Bioconjugate Chem. 2010, 21, 505–512
505
Bioinspired Synthesis and Characterization of Gadolinium-Labeled Magnetite Nanoparticles for Dual Contrast T1- and T2-Weighted Magnetic Resonance Imaging Ki Hyun Bae,† Young Beom Kim,‡ Yuhan Lee,† JinYoung Hwang,‡ HyunWook Park,‡ and Tae Gwan Park*,† Department of Biological Sciences and Graduate School of Nanoscience and Technology, and Department of Electrical Engineering, Korea Advanced Institute of Science and Technology, Daejeon 305-701, South Korea. Received September 23, 2009; Revised Manuscript Received November 24, 2009
Gadolinium-labeled magnetite nanoparticles (GMNPs) were synthesized via a bioinspired manner to use as dual contrast agents for T1- and T2-weighted magnetic resonance imaging. A mussel-derived adhesive moiety, 3,4dihydroxy-L-phenylalanine (DOPA), was utilized as a robust anchor to form a mixed layer of poly(ethylene glycol) (PEG) chains and dopamine molecules on the surface of iron oxide nanoparticles. Gadolinium ions were subsequently complexed at the distal end of the dopamine molecules that were prefunctionalized with a chelating ligand for gadolinium. The resultant GMNPs exhibited high dispersion stability in aqueous solution. Crystal structure and superparamagnetic properties of magnetite nanocrystals were also maintained after the complexation of gadolinium. The potential of GMNPs as dual contrast agents for T1 and T2-weighted magnetic resonance imaging was demonstrated by conducting in vitro and in vivo imaging and relaxivity measurements.
INTRODUCTION Over the past decade, a variety of inorganic nanomaterials, such as gold nanoparticles, iron oxide nanocrystals, and quantum dots, have drawn significant attention because of their unique and controllable optical, magnetic, and electronic properties (1-3). Among them, superparamagnetic magnetite nanocrystals have been utilized as useful platform materials for diverse biomedical applications including magnetic drug targeting, biomolecule separation, and hyperthermal cancer therapy (3-5). Although uniform and highly crystalline magnetite nanoparticles have been recently developed by thermal decomposition processes, they are poorly dispersed in aqueous solution due to their hydrophobic surface coatings (6, 7). For biomedical applications, the surface of the magnetite nanoparticles needs to be suitably engineered to acquire improved colloidal stability in physiological media, biocompatibility, drug encapsulation ability, and specific targetability to ensure desirable interactions with cells or tissues. To this end, magnetite nanocrystals have been coated with hydrophilic polymers (e.g., dextran and polyvinylpyrrolidone) or inorganic shells (e.g., gold and silica) (8, 9) and encapsulated into polymeric micelles or nanoparticles (10, 11). Magnetic resonance imaging (MRI) has been recognized as a powerful noninvasive diagnostic technique to visualize the fine structure of a human body in a high spatial resolution (12). The use of MR contrast agents enables achievement of clear images for accurate diagnosis, by exerting an influence on the longitudinal (T1) or transverse (T2) relaxation time of the surrounding tissue. For example, paramagnetic complexes containing gadolinium (Gd3+) or manganese (Mn2+) ions induce the local relaxation change of the nearby water protons and mainly reduce T1, providing positive contrast (bright signal) on T1-weighted MR image (13). On the other hand, superpara* Corresponding author. Tel: +82-42-350-2621; Fax: +82-42-3502610; E-mail address:
[email protected] (T.G. Park). † Department of Biological Sciences and Graduate School of Nanoscience and Technology. ‡ Department of Electrical Engineering.
magnetic magnetite nanoparticles have been widely utilized as ultrasensitive negative contrast agents for stem cell tracking and early detection of cancers due to their strong T2 shortening effect (14-16). Nevertheless, their clinical applications are quite limited because of the negative contrast effect and magnetic susceptibility artifacts. Since the magnetite nanoparticles represent dark areas in MR images, their negative contrast is often confused with a low-level MR signal arising from adjacent tissues such as bone or vasculature (17, 18). Furthermore, susceptibility artifacts can occur as a result of the steep change of local magnetic field around iron oxide nanoparticles, leading to the locally distorted anatomy of surrounding tissue in MR image (19). In the present study, we synthesize and characterize gadolinium-labeled magnetite nanoparticles (GMNPs) using 3,4dihydroxy-L-phenylalanine (DOPA) as a bioinspired adhesive, and demonstrate their utility as dual contrast agents for T1- and T2-weighted MRI. DOPA is a modified amino acid abundantly found within adhesive proteins, which are secreted by marine mussels (Mytilus edulis) (20). DOPA is known to play an important role in the strong attachment of marine mussels on a wide range of organic and inorganic substrates (21, 22). Recently, it was discovered that DOPA and its analogue dopamine can be effectively utilized for anchoring small organic molecules or polymers onto the surface of metal oxides such as TiO2, Al2O3, and Fe3O4 (23-25). Taking advantage of its strong and versatile binding ability, magnetite nanoparticles were surface-coated by a mixed layer of DOPA-terminated poly(ethylene glycol) chains and dopamine molecules, resulting in high water dispersibility and good biocompatibility. The surface of magnetite nanoparticles was further modified with multiple chelating ligands for complexing gadolinium ions. The resultant GMNPs are expected to induce a simultaneous positive and negative contrast enhancement according to the MR imaging protocols employed, making them useful as dual-functional MRI contrast agents (26). Depending on the tissue site of interest, GMNPs can be selectively visualized by T1- or T2-weighted MRI in order to achieve complementary information that cannot be obtained by the use of only one type of contrast agent, thereby
10.1021/bc900424u 2010 American Chemical Society Published on Web 02/18/2010
506 Bioconjugate Chem., Vol. 21, No. 3, 2010
leading to more precise diagnosis. We examined the stability, morphology, and crystalline structures of GMNPs by using transmission electron microscopy (TEM) and X-ray diffraction (XRD) analysis. The capability of GMNPs to generate dual contrast for T1- and T2-weighted MRI was also validated by performing in vitro and in vivo imaging and relaxivity measurements.
