then, a wafer was placed in a vacuum system (∼5 106 Torr, room temperature) and gadolinium(III) oxide pellets (99.9% purity, Alfa Aesar, MA, USA) were put in a copper crucible directly facing the wafer. An electron beam was focused 15 min onto the oxide pellets to produce a thin layer of gadolinium oxide on the wafer (Figure S1 Supporting Information). The flatness of the Gd2O3 deposits was assessed by 775
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using a Nicolet Magna 550 Fourier transform infrared spectrometer (Thermo-Nicolet, WI, USA) equipped with a germanium-coated KBr beamsplitter and a DTGS/KBr detector. The pure polymer (PEGphosphate) as well as aqueous suspensions of DEG-Gd2O3 and PEGphosphate-Gd2O3 were directly deposited on the Si crystal. Spectra were recorded in absorbance mode and 250 scans were recorded with a spectral resolution of 4 cm1. The contribution of water was subtracted from each spectrum. Nanoparticle Characterization. Particle Size Studies. The hydrodynamic diameter of as-dialyzed aqueous suspensions of DEGGd2O3 and PEG-phosphate-Gd2O3 was measured by dynamic light scattering (DLS, Malvern Zetasizer, 173°). The analyses were performed at 25 °C. The viscosity and refractive index of water were fixed at 0.8872 cP and 1.33. A refractive index of 1.59 was considered for Gd2O3. Five measurements were completed for each sample. Relaxometric Performance and MRI Signal Enhancement. 400 μL aliquots of as-dialyzed and diluted aqueous suspensions of DEG-Gd2O3 and PEG-phosphate-Gd2O3 were distributed in 6.0 mm NMR tubes. Longitudinal and transversal relaxation times (T1 and T2) were measured with a dedicated TD-NMR relaxometer (Bruker Minispec 60 mq, 60 MHz, 20 °C). The relaxation rates (1/T1 and 1/T2) were then plotted against Gd concentration values, and relaxivities (r1 and r2) were calculated from the slope of the graphs. To quantify the amount of Gd in each suspension, Gd2O3 samples were digested at high temperature in trace metal-grade nitric acid (Fisher, Canada) and 30% H2O2, and analyzed by inductively coupled plasma mass spectrometry (ICP-MS, Perkin-Elmer Elan 6000). The particle suspensions were also imaged with a 1 T small-animal MRI system (M2M, Aspect Imaging, Israel) using T1-weighted spinecho sequences (repetition time, 635 ms; echo time, 10 ms; dwell time, 20; matrix, 248 250; slice thickness, 0.9 mm; interslice, 0.1 mm; field of view, 90 mm; 4 excitations; 25 °C). Leaching Assay. Samples (0.5 mL) of 48 h-dialyzed PEG-phosphateGd2O3 suspensions were dialyzed for 7 days more against water (membrane pore size of 1000 MW, Spectra/Por no. 6). The sampleto-volume ratio was kept to 1:1000 and the water was refreshed every day. Samples of the dialysis water (2 mL) were taken daily and analyzed by ICP-MS to assess the leaching of rare-earth ions from the PEGphosphate-Gd2O3 suspensions.
