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Preparation and Characterization of Two New Water-Soluble Endohedral Metallofullerenes as Magnetic Resonance Imaging Contrast Agents Er-Yun Zhang,†,‡ Chun-Ying Shu,†,‡ Lai Feng,*,† and Chun-Ru Wang*,† Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, China, and Graduate School of Chinese Academy of Sciences, Beijing, China ReceiVed: July 15, 2007; In Final Form: October 5, 2007
Two new water-soluble Gd-containing endohedral metallofullerenes [ScxGd3-xN@C80Om(OH)n (x ) 1, 2; m ≈ 12; n ≈ 26)] were synthesized in a simple one-step reaction and characterized by Fourier transform (FT)IR as well as X-ray photoelectron spectroscopy (XPS). Their observed longitudinal relaxivities (R1) for water protons are 20.7 and 17.6 mM-1 s-1, respectively, which are significantly higher than that of the commercial magnetic resonance imaging (MRI) contrast agent (Gd-DTPA, 3.2 mM-1 s-1). These results indicate these trimetallic nitride endohedral fullerenols are potential next-generation high-efficiency MRI contrast agents.
1. Introduction In the past 20 years, the applications of water-soluble fullerene derivatives have attracted great interest.1-4 Specifically in the fields of medicine and biological sciences, fullerene derivatives are expected to be widely used as DNA cleavers,5 human immunodeficiency virus (HIV) inhibitors,6,7 and neuroprotective agents.8 Endohedral metallofullerenes, as the new members of the fullerene family, have great potential to be applied as radiopharmaceuticals,9-11 X-ray diffraction contrast agents,12 magnetic resonance imaging (MRI) contrast agents,13-17 and so forth. Recently, gadolinium-containing endohedral fullerene-based MRI contrast agents have become quite attractive because of their high MRI contrast efficiency and low toxicity. Several water-soluble derivatives of Gd@C60 and Gd@C82 were revealed to be excellent MRI contrast agents.13-17 However, the low yield of Gd@C82 and the poor solubility of Gd@C60 in common fullerene solvents largely hampered their clinical applications. Compared with traditional metallofullerenes M@C2n (2n ) 60, 82), a family of trimetallic nitride template (TNT) endohedral fullerenes M3N@C80 (M ) Sc, Y, lanthanide metals)18 were mostly revealed to have higher yields and reasonable solubilities. Very recently, Dorn and co-workers reported a new MRI contrast agent based on the multi-Gd encapsulated endohedral fullerene Gd3N@C80, which is water-soluble and expressed as Gd3N@C80[DiPEG5000(OH)x].19 Its longitudinal relaxivity (R1 ) 143 mM-1 s-1 at 2.4 T) is much higher than other Gd-containing fullerene derivatives and far higher than commercial MRI contrast agents. This result definitely proved that the M3N@C80 family can also be used as MRI contrast agents, and their efficiency may be as good as or even much higher than that of Gd@C2n (2n ) 60, 82). However, the yield of Gd3N@C80 is quite low, so we considered exploring other more abundantly yielded members in the M3N@C80 family as new MRI contrast agents. * Corresponding authors. Phone, Fax: (+86)10-62652120. E-mail:
[email protected] (C.-R.W.). † Institute of Chemistry. ‡ Graduate School of Chinese Academy of Sciences.