EXPERIMENTAL PROCEDURES Materials. Methoxy-poly(ethylene glycol)-succinic acid (mPEG-SA, Mw ) 2000) was obtained from Sunbio Co. (Anyang, Korea). Dopamine hydrochloride (3-hydroxytyramine hydrochloride), 3,4-dihydroxy-L-phenylalanine (DOPA), N,N′dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), and gadolinium(III) chloride hexahydrate (GdCl3 · 6H2O) were purchased from Sigma Chemical Co. (St. Louis, MO). 2-(pIsothiocyanatobenzyl)diethylene triamine pentaacetic acid (pSCN-benzyl-DTPA) was purchased from Macrocyclics (Dallas, TX). Ferumoxides suspension (Feridex) and gadopentetate dimeglumine (Magnevist, Gd-DTPA) were supplied by Berlex Laboratories (Wayne, NJ). Extracel hydrogel kit obtained from Glycosan BioSystems, Inc. (Salt Lake City, UT) was used according to the manufacturer’s instruction. All other chemicals were of analytical grade. Synthesis of DOPA-Conjugated Poly(ethylene glycol) (mPEG-DOPA). mPEG-DOPA was synthesized by conjugating an amino group of DOPA with a carboxylic acid group of mPEG-SA using DCC/NHS succinimidyl carbonate chemistry (23). Briefly, mPEG-SA (2116 mg) and DCC (654 mg) were first dissolved in 20 mL of distilled dichloromethane. Then, NHS (183 mg) dissolved in 5 mL of acetone was slowly added to the above solution. After 12 h of reaction under nitrogen atmosphere, the solution was precipitated into excess of cold ethyl ether and then evaporated under vacuum for 2 day. The activated PEG (1120 mg) dissolved in 10 mL of methanol was added to 30 mL of 0.1 M Na2B4O7 solution (pH ) 9.32) containing 313.5 mg of DOPA. After reaction for 12 h, the solution was dialyzed against acidified deionized water (pH 4) by a Spectra/Por dialysis membrane with a MW cutoff of 1 kDa and then lyophilized. The chemical structure of the product was confirmed by a Bruker DRX 400 1H NMR spectrometer operating at 400 MHz. The degree of substitution was estimated to be ca. 80% by comparing the relative peak area ratio between two methylene protons from an ethylene oxide group (3.6 ppm) and three protons on a DOPA phenyl ring (6.5-6.9 ppm). Synthesis of mPEG-DOPA/Dopamine Stabilized Magnetite Nanoparticles. Oleic acid-coated magnetite nanoparticles with an average diameter of 17 nm were produced by thermal decomposition of Fe-oleate complex (6, 11). Oleic acidcoated magnetite nanoparticles (5 mg) dispersed in 3 mL of dichloromethane were mixed with 0.5 mL of dimethylformamide containing mPEG-DOPA (13.2 mg) and dopamine hydrochloride (3.3 mg). The solution was added to 10 mL of deionized water, and then sonicated for 2 min by using a Branson sonifier 450 (20 kHz, output control 3, duty cycle 40%). The oil-inwater emulsion was subjected to rotary evaporation at 40 °C for 10 min to remove residual solvent under reduced pressure. The product was purified by dialysis against deionized water for 1 day (MW cutoff of 12 kDa) and then lyophilized. Synthesis of Gadolinium-Labeled Magnetite Nanoparticles (GMNPs). Five milligrams of the dried mPEGDOPA/dopamine stabilized magnetite nanoparticles were dispersed in 5 mL of dimethylformamide, and then added in a dropwise manner to a stirred solution of dimethylformamide containing 22.6 mg of p-SCN-benzyl-DTPA. After reaction for 16 h, the excess of unreacted DTPA was removed by dialysis against deionized water for 1 day (MW cutoff of 12 kDa). To
Bae et al.