scanning electron microscopy (SEM, JSM-840A, Jeol; 15 kV, secondary electron imaging) and atomic force microscopy (AFM, Dimension 3100, Veeco, USA) in tapping mode (etched silicon tip, OTESPA, tip radius 10 nm, Figure S3; Supporting Information) of the thin films were confirmed similar to the crystallinity and diameter of the USGd2O3 particles, respectively. Finally, XPS analyses (Figure S3c; Supporting Information) revealed that the surfaces were highly hydroxylated. In fact, many studies have demonstrated the necessity of hydroxyl groups for the grafting of carboxyl, silane, and phosphate groups at the surface of metal oxides, leading to the establishment of strong and stable bonds.34,4345 From all the aforementioned evidence, the thin films were considered similar to the nanoparticles, and therefore were used for comparative surface functionalization studies. 776
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Table 1. XPS Survey Data of Gd2O3 Thin Films Following Their Immersion in Different Solutions for 24 h atomic concentrations (%) sample
solvent
unmodified Gd2O3/Si (control)
water
C1s
O1s
P2s
Si2p
Gd4d
41.3 ( 0.6
41.0 ( 0.5
-
4.9 ( 0.2
12.9 ( 0.1
4.5% CO ethanol
SiO2/Si
41.5 ( 1.0
43.4 ( 0.5
-
-
15.1 ( 0.5
8.0% CO PEG-phosphatea on Gd2O3/Si
water pH 7.0
28.7 ( 1.3
53.0 ( 1.1
7.3 ( 0.8
-
11.0 ( 0.5
ethanol
3.6% CO 20.3 ( 2.0
58.6 ( 1.1
5.1 ( 0.5
-
16.0 ( 0.8
2.0% CO a
Theoretical composition and ratio: CH3-(OCH2CH2)n-OPO3Na2: 65.8% C (65.8% CO and COP), 33.9% O, and 0.3% P; CO/P: 221.
Figure 1. Gd4d HRXPS spectra of Gd2O3 thin films immersed in PEG-phosphate ethanol solution (a); Gd4d of thin films immersed in ethanol only (b); P2s HRXPS spectra of PEG-phosphate-treated Gd2O3 thin films in ethanol (c); P2s of pure PEG-phosphate (d).
PEG-Phosphate Grafting on Gd2O3 Thin Films. The grafting efficiency of PEG-phosphate on hydroxylated Gd2O3 thin films, performed both in water and in ethanol, was clearly evidenced by the strong presence of phosphorus on the surfaces (7.3% and 5.1%, respectively). There was also a significant increase of oxygen (Table 1). However, we noticed a deficit of carbon contribution on these surfaces (Table 1). To discriminate between carbon from organic contamination and that from PEG molecules, comprehensive high-resolution XPS analyses were performed. Indeed, on C1s spectra (Figure S4a; Supporting Information), the binding energies were assigned as follows: 285 eV to CC and CH bonds from organic contamination, 286.5 eV to CO, and 289 eV to COO groups.46,47 The fourth contribution at 291 eV was attributed to the Gd4p1/2 band. The main carbon contribution in the C1s HRXPS spectra of the PEGphosphate-treated Gd2O3 thin films appeared at 285.0 eV (organic contamination, CC and CH, Figure S4b and f; Supporting Information). Moreover, the total CO percentages and the CO/P ratios were very low (2.8% CO instead of 65.8% and CO/P = 0.44 instead of 221). Therefore, these results suggest the detachment of PEG chains from the phosphate head upon reaction with Gd2O3. Although the stability of such phosphate ester bonds has been scarcely documented in the past, evidence can be found of cleavage in either acidic or alkaline media.48 Further studies in weakly alkaline medium at pH 89 provided evidence of an
acceleration of the breakdown of the phosphate ester bond in presence of lanthanum salts.48 In fact, the lanthanide ions have attracted attention in biochemical studies over the last years, owing to their excellent catalytic effects in phosphate ester hydrolysis.4955 It has been suggested that the hydrolysis reaction is promoted by cooperative interactions between hydroxide groups and lanthanide ions.52,53,55 Such catalytic activity is in agreement with the very low C and CO percentages, as well as reduced CO/P ratios measured in this study by XPS on PEGphosphate-treated Gd2O3 thin films. Reactivity of Functional Groups with Gd2O3 Thin Films. As a comparative assessment with PEG-phosphate, Gd2O3 thin films were also incubated with PEG-silane and PEG diacid, followed by XPS analyses (Table S1, Figures S4 and S5; Supporting Information). In order to compare the affinity of these three head groups for Gd2O3, Gd4d HRXPS spectra were recorded (Figure 1a,b and Figure S5; Supporting Information). However, the Gd4d peak is complex: it consists of a multiplet splitting of the 4d hole with the 4f7 valence electrons, forming 9D and 7D final ionic states. Therefore, the calculation of elemental ratios based on the deconvolution of this peak is hazardous.56,57 However, a positive peak shift was observed in Gd4d HR spectra, suggesting a charge transfer from the Gd present in Gd2O3 thin films to a binding element. As shown in Figure 1, the PEGphosphate-treated Gd2O3 surfaces (Figure 1a) showed a significant shift (∼0.5 eV) toward higher binding energies compared to 777