In the present study, we investigated the other two Gdcontaining TNT endohedral metallofullerenes, Sc2GdN@C80 and ScGd2N@C80, which are expressed as ScxGd3-xN@C80 (x ) 1, 2). Their yields are as high as about 10 times that of Gd@C82 and 50 times that of Gd3N@C80, only inferior to C60 and C70. After multi-hydroxyl modification, their properties as MRI contrast agents were studied, and satisfactory results were achieved. 2. Experimental Section Production and Separation of ScxGd3-xN@C80 (x ) 1, 2). The synthesis and isolation of ScxGd3-xN@C80 (x ) 1, 2) were reported in our previous paper.20 Briefly, soot containing ScxGd3-xN@C80 (x ) 1, 2) was produced by the traditional direct current arc evaporation of Sc/Gd/C composite rods (at a molar ratio of 1:1:24) under a mixed gas atmosphere (3 Torr N2 and 300 Torr He). Then, the as-produced ScxGd3-xN@C80 (x ) 1, 2)-containing soot was Soxhlet-extracted with toluene for 24 h. The isolation and purification of ScxGd3-xN@C80 (x ) 1, 2) were fulfilled by the three-stage high performance liquid chromatography (HPLC). Two complementary columns, Cosmosil Buckyprep column (Φ20 × 250 mm, Nacalai Tesque) and Cosmosil Buckyprep-M column (Φ20 × 250 mm, Nacalai Tesque), were used with toluene as eluent. The purity of ScxGd3-xN@C80 (x ) 1, 2) (g98%) was confirmed by both HPLC and matrix-assisted laser desorption and ionization timeof-flight (MALDI-TOF) mass spectrometry (BIFLEXIII, Bruker), as shown in Figure 1. Synthesis of ScxGd3-xN@C80Om(OH)n (x ) 1, 2). ScxGd3-xN@C80Om(OH)n (x ) 1, 2) were synthesized according to the method of Iezzi and co-workers.21 Briefly, a highly concentrated toluene solution of pure ScxGd3-xN@C80 (x ) 1, 2) (ca. 0.1 mg/mL) was refluxed with sodium metal for 3 h, and a black precipitate appeared in the solution. Then, 50 mL of water was added with vigorous stirring. The water phase immediately became gold-colored; meanwhile, the upper toluene solution turned colorless. After continuous stirring for 24 h, the toluene was removed. The remaining water solution was concentrated. The products were separated on a Sephadex G-25 (Pharmacial) size-exclusion gel column with distilled water as eluent. The brown fraction at pH ) 6∼7 was collected.
10.1021/jp075529y CCC: $37.00 © 2007 American Chemical Society Published on Web 11/30/2007
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Figure 1. HPLC chromatogram (1 mL injection, 12 mL/min toluene flow rate, UV detection at 310 nm, Buckyprep column) and negative MALDI-TOF mass spectra (inset) of the purified (a) Sc2GdN@C80 and (b) ScGd2N@C80.
Figure 3. XPS spectra of the C 1s binding energy of (a) Sc2GdN@C80Om(OH)n and (b) ScGd2N@C80Om(OH)n.
Figure 2. FT-IR spectra (KBr pellets) of (a) Sc2GdN@C80Om(OH)n and (b) ScGd2N@C80Om(OH)n.
Spectroscopic Characterizations of ScxGd3-xN@C80Om (OH)n (x ) 1, 2). For the Fourier transform infrared (FT-IR) spectrum experiment, the mixed powders of ScxGd3-xN@C80 (x ) 1, 2) and potassium bromide (KBr) were pressed into transparent pellets and then recorded on a Perkin-Elmer FT-IR (Figure 2) spectrometer with a resolution of 0.5 cm-1. X-ray photoelectron spectroscopy (XPS) was used to determine the formula of the water-soluble TNT metallofullerenol (Figure 3). First, several drops of highly concentrated aqueous solution of ScxGd3-xN@C80Om(OH)n were transferred to a clean platinum foil. After evaporation of the solvent, uniform films of ScxGd3-xN@C80Om(OH)n were formed, which were further dried in vacuum at 50 °C for 24 h. The consequent measurement was carried out on an ESCALab220I-XL electron spectrometer from VG Scientific using 300 W Al KR radiation. Proton Relaxivity Measurements. The proton relaxivities (R1) of the two TNT metallofullerenols in aqueous solutions were determined at pH ) 7 at five different concentrations (as shown in Figures 4 and 5). The concentrations of the samples were determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES, PE, U.S.A.). The measurements for longitudinal relaxation time (T1) were performed on a Bruker Advance 600 NMR spectrometer (14.1 T).