the colloidal solution of DTPA-grafted magnetite nanoparticles, 25.8 mg of GdCl3 · 6H2O was added. After incubation for 24 h, the resultant GMNPs were purified by extensive dialysis against deionized water for 2 days (MW cutoff of 12 kDa), and then stored at 4 °C until use. Particle Characterization. The core size and dispersion stability of magnetite nanoparticles were investigated by transmission electron microscopy (TEM). The solution of nanoparticles was air-dried on a 300 mesh Formvar/carboncoated copper grid, and then observed by a Philips F20 transmission electron microscope. The average particle size was determined by measuring the diameters of more than 50 particles in the TEM image. The hydrodynamic diameter of the magnetite nanoparticles was analyzed by using a dynamic light scattering instrument (Zeta-Plus, Brookhaven, NY) equipped with a He-Ne laser at a wavelength of 632 nm. The ζ potential values of the particles dispersed in 0.1 M phosphate-buffered saline solution (PBS, pH 7.4) were also measured in triplicate at a concentration of 0.5 mg/mL at 37 °C. Fourier transform infrared (FT-IR) spectra were recorded in the 500-4000 cm-1 region using an EQUINOX55 spectrometer (Bruker, Germany). Lyophilized samples were mixed with dried potassium bromide (KBr) and then compressed to prepare a pellet. For thermogravimetric analysis, dried samples were placed in aluminum sample cells, and a thermogram for each sample was obtained using a TG 209 F3 Tarsus thermo-microbalance (NETZSCH Instruments, Inc., Burlington, MA). Samples were heated at a rate of 10 °C/min up to 700 °C under a flow of nitrogen gas. Elemental analysis data were obtained using a FlashEA 1112 element analyzer (Thermo Finnigan, Italia). The metal concentration of the nanoparticles was quantified using inductively coupled plasma absorption emission spectroscopy (ICP-AES). X-ray powder diffraction profiles were measured using Rigaku D/max-IIIC diffractometer with CuKR radiation (40 kV, 80 mA). The field-dependent magnetization curves of samples were measured at room temperature with a vibrating sample magnetometer (VSM). All the characterizations were performed within 3 months after sample preparation. T1- and T2-Weighted MRI Experiments. To verify contrast characteristics of the nanoparticles as MRI contrast agents, longitudinal (1/T1) and transverse (1/T2) relaxation rates were calculated from the magnetic resonance images. The synthesized GMNPs were compared with aqueous GdCl3 solution and Magnevist (Gd-DTPA) at various gadolinium concentration (50, 100, 150, 200, and 250 µM), as well as with mPEG-DOPA/ dopamine stabilized magnetite nanoparticles and Feridex at equivalent iron concentration. All samples were diluted in deionized water and compared to pure water as a control. In addition, normal saline (0.9% w/v NaCl, pH 7.4) solution was also used to simulate the relaxation properties of the samples in the physiological environment. All MRI measurements were performed on a whole body scanner (ISOL Technology, South Korea) with a human head coil at a clinically relevant field strength of 3.0 T. Conventional spin-echo pulse sequence was used for both T1 and T2 measurements with a single slice of 4 mm thickness, a flip angle of 90°, the number of signal averages of 2, 128 × 128 mm field of view, and 128 × 128 matrices. T1 relaxation measurements were acquired with repetition time (TR) of 100, 300, 600, 1000, 2000, and 3000 ms with an echo time (TE) of 12 ms, and T2 relaxation measurements with TE of 12, 30, 60, 100, 200, and 300 ms with a TR of 3000 ms. The longitudinal (T1) and transverse (T2) relaxation time were obtained by calculating the mean signal intensity of each sample individually in the respective region of interest of MR images. The specific relaxivities (r1 and r1) of the samples were determined by measuring the relaxation rates based on the metal concentration (Supporting Information).
Bioinspired Synthesis of Gd-Labeled Magnetite NPs
Bioconjugate Chem., Vol. 21, No. 3, 2010 507
Figure 1. Schematic illustration for the synthesis of gadolinium-labeled magnetite nanoparticles (GMNPs) as dual contrast agents for T1- and T2-weighted magnetic resonance imaging.
To evaluate the dual contrast ability of GMNPs in a live animal model, in vivo MRI experiments were performed using BALB/c nude mice. All animal experiments were conducted based on the guidelines provided by Korea Advanced Institute of Science and Technology. GMNPs were suspended in Extracel hydrogel solution with a gadolinium concentration of 0.379 mM in order to prevent leakage from the injection site. A 200 µL aliquot of the suspension was injected subcutaneously into dorsal flanks of nude mice. For comparison, mice were also injected with Magnevist or Feridex at equivalent gadolinium or iron concentration. As a control, only the hydrogel solution was applied for injection. Two mice were included in each group. In vivo MRI was obtained from the same scanner, which was used in in vitro imaging, using a customized quadrature radio frequency (RF) coil with a diameter of 9 cm. T1-weighted MR images were acquired using a conventional spin-echo sequence using the following parameters: TR/TE ) 600/17 ms, 256 × 256 matrices, 100 × 100 mm field of view, a slice thickness of 3 mm, and the number of signal averages of 1. For T2-weighted MR images, a fast spin-echo sequence was used to reduce acquisition time with the same parameters of T1-weighted MRI, except for TR/TE ) 3500/114 ms and the number of signal averages of 2. Each in vivo scan was completed within an hour.
RESULTS AND DISCUSSION Figure 1 illustrates a synthetic scheme of gadolinium-labeled magnetite nanoparticles (GMNPs). Oleic acid-coated magnetite nanoparticles with a uniform and highly crystalline structure were first synthesized by the thermal decomposition of iron oleate complexes, as previously reported (6, 11). These nanoparticles can only be dispersed in an organic solvent because an oleic acid layer ionically adsorbed on the surface acts as a hydrophobic shell around the iron oxide core (7). However, for biomedical applications, it is necessary to make them hydrophilic and well-dispersed in aqueous solution. To prepare waterdispersible magnetite nanoparticles, we utilized DOPA as a bioinspired adhesive to immobilize hydrophilic polymers on the surface of ion oxide structures. Previously, we surface-coated iron oxide nanoparticles with hyaluronic acid (HA) by using dopamine-grafted HA. They showed an excellent water dispersibility while maintaining a superparamagnetic property, useful for HA receptor-specific MRI of cancer cells (27). The surface modification process was accomplished by simply incubating oleic acid-coated magnetite nanoparticles with a mixture of DOPA-conjugated poly(ethylene glycol) (mPEGDOPA) and dopamine in an organic solvent. Since the ortho-
Figure 2. TEM images of (A) oleic acid-coated magnetite nanoparticles, (B) mPEG-DOPA/dopamine stabilized magnetite nanoparticles, and (C) GMNPs. The inset photographs show the dispersion of the magnetite nanoparticles in deionized water. (D) Hydrodynamic diameters of GMNPs dispersed in deionized water and in PBS solution.