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the control (Figure 1b), and PEG-silane and PEG diacid-treated Gd2O3 surfaces (Figure S5a and b; Supporting Information), indicating the strong affinity of the phosphate group for Gd2O3. A chemical shift of ∼0.4 eV was also evidenced in P2s HRXPS spectra between the phosphate group present on the unreacted PEG molecule and the PEG-phosphate grafted on Gd2O3 thin films (190.9 to 191.3 eV, respectively; Figure 1c and d). This observation is in accordance with the reduction of the net charge of the phosphate group, which leads to a peak shift toward higher binding energies. Similar shifts were observed for samples reacted in ethanol and in water (data not shown). Phosphate Binding on Gd2O3 Surfaces. Gd4d and P2s peak shifts measured in HRXPS studies are characteristic of a strong interaction between the metal oxide and the highly electronegative phosphate group. Gresch et al.58 measured P2s binding energies (EB) of 190.8 and 191.8 eV in the phosphate structures Na4P2O7 (PO3.52) and NaPO3 (PO3), respectively. According to these values, the type of interaction between the phosphate group and Gd2O3 can be deduced. In the present study, after PEG-phosphate grafting on Gd2O3 thin films, the binding energy
of P2s peaks appeared shifted to 191.3 eV. This is an intermediate value between experimental EB values attributed to PO3.52 and PO3 groups. Since evidence were found in this study of the detachment of PEG chains from the phosphate head upon reaction with Gd2O3 thin films, the remaining charges (2 and 1) on the reacted phosphate groups can only be explained by a combination of monodentate and bidentate bonds (Scheme 1). PEG-Phosphate Grafting on Gd2O3 Nanoparticles. The detachment of PEG from the phosphate head was not clearly observed with PEG-phosphate reacted US-Gd2O3 nanoparticles. Therefore, the occurrence of a catalytic reaction between Gd2O3 and phosphate ester bonds in PEG-phosphate molecules appears much more limited than on Gd2O3 thin films. In fact, both XPS and FTIR analyses performed with thoroughly dialyzed PEGphosphate-treated Gd2O3 nanoparticles revealed (1) the strong contribution of CO links and (2) the absence of other contaminating CO contributions (such as from diethylene glycol). The precursor nanoparticle suspension (DEG-Gd2O3) was first dialyzed against nanopure water or ethanol prior to the grafting with PEG-phosphate. These suspensions were used as controls in both XPS and FTIR studies. The dialysis step eliminates Gd3+ ions from the precursor solution; however, a DEG coating is preserved at the surface of particles, preventing aggregation. XPS analyses clearly demonstrated the efficiency of dialysis against water because low C, CO (DEG), and N (from the Gd nitrate precursor salt) contents were detected after the purification (Table 2). After the reaction with PEG-phosphate (in water or in ethanol), the particles were thoroughly dialyzed (48 h) against nanopure
Scheme 1. Illustration of the Most Probable Reaction Mechanism between Phosphate Groups and Gd2O3 Thin Films
Table 2. XPS Survey Data of DEG-Gd2O3 and PEG-Phosphate-Gd2O3 Nanoparticles atomic concentrations (%) sample DEG-Gd2O3a PEG-phosphate- Gd2O3b
conditions of dialysis or reaction
C1s
O1s
Dialysis in water
31.6 ( 1.3
52.2 ( 1.2
Reaction in water
5% CO 65.3 ( 0.7
33.9 ( 0.8 32.2 ( 1.3
P2s
Gd4d
N1s
15.6 ( 0.1
∼0.6
0.4 ( 0.1
0.3 ( 0.1
-
0.8 ( 0.1
0.9 ( 0.1
-
-
52% CO PEG-phosphate- Gd2O3
Reaction in ethanol
66.1 ( 1.4 49% CO
a
Theoretical composition and ratio: HO-(CH2)2-O-(CH2)2-OH: 57.1% C (57.1% CO) and 42.9% O. b Theoretical composition and ratio: CH3-(OCH2CH2)n-OPO3Na2: 65.8% C (65.8% CO and COP), 33.9% O and 0.3% P; CO/P: 221.