Figure 4. Linear-fitting of 1/T1 to concentration of Sc2GdN@C80O12(OH)26.
3. Results and Discussion Spectroscopic Studies. The FT-IR spectra of Sc2GdN@C80Om(OH)n and ScGd2N@C80Om(OH)n show nearly identical results, both of which comprise five characteristic peaks at almost the same positions, as shown in Figure 2. Obviously, peak II suggests the presence of hydroxyl groups on the fullerene cage. Peak II can be assigned to the remaining π bonded carbons (CdC). Peaks III and IV correspond to the O-H deformation and C-O stretching vibrations, respectively. It is noteworthy that the weak peak IV is due to the internal Gd-N stretching, which agrees well with the reported results for Sc2GdN@C80 (647 cm-1) and ScGd2N@C80 (649 cm-1).22 The decreased νGd-N compared with that of pristine fullerene may be derived from the modification-caused distortion of the fullerene cage, which induces the elongation of the Gd-N bond. No Sc-N stretching can be observed because of the low resolution of the spectra. The XPS spectrum of Sc2GdN@C80Om(OH)n (Figure 3a) demonstrates that there exist different types of carbons on the hydroxylated fullerene cages. At least three peaks are required
Two Water-Soluble Endohedral Metallofullerenes
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Figure 5. Linear-fitting of 1/T1 to concentration of ScGd2N@C80O12(OH)26.
to fit the asymmetric curve in the C 1s region. The main peak at 284.8 eV (52.0%) corresponds to the sp2 carbons of the cage. The other two peaks at 286.4 eV (32.5%) and 288.1 eV (15.5%) are attributed to the hydroxylated carbons (-C-OH) and highly oxygenated carbons (carbonyl or hemiketal), respectively. Accordingly, the hydroxylated and highly oxygenated carbons were calculated to be 26 and 12, respectively, suggesting a possible molecular formula as Sc2GdN@C80O12(OH)26. As to the water-soluble derivatives of ScGd2N@C80 (Figure 3b.), three fitting peaks for C 1s were found at 284.7 eV (50.1%), 286.2 eV (33.6%), and 287.9 eV (16.3%). Therefore, its molecular formula is proposed as ScGd2N@C80O12(OH)26. Proton Relaxivity Analysis. For paramagnetic aqueous solutions, the longitudinal and transverse relaxation rates (1/T1 and 1/T2, respectively) of the solvent proton spin are known to be expressed by the term relaxivity Ri as denoted in eq 1.
(1/Ti)obsd ) (1/Ti)d + Ri × [M]
i ) 1, 2
(1)
Here, (1/Ti)obsd and (1/Ti)d are the observed relaxation rates of water protons with and without the presence of paramagnetic species, respectively, while [M] is the concentration of the paramagnetic species. Thus, a plot of (1/T1)obsd versus concentration [M] gives the relaxivity as the slope. The relaxivity (Ri) commonly expressed in units of mM-1 s-1, reflects the relaxation enhancement ability of a paramagnetic compound. As calculated in Figures 4 and 5, the R1 values of Sc2GdN@C80O12(OH)26 and ScGd2N@C80O12(OH)26 are 20.7 and 17.6 mM-1 s-1, respectively. These R1 values are much higher than that of Gd-DTPA (a commercial MRI contrast agent; R1 ) 3.2 mM-1 s-1) but lower than that of Gd@C82O8(OH)30 (R1 ) 58.2 mM-1 s-1), which were all determined under the same conditions. Herein, it is interesting to find that previously reported Gd@C82O8(OH)30 possesses less Gd atoms but affords a higher R1 value than ScGd2N@C80O12(OH)26. The possible explanation might derive from the work of Bolskar and coworkers16 as follows. The unpaired f electrons of the Gd3+ ion in Gd@C82O8(OH)30 magnetically couple with the unpaired electron in the fullerene-centered molecular orbital, and some spin density is transferred from Gd3+ to the substituents. However, there is no unpaired electron in the fullerene-centered molecular orbital of ScxGd3-xN@C80. Therefore, the spin is only localized inside the fullerene cage, which is not beneficial to the spin exchange between ScGd2N@C80O12(OH)26 and water. Also, the type and the degree of functionality are other possible factors that influence the R1 values of these Gd-containing fullerenols. In addition, intermolecular aggregation of MRI contrast agents is known to enhance their relaxivities because of prolonged
Figure 6. Size distributions of the as-prepared samples in aqueous solution: (A) Sc2GdN@C80O12(OH)26 and (B) ScGd2N@C80O12(OH)26.