dihydroxylphenyl (catechol) group of DOPA and its analogue dopamine forms strong coordination bonds with the surface of iron oxides (e.g., γ-Fe2O3, Fe3O4) (25, 28), it was anticipated that the magnetite nanoparticles was decorated with a mixed layer of mPEG chains and dopamine molecules. The resulting magnetite nanoparticles dispersed in an organic phase could be readily dispersed into an aqueous phase after solvent evaporation. For synthesis of GMNPs, these magnetite nanoparticles were further derivatized with diethylene triamine pentaacetic acid (DTPA), a chelating ligand for binding a paramagnetic gadolinium (Gd3+) ion (29). The conjugation reaction of DTPA was carried out through the formation of an isothiourea bond between an isothiocyanate derivative of DTPA (p-SCN-benzyl DTPA) and a primary amine group in the dopamine molecule bound on the nanoparticles. Thereafter, DTPA-grafted magnetite nanoparticles were reacted with excess amount of GdCl3 · 6H2O, resulting in the direct complexation of Gd3+ into the particles by chelation. Once nonchelated Gd3+ was removed by extensive dialysis against deionized water, GMNPs comprising magnetite nanoparticles carrying numerous gadolinium chelates were successfully produced. We investigated the size distribution and dispersion stability of GMNPs in aqueous solution by using transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis. As shown in Figure 2A, oleic acid-coated magnetite nanoparticles were produced with a spherical morphology and an average diameter of 17.2 ( 1.3 nm. The TEM images obviously showed that the core size and shape of magnetite nanoparticles did not significantly change after surface modification with mPEG-DOPA and dopamine, even after the incorporation of paramagnetic gadolinium chelates. It should be noted that the dispersion stability of the oleic acid-coated magnetite nanoparticles was greatly changed after the ligand exchange reaction on the surface from oleic acid to mPEG-DOPA and dopamine (inset photographs). While the oleic acid-coated magnetite nanoparticles were not dispersible in deionized water and finally precipitated out of solution, mPEG-DOPA/dopamine stabilized magnetite nanoparticles and GMNPs exhibited excellent stability
508 Bioconjugate Chem., Vol. 21, No. 3, 2010
in deionized water, forming a transparent brown solution. This dramatic difference was probably attributed to the hydrophilic nature of PEG chains anchored on the surface of the magnetite nanoparticles. It has been shown previously that the immobilization of PEG on various polymeric and inorganic nanoparticles improves their colloidal stability in aqueous physiological environment due to the steric stabilization effect of surfaceexposed PEG chains (16, 23). Additionally, it was likely that the surface coating of dopamine molecules might partially contribute to the improved dispersion stability through the charge stabilization of magnetite nanoparticles (27, 30). As shown in Figure 2B and C, the magnetite nanoparticles were wellseparated from each other without observable aggregation, suggesting that they were effectively stabilized in aqueous solution by the hydrophilic protective shell layer comprising hydrophilic PEG chains and dopamine molecules. Figure 2D presents the hydrodynamic diameters of GMNPs dispersed in deionized water and in PBS solution. The size of GMNPs (73.8 ( 7.6 nm) measured by DLS analysis was larger than the value obtained from TEM, probably due to the swelling of the PEG shell layer on the magnetite nanoparticles in water (28). It was also noteworthy that the hydrodynamic diameters and dispersion stability of GMNPs were not greatly changed after the incubation in PBS solution, suggesting that the adhesive properties of DOPA and dopamine molecules bound on the magnetite nanoparticles were significantly maintained in aqueous physiological environment. Fourier transform infrared (FT-IR) spectroscopy was performed in order to examine the ligand exchange on the surface of magnetite nanoparticles (Figure 3A). The FT-IR spectrum of oleic acid-coated magnetite nanoparticles showed strong absorption bands at 2852 and 2923 cm-1, arising from symmetric and asymmetric C-H stretch in the oleyl chains, respectively. The characteristic bands of oleic acid remarkably decreased after the ligand exchange, whereas new absorption bands centered at 1110 and 1635 cm-1 appeared. Since these absorption bands can be assigned to the C-O-C ether stretch of PEG and CdC vibration of catechol rings, respectively, it was evident that the oleic acid on the surface of magnetite nanoparticles was efficiently replaced by PEG chains and dopamine molecules. In the FT-IR spectrum of DTPA-grafted magnetite nanoparticles, an absorption band associated with the O-H stretch in carboxylic acid groups appeared at 3447 cm-1, accounting for the conjugation of DTPA to the dopamine molecule bound on the nanoparticles. The differential thermograms of oleic acid-coated magnetite nanoparticles indicated that the thermal decomposition of oleic acid took place mainly at 282 and 453 °C (Figure 3B). After the ligand exchange, however, the magnetite nanoparticles exhibited a strong mass loss at 405 °C without any signs of the decomposition of oleic acid. This result revealed that the organic layer was completely removed from the particle surface, and mPEG-DOPA chains were anchored onto them concomitantly. It was also found that the weight loss of DTPA-grafted magnetite nanoparticles occurred over a broad range of temperature around 375 °C, presumably due to the desorption of DTPA molecules from the particles. Taken together, these results demonstrated that PEG chains and DTPA molecules were stably immobilized onto the surface of the magnetite nanoparticles via a catechol-metal coordination interaction. The surface modification processes were further investigated by examining surface charge properties of magnetite nanoparticles. As shown in Figure 4, magnetite nanoparticles coated with only mPEG-DOPA showed a slightly negative ζ potential of -2.9 ( 2.2 mV, which was in agreement with the previous finding (30). In contrast, mPEG-DOPA/dopamine stabilized magnetite nanoparticles had a positively charged surface (10.5
Bae et al.