Figure 2. Gd4d HRXPS spectra of PEG-phosphate-Gd2O3 nanoparticles after reaction in ethanol (a); Gd4d of DEG-Gd2O3 (before reaction in ethanol, b); P2s of PEG-phosphate-Gd2O3 particles after reaction in ethanol (c); P2s of pure PEG-phosphate (d). 778
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water to eliminate excess PEG. By opposition to the results obtained by using Gd2O3 thin films (Table 1 and Figure S4; Supporting Information), the presence of PEG-phosphate at the surface of the particles was unambiguously confirmed by XPS (Table 2). In both solvents, the contribution of CO (PEG) increased by 1 order of magnitude, from 5% (DEG-Gd2O3) to 52% and 49% (PEG-phosphate-Gd2O3). This is much closer to the expected theoretical value of 65.8% CO. Also, CO/P ratios of 60 and higher indicate the strong presence of PEGphosphate. The CO contribution and CO/P ratio are lower than expected, but the values are by no means as low as what were observed on Gd2O3 thin films. The strength of the interaction between the phosphate head and Gd2O3 was studied by Gd4d and P2s HRXPS (Figure 2). As for the Gd2O3 thin films (Figure 1), a shift toward higher binding energies was evidenced for both Gd4d (ΔE ≈ 0.6 eV) and P2s (ΔE ≈ 0.3 eV) peaks (Figure 2a,b and c,d). Similar shifts were observed after reaction in water as the reaction solvent (data not shown). Due to the chemical similarity of DEG and PEG, it was not possible to differentiate between these two molecules based on
C1s and O1s HRXPS spectra only. In order to confirm the ligand reaction efficiency, resulting in the elimination of DEG, attenuated total reflectanceFourier transform infrared spectroscopy (ATR-FTIR) was used. Infrared spectra acquired with PEGphosphate-Gd2O3 (Figure 3c) were similar to that of pure PEGphosphate (Figure 3b). All spectra showed a high-intensity band near 1080 cm1, attributable to the asymmetric elongation of the OPO32 group and to the elongation of the COC bonds (10751080 cm1).59 The spectral signature of DEG-Gd2O3 (11501050 cm1, Figure 3a) was not evidenced in any of the infrared spectra acquired with PEG-phosphate-Gd2O3 (Figure 3c), confirming the efficient exchange of DEG by PEG-phosphate at the surface of the particles. Similar results were obtained using water as the reaction solvent (data not shown). PEG-Phosphate Binding on Gd2O3 Nanoparticles. O1s HRXPS spectra were used to identify the type of coordination of the phosphate head groups with the metal oxide, based on a previously reported methodology.34 The binding energies of O1s HRXPS peaks were assigned as follows: 531.2 eV to GdOP and PdO bonds, 532.9 eV to CO bonds in the PEG structure, and 534.2 eV to POC and POH (data not shown).34,46 Consequently, for the monodentate coordination, the ratio O1s (531.2 eV):O1s (534.2 eV) is 2:2, whereas for the bidentate and the tridentate mode, this ratio is 3:1. Once covered with PEGphosphate (reaction in ethanol), the nanoparticles exhibited a ratio of ∼1.6 (Table 3). Moreover, the PdO band was consistently detected in FTIR (Figure 3c). These results indicate that phosphate head groups are not coordinated in a tridendate manner, but rather according to monodendate and bidendate bonds (Scheme 2). Size and Relaxometric Properties of US-Gd2O3 Nanoparticles. Table 4 shows the size and the relaxometric properties of aqueous suspensions of DEG-Gd2O3 and PEG-phosphateGd2O3 nanoparticles. The hydrodynamic diameter (Table 4 and Figure 4) increased after reaction with PEG-phosphate, in particular when using water as the reaction solvent. Increased hydrodynamic diameters can be explained by a possible reaction of phosphate heads with more than one Gd2O3 nanoparticle, and by the resulting entanglement of the PEG chains. The longScheme 2. Illustration of the Most Probable Reaction Mechanism between PEG-Phosphate and Gd2O3 Nanoparticles
Figure 3. Infrared spectra of aqueous suspensions of DEG-Gd2O3 particles (a), pure PEG-phosphate (b), and PEG-phosphate-Gd2O3 nanoparticles after reaction in ethanol (c).