rotational correlation times of aggregation.23 So far, cluster formation or aggregation has been observed in the solutions of various fullerene derivatives, such as multi-hydroxylated derivatives and multi-carboxylated derivatives.16,17,24,25 Aggregation behavior of our species were also studied by dynamic light scattering (DLS). As shown in Figure 6, both Sc2GdN@ C80O12(OH)26 and ScGd2N@C80O12(OH)26 undergo severe aggregation. The hydrodynamic diameters of their aggregations ranging from tens to 1000 nm could be detected. These serious aggregates were considered to also contribute to the large R1 values of our products. 4. Conclusion Two new water-soluble endohedral metallofullerenes were synthesized and characterized by a variety of spectroscopic techniques including FT-IR and XPS. The general molecular formula was proposed as ScxGd3-xN@C80O12(OH)26 (x ) 1, 2). Their properties as MRI contrast agents were investigated. The R1 values of Sc2GdN@C80O12(OH)26 and ScGd2N@C80O12 (OH)26 were determined to be 20.7 and 17.6 mM-1 s-1, respectively. Compared with other Gd-containing metallofullerenes, the abundance of ScxGd3-xN@C80 (x ) 1, 2) makes them very competitive candidates for next-generation highefficiency MRI contrast agents. Acknowledgment. C.-R.W. thanks NSFC (Nos. 20573121, 20121301) and the Major State Basic Research Program of China “Fundamental Investigation on Micro-Nano Sensors and Systems based on BNI Fusion” (Grant 2006CB300402) for financial supports. References and Notes (1) Jensen, A. W.; Wilson, S. R.; Schuster, D. I. Bioorg. Med. Chem. 1996, 4, 767-779. (2) Da Ros, T.; Prato, M. Chem. Commun. 1999, 663-669. (3) Bosi, S.; Da Ros, T.; Spalluto, G.; Prato, M. Eur. J. Med. Chem. 2003, 38, 913-923. (4) Nakamura, E.; Isobe, H. Acc. Chem. Res. 2003, 36, 807-815. (5) Tokuyama, H.; Yamago, S.; Nakamura, E.; Shiraki, T.; Sugiura, Y. J. Am. Chem. Soc. 1993, 115, 7918-7919. (6) Sijbesma, R.; Srdanov, G.; Wudl, F.; Castoro, J. A.; Wilkins, C.; Friedman, S. H.; Decamp, D. L.; Kenyon, G. L. J. Am. Chem. Soc. 1993, 115, 6510-6512. (7) Friedman, S. H.; Decamp, D. L.; Sijbesma, R. P.; Srdanov, G.; Wudl, F.; Kenyon, G. L. J. Am. Chem. Soc. 1993, 115, 6506-6509. (8) Dugan, L. L.; Turetsky, D. M.; Du, C.; Lobner, D.; Wheeler, M.; Almli, C. R.; Shen, C. K. F.; Luh, T.-Y.; Choi, D. W.; Lin, T.-S. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 9434-9439. (9) Wilson, L. J.; Cagle, D. W.; Thrash, T. P.; Kennel, S. J.; Mirzadeh, S.; Alford, J. M.; Ehrhardt, G. J. Coord. Chem. ReV. 1999, 192, 199-207.
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