Figure 3. (A) FT-IR spectra and (B) differential thermograms of (i) oleic acid-coated magnetite nanoparticles, (ii) mPEG-DOPA/dopamine stabilized magnetite nanoparticles, and (iii) DTPA-grafted magnetite nanoparticles.
Figure 4. The ζ potential values of (A) mPEG-DOPA/dopamine stabilized magnetite nanoparticles, (B) magnetite nanoparticles coated with mPEG-DOPA, (C) GMNPs, and (D) DTPA-grafted magnetite nanoparticles.
( 2.8 mV) owing to the presence of dopamine molecules bearing free amine groups. It was also found that DTPA-grafted magnetite nanoparticles displayed a negative ζ potential of -21.3 ( 4.9 mV, which can be explained by the surface coverage of the particles with negatively charged DTPA
Bioinspired Synthesis of Gd-Labeled Magnetite NPs
Figure 5. (A) X-ray diffraction pattern of GMNPs. (B) Field-dependent magnetization curve of mPEG-DOPA/dopamine stabilized magnetite nanoparticles (black dots) and GMNPs (hollow dots) at room temperature.
molecules. However, their ζ potential value was dramatically changed to -5.5 ( 3.1 mV upon incubating with an aqueous GdCl3 solution, presumably because cationic Gd3+ ions were coordinated to anionic carboxylate groups in DTPA molecules, resulting in the neutralization of the negative surface charge (31). ICP-AES analysis of GMNPs indicated that each particle contained 11.5 wt % of Gd atoms, indicating that a single magnetite nanoparticle was covered with ca. 7010 gadolinium chelates. Taking into account the number of the conjugated dopamine molecules determined by elemental analysis, almost 68% of the dopamine molecules immobilized on the surface were estimated to participate in the incorporation of the gadolinium chelates (Supporting Information). It was previously reported that the surface density of Gd3+ paramagnetic species might have a profound impact on the positive contrast enhancement of Gd3+-labeled nanoparticles (32). In the present study, the surface density of the gadolinium chelates could be controlled by simply changing the ratio of mPEG-DOPA and dopamine, resulting in the formation of GMNPs with tunable compositions and contrast properties. The above results revealed that GMNPs could hold a large number of Gd3+ ions within the DTPA-functionalized shell layer around the iron oxide core, and hence they were advantageous for generating gadoliniumenhanced contrast in MRI images. X-ray diffraction (XRD) pattern was examined to confirm the crystal structure of magnetite nanoparticles (Figure 5A). The XRD pattern of GMNPs displayed characteristic diffraction peaks corresponding to a face-centered cubic (fcc) lattice of magnetite crystals (9). This was consistent with the XRD pattern of oleic acid-coated magnetite nanoparticles, suggesting that the
Bioconjugate Chem., Vol. 21, No. 3, 2010 509
surface modification with mPEG-DOPA and gadolinium chelates did not induce any change on the crystallinity of the iron oxide core. Figure 5B shows the field-dependent magnetization curve of the magnetite nanoparticles before and after the functionalization with gadolinium chelates. GMNPs had a lower saturation magnetization value (6.85 emu/g) as compared with that of mPEG-DOPA/dopamine stabilized magnetite nanoparticles (8.12 emu/g), which was probably attributed to the decrease in iron oxide content of the particles (30, 33). It was noteworthy that GMNPs exhibited no hysteresis in the magnetization curve, suggesting that the magnetite nanoparticles maintained their superparamagnetic behavior after paramagnetic Gd3+ ions were coordinated onto the surface. To validate the potential of GMNPs as a dual contrast agent, we acquired the T1- and T2-weighted MR images and measured the relaxation rates as a function of metal concentration using a 3.0 T MRI scanner. As shown in Figure 6A, GMNPs induced a bright signal enhancement in a concentration-dependent manner on the T1-weighted images. It should be noted that they exhibited better T1 positive contrast compared to a clinically available Gd-DTPA-based contrast agent (Magnevist), while there was very little signal enhancement from the magnetite nanoparticles without Gd3+ ions. Moreover, the specific relaxivity (r1) of GMNPs (11.17 mM-1 s-1) was markedly higher than r1 of Magnevist (5.39 mM-1 s-1), suggesting that they were much more efficient in enhancing T1 positive contrast than Magnevist (Figure 6B). It was reported previously that the r1 values of Gd-based contrast agents were strengthened when bound to large molecules such as proteins and polymers due to the limited molecular motion of Gd3+ ions (29, 34). Hence, it was conceivable that the remarkably high r1 value of GMNPs resulted from the reduced mobility of Gd3+ ions tightly incorporated into nanometer-scale particles. Despite the high r1 value (10.15 mM-1 s-1), aqueous GdCl3 solution is not approved for clinical use due to toxicity of free Gd3+ ions (13). Since the covalently immobilized DTPA shell on GMNPs allowed for the strong coordination of Gd3+ ions onto the particles, GMNPs were considered to be