Table 3. O1s Binding Energies and Ratios for Pure PEG-Phosphate and for PEG-Phosphate-Gd2O3 Particles Atomic ratio of O1s(1)/O1s(2)
a
sample
O1s (1) (eV)
O1s (2) (eV)
O1s (3) (eV)
experimental
theoretical
PEG-phosphate PEG-phosphate-Gd2O3 (reaction in ethanol)
531.2 531.2
533.9 534.2
532.7 532.9
0.54 ( 0.15 1.60 ( 0.59
0.33 1.0a or 3.0b
Monodentate coordination. b Bidentate or tridentate coordination. 779
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itudinal and transversal relaxivities (r1 and r2) are significantly higher than those of DEG-Gd2O3 nanoparticles and Gd-DTPA, provided here for comparison (Table 4 and Figure 5a). In the case of DEG-Gd2O3, the presence of a thick and dense DEG layer at the surface of the particles affects the accessibility of 1H protons to paramagnetic atoms, resulting in lower relaxivities.12 Elimination of the DEG coating by reaction with phosphate strongly enhances the relaxometric performance of the contrast agent, whereas hydrophilic and intricate PEG chains facilitate the accessibility of water to the paramagnetic surface atoms. The better longitudinal relaxivity can also be explained by the longer water residency time in the inner sphere of PEG-coated Gd2O3, as well as by the enhanced hydrodynamic diameter of the paramagnetic nanoparticle, which affects the tumbling rate of the contrast agent. The r2/r1 ratios remain close to unity after reaction with PEG-phosphate, establishing this product among efficient “positive” MRI contrast agents. According to Table 4, smaller hydrodynamic diameters, higher longitudinal relaxivities, and lower relaxometric ratios were achieved by performing the ligand exchange reaction in ethanol. The gadolinium recovery is also about three times higher in ethanol than in water (data not shown); this was also noticed in the XPS analyses (Table 2). After phosphate grafting in ethanol and dialysis in nanopure water, PEG-phosphate-Gd2O3 particles were also imaged with MRI (1 T) using standard T1-weighted spinecho sequences. DEG-Gd2O3 nanoparticles were imaged as controls. Figure 5b,c represents MRI longitudinal cross sections of contrast agent tubes. For the same gadolinium concentration, the MRI signal of PEG-phosphate-Gd2O3 suspension was higher than that of DEG-Gd2O3 suspension, confirming the superior signal enhancement performance of the PEG-treated particles to be used as a “positive” contrast agent.
Finally, samples containing 0.8 mM PEG-phosphate-Gd2O3 were dialyzed for 7 days more against nanopure water, which is a severe purification protocol. Samples of the dialysis water (2 mL) were taken daily and analyzed by ICP-MS. Each one of the samples collected from the dialysis water was under the ppb scale. Therefore, the daily leaching of Gd3+ ions (