safer than aqueous GdCl3 solution. As shown in the T2-weighted images (Figure 6C), GMNPs also displayed a significant signal reduction with increasing the particle concentration. It was found that the r2 value of magnetite nanoparticles decreased from 225.81 to 30.32 mM-1 s-1 after the labeling with Gd3+ ions (Figure 6D), suggesting that the paramagnetic gadolinium chelates interfered with the T2 relaxation processes of the neighboring magnetite nanoparticles (35, 36). The observed decrease in the r2 value was partially attributed to the reduced magnetization of the particles (7, 37). Although GMNPs had a lower r2 compared to that of commercially available Feridex (148.95 mM-1 s-1), their T2 contrast effect was sufficiently strong to generate negative contrast enhancement on the MR images. Since the relaxivities of GMNPs were not diminished in normal saline (r1 ) 14.45 mM-1 s-1, r2 ) 36.68 mM-1 s-1), it was anticipated that their relaxation properties were maintained in physiological environment. We performed in vivo MRI experiments to demonstrate the dual contrast ability of GMNPs in a live animal model. In order to prevent leakage from the injection site, GMNPs suspended in Extracel hydrogel solution were injected subcutaneously into dorsal flanks of nude mice, and then, the hydrogel formed at the injection site was examined by using a 3.0 T MRI scanner. Figure 7A shows that the hydrogel containing Magnevist appears as a bright area in the T1-weighted MRI. In contrast, a dark signal void can be observed at the injection site of Feridex, indicative of its strong T2 negative contrast (Figure 7B). As shown in Figure 7C and D, GMNPs displayed a bright signal enhancement
510 Bioconjugate Chem., Vol. 21, No. 3, 2010
Bae et al.
Figure 6. (A) T1-weighted and (C) T2-weighted magnetic resonance images of (i) aqueous GdCl3 solution, (ii) Magnevist (Gd-DTPA), (iii) mPEGDOPA/dopamine stabilized magnetite nanoparticles, (iv) GMNPs, and (v) pure deionized water. The mPEG-DOPA/dopamine-stabilized magnetite nanoparticles were compared to GMNPs at equivalent Fe concentration. The magnetic resonance images of Feridex are not shown here for a clear presentation. (T1-weighted images: repetition time ) 600 ms, echo time ) 12 ms; T2-weighted images: repetition time ) 3000 ms, echo time ) 200 ms). (B) Longitudinal relaxation rates of aqueous solutions of GMNPs (squares), GdCl3 (triangles), and Magnevist (circles). (D) Transverse relaxation rates of mPEG-DOPA/dopamine stabilized magnetite nanoparticles (diamonds), Feridex (circles), and GMNPs (squares).
Figure 7. (A) T1-weighted and (B) T2-weighted magnetic resonance images of a mouse injected with Feridex and Magnevist (the orange arrows indicate the injection sites of Feridex, and the green arrows indicate the injection sites of Magnevist). (C) T1-weighted and (D) T2-weighted magnetic resonance images of a mouse injected with GMNPs and the hydrogel solution as a control (the white arrow indicates the injection site of the hydrogel solution, and the blue arrows indicate the injection sites of GMNPs).
and a marked signal drop at the injection site on the T1- and T2-weighted MR image, respectively, while no apparent contrast was detected from the control site containing only the gel matrix. Consequently, the above results demonstrated that GMNPs having high r1 and r2 relaxivities exhibited both positive and negative contrast enhancement on the MR images. Due to their dual contrast effect, GMNPs can be utilized to achieve both T1- and T2-weighted MR images of the tissue site of interest, and hence increase the accuracy of diagnosis. Furthermore, this dual contrast approach may help to overcome the limitations of traditional iron oxide-based and gadolinium-based contrast agents, thereby expanding
opportunities of MRI for diverse therapeutic and diagnostic applications.
CONCLUSIONS Gadolinium-labeled magnetite nanoparticles were developed as dual contrast agents for T1- and T2-weighted magnetic resonance imaging. These nanoparticles were produced through the formation of a mixed layer of poly(ethylene glycol) chains and dopamine molecules via strong coordination bonds of DOPA to iron oxides, and subsequent immobilization of gadolinium chelates onto the nanoparticle surface. The surface modification process did not disturb the crystal structure and
Bioinspired Synthesis of Gd-Labeled Magnetite NPs
superparamagnetic properties of the magnetite nanoparticles, suggesting that the current strategy would afford stabilization and functionalization of various inorganic nanomaterials, which are poorly soluble and processable in aqueous solution. Moreover, we demonstrated that GMNPs not only had the ability to improve surrounding water proton signals on the T1-weighted image, but also could induce significant signal reduction on the T2-weighted image. These novel nanomaterials having both positive and negative contrast could be potentially utilized as molecular imaging probes for a wide range of diagnostic and therapeutic applications.
ACKNOWLEDGMENT This research was partly supported by the Ministry for Health, Welfare and Family Affairs, the Ministry of Knowledge Economy, and the World Class University project and the National Research Laboratory program from the Ministry of Education, Science and Technology, Republic of Korea. We would like to thank Jaewon Lee and Prof. Jung Hee Lee in Samsung Medical Center for helpful discussion and synthesis of iron oxide nanocrystals, and Myung Wook Song for VSM measurements. Supporting Information Available: 1H NMR spectrum of DOPA-conjugated poly(ethylene glycol) (mPEG-DOPA), the elemental analysis results of mPEG-DOPA coated magnetite nanoparticles, and the method for the calculation of the Gd chelation efficiency and the relaxation time (T1 and T2). This material is available free of charge via the Internet at http:// pubs.acs.org.
LITERATURE CITED (1) Alivisatos, A. P. (2004) The use of nanocrystals in biological detection. Nat. Biotechnol. 22, 47–52. (2) Nie, S., Xing, Y., Kim, G. J., and Simons, J. W. (2007) Nanotechnology applications in cancer. Annu. ReV. Biomed. Eng. 9, 257–88. (3) Rosi, N. L., and Mirkin, C. A. (2005) Nanostructures in biodiagnostics. Chem. ReV. 105, 1547–62. (4) Sonvico, F., Mornet, S., Vasseur, S., Dubernet, C., Jaillard, D., Degrouard, J., Hoebeke, J., Duguet, E., Colombo, P., and Couvreur, P. (2005) Folate-conjugated iron oxide nanoparticles for solid tumor targeting as potential specific magnetic hyperthermia mediators: synthesis, physicochemical characterization, and in vitro experiments. Bioconjugate Chem. 16, 1181–8. (5) Sun, C., Lee, J. S. H., and Zhang, M. (2008) Magnetic nanoparticles in MR imaging and drug delivery. AdV. Drug DeliVery ReV. 60, 1252–65. (6) Park, J., An, K., Hwang, Y., Park, J.-G., Noh, H.-J., Kim, J.Y., Park, J.-H., Hwang, N.-M., and Hyeon, T. (2004) Ultra-largescale syntheses of monodisperse nanocrystals. Nat. Mater. 3, 891–5. (7) Jun, Y.-W., Lee, J.-H., and Cheon, J. (2008) Chemical design of nanoparticle probes for high-performance magnetic resonance imaging. Angew. Chem., Int. Ed. 47, 5122–35. (8) Gupta, A. K., and Gupta, M. (2005) Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications. Biomaterials 26, 3995–4021. (9) Lee, J., Yang, J., Ko, H., Oh, S. J., Kang, J., Son, J.-H., Lee, K., Lee, S.-W., Yoon, H.-G., Suh, J.-S., Huh, Y.-M., and Haam, S. (2008) Multifunctional magnetic gold nanocomposites: human epithelial cancer detection via magnetic resonance imaging and localized synchronous therapy. AdV. Funct. Mater. 18, 258–64. (10) Herdt, A. R., Kim, B.-S., and Taton, T. A. (2007) Encapsulated magnetic nanoparticles as supports for proteins and recyclable biocatalysts. Bioconjugate Chem. 18, 183–9. (11) Kim, J., Lee, J. E., Lee, S. H., Yu, J. H., Lee, J. H., Park, T. G., and Hyeon, T. (2008) Designed fabrication of a multi-
Bioconjugate Chem., Vol. 21, No. 3, 2010 511 functional polymer nanomedical platform for simultaneous cancer-targeted imaging and magnetically guided drug delivery. AdV. Mater. 20, 478–83. (12) Rudin, M., and Weissleder, R. (2003) Molecular imaging in drug discovery and development. Nat. ReV. Drug DiscoVery 2, 123–31. (13) Meade, T. J., Taylor, A. K., and Bull, S. R. (2003) New magnetic resonance contrast agents as biochemical reporters. Curr. Opin. Neurobiol. 13, 597–602. (14) Bulte, J. W. M., Zhang, S.-C., Van Gelderen, P., Herynek, V., Jordan, E. K., Duncan, I. D., and Frank, J. A. (1999) Neurotransplantation of magnetically labeled oligodendrocyte progenitors: Magnetic resonance tracking of cell migration and myelination. Proc. Natl. Acad. Sci. U.S.A. 96, 15256–61. (15) Josephson, L., Tung, C. H., Moore, A., and Weissleder, R. (1999) High-efficiency intracellular magnetic labeling with novel superparamagnetic-Tat peptide conjugates. Bioconjugate Chem. 10, 186–91. (16) Lee, H., Lee, E., Kim, D. K., Jang, N. K., Jeong, Y. Y., and Jon, S. (2006) Antibiofouling polymer-coated superparamagnetic iron oxide nanoparticles as potential magnetic resonance contrast agents for in vivo cancer imaging. J. Am. Chem. Soc. 128, 7383– 9. (17) Bulte, J. W. M., and Kraitchman, D. L. (2004) Iron oxide MR contrast agents for molecular and cellular imaging. NMR Biomed. 17, 484–99. (18) Kim, Y. B., Bae, K. H., Yoo, S.-S., Park, T. G., and Park, H. W. (2009) Positive contrast visualization for cellular magnetic resonance imaging using susceptibility-weighted echo-time encoding. Magn. Reson. Imaging 27, 601–10. (19) Zhuo, J., and Gullapalli, R. P. (2006) MR artifacts, safety, and quality control. RadioGraphics 26, 275–97. (20) Deming, T. J. (1999) Mussel byssus and biomolecular materials. Curr. Opin. Chem. Biol. 3, 100–5. (21) Lee, H., Scherer, N. F., and Messersmith, P. B. (2006) Singlemolecule mechanics of mussel adhesion. Proc. Natl. Acad. Sci. U.S.A. 103, 12999–13003. (22) Holten-Andersen, N., Mates, T. E., Toprak, M. S., Stucky, G. D., Zok, F. W., and Waite, J. H. (2009) Metals and the integrity of a biological coating: The cuticle of mussel byssus. Langmuir 25, 3323–6. (23) Dalsin, J. L., Lin, L., Tosatti, S., Vo¨ro¨s, J., Textor, M., and Messersmith, P. B. (2005) Protein resistance of titanium oxide surfaces modified by biologically inspired mPEG-DOPA. Langmuir 21, 640–6. (24) Lee, H., Dellatore, S. M., Miller, W. M., and Messersmith, P. B. (2007) Mussel-inspired surface chemistry for multifunctional coatings. Science 318, 426–30. (25) Xu, C., Xu, K., Gu, H., Zheng, R., Liu, H., Zhang, X., Guo, Z., and Xu, B. (2004) Dopamine as a robust anchor to immobilize functional molecules on the iron oxide shell of magnetic nanoparticles. J. Am. Chem. Soc. 126, 9938–9. (26) Choi, D., Han, A., Park, J. P., Kim, J. K., Lee, J. H., Kim, T. H., and Kim, S. W. (2009) Fabrication of MnxFe1-xO colloidal solid solution as a dual magnetic-resonance-contrast agent. Small 5, 571–3. (27) Lee, Y., Lee, H., Kim, Y. B., Kim, J., Hyeon, T., Park, H. W., Messersmith, P. B., and Park, T. G. (2008) Bioinspired surface immobilization of hyaluronic acid on monodisperse magnetite nanocrystals for targeted cancer imaging. AdV. Mater. 20, 4154– 7. (28) Xie, J., Xu, C., Kohler, N., Hou, Y., and Sun, S. (2007) Controlled PEGylation of monodisperse Fe3O4 nanoparticles for reduced non-specific uptake by macrophage cells. AdV. Mater. 19, 3163–6. (29) Turner, J. L., Pan, D., Plummer, R., Chen, Z., Whittaker, A. K., and Wooley, K. L. (2005) Synthesis of gadolinium-labeled shellcrosslinked nanoparticles for magnetic resonance imaging applications. AdV. Funct. Mater. 15, 1248–54.
512 Bioconjugate Chem., Vol. 21, No. 3, 2010 (30) Yuan, J.-J., Armes, S. P., Takabayashi, Y., Prassides, K., Leite, C. A. P., Galembeck, F., and Lewis, A. L. (2006) Synthesis of biocompatible poly[2-(methacryloyloxy)ethyl phosphorylcholine]-coated magnetite nanoparticles. Langmuir 22, 10989–93. (31) Debouttie`re, P.-J., Roux, S., Vocanson, F., Billotey, C., Beuf, O., Favre-Re´guillon, A., Lin, Y., Pellet-Rostaing, S., Lamartine, R., Perriat, P., and Tillement, O. (2006) Design of gold nanoparticles for magnetic resonance imaging. AdV. Funct. Mater. 16, 2330–9. (32) Voisin, P., Ribot, E. J., Miraux, S., Bouzier-Sore, A.-K., Lahitte, J.-F., Bouchaud, V., Mornet, S., Thiaudie’re, E., Franconi, J.-M., Raison, L., Labruge’re, C., and Delville, M.-H. (2007) Use of lanthanide-grafted inorganic nanoparticles as effective contrast agents for cellular uptake imaging. Bioconjugate Chem. 18, 1053–63. (33) Fan, Q.-L., Neoh, K.-G., Kang, E.-T., Shuter, B., and Wang, S.-C. (2007) Solvent-free atom transfer radical polymerization for the preparation of poly(poly(ethyleneglycol) monomethacrylate)-grafted Fe3O4 nanoparticles: synthesis, characterization and cellular uptake. Biomaterials 28, 5426–36.
Bae et al. (34) Nakamura, E., Makino, K., Okano, T., Yamamoto, T., and Yokoyama, M. (2006) A polymeric micelle MRI contrast agent with changeable relaxivity. J. Controlled Release 114, 325–33. (35) Vilringer, A., Rosen, B. R., Belliveau, J. W., Ackerman, J. L., Lauffer, R. B., Buxton, R. B., Chao, Y.-S., Wedeenand, V. J., and Brady, T. J. (1988) Dynamic imaging with lanthanide chelates in normal brain: Contrast due to magnetic susceptibility effects. Magn. Reson. Med. 6, 164–74. (36) Loubeyre, P., De Jaegere, T., Bosmans, H., Miao, Y., Ni, Y., Landuyt, W., and Marchal, G. (1999) Comparison of iron oxide particles (AMI 227) with a gadolinium complex (Gd-DOTA) in dynamic susceptibility contrast MR imagings (FLASH and EPI) for both phantom and rat brain at 1.5 T. J. Magn. Reson. Imaging 9, 447–53. (37) Seo, S.-B., Yang, J., Lee, T.-I., Chung, C.-H., Song, Y. J., Suh, J.-S., Yoon, H.-G., Huh, Y.-M., and Haam, S. (2008) Enhancement of magnetic resonance contrast effect using ionic magnetic clusters. J. Colloid Interface Sci. 319, 429–34. BC900424U
