High Relaxivity Trimetallic Nitride (Gd3N) Metallofullerene MRI ContrastAgents with Optimized Functionality

Jianfei Zhang,† Panos P. Fatouros,‡ Chunying Shu,† Jonathan Reid,† Lesley Shantell Owens,† Ting Cai,†

Harry W. Gibson,† Gary L. Long,† Frank D. Corwin,‡ Zhi-Jian Chen,§ and Harry C. Dorn*,†

Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24060, and Department ofRadiology and Department of Neurosurgery, Virginia Commonwealth University, Richmond, Virginia 23298. ReceivedAugust 23, 2009; Revised Manuscript Received November 20, 2009

Water-soluble poly(ethylene glycol) (PEG) functionalized and hydroxylated endohedral trimetallic nitridemetallofullerene derivatives, Gd3N@C80[DiPEG(OH)x], have been synthesized and characterized. The 1H MRIrelaxivities in aqueous solution were measured for the derivatives with four different molecular weights of PEG(350-5000 Da) at 0.35, 2.4, and 9.4 T. The 350/750 Da PEG derivatives have the highest relaxivities among thederivatives, 237/232 mM-1 s-1 for r1 and 460/398 mM-1 s-1 for r2 (79/77 mM-1 s-1 and 153/133 mM-1 s-1

based on Gd3+ ion), respectively, at a clinical-range magnetic field of 2.4 T. These represent some of the highestrelaxivities reported for commercial or investigational MRI contrast agents. Dynamic light scattering results confirma larger average size for 350/750 Da PEGs derivatives (95/96 nm) relative to longer chain length derivatives,5000 Da PEG derivatives (37 nm). Direct infusion of the optimized 350 Da PEG derivatives into live tumor-bearing rat brains demonstrated an initial uniform distribution, and hence, the potential for effective brachytherapyapplications when the encapsulated Gd3+ ions are replaced with radioactive 177Lu.

INTRODUCTION

Magnetic resonance imaging (MRI) is widely used in theclinical diagnosis of cancer and other diseases. Improvedcontrast can be obtained by the use of intravenously injectedparamagnetic contrast agents due to their extravasation acrossleaking vasculature and subsequent accumulation in lesions.Since contrast agents are widely employed in clinical MRIstudies, these agents must be biologically safe and have a highrelaxivity in order to minimize the amount delivered. Currently,the most widely used MRI contrast agents are gadoliniumchelates: the large number of unpaired electrons (seven) perGd3+ ion and favorably long electron spin-lattice relaxationtime significantly increase the relaxation rate of water hydro-gens (1, 2). However, it has been recently reported thatnephrogenic systemic fibrosis (NSF), a painful and debilitatingdisorder of the skin and systemic tissues in patients with renalinsufficiency, is associated with exposure to Gd3+ ions fromcontrast agents (3, 4). New contrast agents with higher relaxivityand lower toxicity are an important target of current investigations.

Because of their shape, capacity for multiple endo encapsu-lants and isolation of the Gd3+ ions from the bioenvironment,endohedral metallofullerenes (EMFs) represent ideal nextgeneration nanoparticles as diagnostic biomedical nanoprobes.Several new classes of MRI contrast agents, water-solubleendohedral metallofullerenes, have been reported (5-17). EMFshave been functionalized with hydrophilic groups, e.g., -OHor -COOH (8-12). These water-soluble EMF derivatives showsignificantly greater relaxivities than the chelated Gd3+ com-pounds in current clinical use.

We have previously reported the synthesis of trimetallicnitride templated (TNT) EMFs, AxB3-xN@C80 (A,B ) metal, x) 0-3) (18). The TNT EMFs have an additional advantage:they can encapsulate up to three Gd3+ ions inside the cage,resulting in higher relaxivity. If different metal atoms/ions areencapsulated, e.g., Lu, Tb, Ho, and so forth, TNT EMFs canalso act as multimodal (MRI and radiotherapy) platforms withthe same biodistribution. We previously reported the synthesisof Gd3N@C80[DiPEG5000(OH)x] (PEG ) poly(ethylene gly-col)) (7), which is water-soluble and exhibits high relaxivity.In Vitro and in ViVo studies demonstrated the potential ap-plicability of this MRI contrast agent. More recent studies havealso demonstrated high relaxivity for related TNT EMFs withdifferent functionalities (13, 15, 16). The high relaxivity of theTNT EMFs derivative arises from the following factors: (1) thelarge number of exchangeable water molecules coordinated tothe hydroxyl groups which are attached to the carbon cage(Figure 1) and are simultaneously relaxed by the (Gd3N)6+

* Corresponding author. Harry C. Dorn, Department of Chemistry,Virginia Polytechnic Institute and State University, Blacksburg, VA,24060; E-Mail: hdorn@vt.edu; Phone: 540-231-5953; Fax: 540- 231-3255.

† Virginia Polytechnic Institute and State University.‡ Department of Radiology, Virginia Commonwealth University.§ Department of Neurosurgery, Virginia Commonwealth University.

Figure 1. The Gd3N@C80[DiPEG(OH)x] nanoparticle with severalhydrogen-bonded water molecules shown.

Bioconjugate Chem. 2010, 21, 610–615610

10.1021/bc900375n 2010 American Chemical SocietyPublished on Web 03/10/2010

endohedral cluster, (2) the slow rotational correlation timecaused by the long PEG chain and aggregation effects.

In the current study, we synthesized pegylated and hydroxy-lated Gd3N@C80 with different PEG chain lengths (350-5000MW), and the relaxivities of these derivatives were measuredat three different magnetic field strengths. The optimum PEGchain of these pegylated and hydroxylated Gd3N@C80 deriva-tives is an important structural property for potential MRI studiesat different magnetic field strengths. To demonstrate the in ViVoapplicability of these high-relaxivity contrast agents, a convec-tion-enhanced delivery (CED) method was used to infuseGd3N@C80[DiPEG(OH)x] directly into a brain-tumor-bearingrat.

MATERIALS AND METHODS

Synthesis of Gd3N@C80[DiPEG(OH)x]. Gd3N@C80 wasproduced by the standard Kratschmer-Huffman (K-H) methodwith the introduction of nitrogen gas into the K-H generator(18). The product was purified by HPLC as previously reported(19). 1,8-Diazabicyclo(5.4.0)undec-7-ene (DBU, FW 152.24),98%, was purchased from Sigma-Aldrich; 99% CBr4, 99% 18-crown-6, 50% sodium hydroxide solution, and 30 wt %hydrogen peroxide were used as obtained from Aldrich Chemi-cal Co. Di{ω-methyl-poly(ethylene glycol)} malonate (DiPEG)350, 750, 2000, and 5000 (MW. 768, 1568, 4068, 10 068,respectively) were prepared from ω-methyl-poly(ethylene gly-col)s (PEGs) as follows: To a solution of PEG and triethylamine(TEA) in CH2Cl2, a solution of malonyl dichloride in CH2Cl2

was added dropwise. The molar ratio of PEG/TEA/malonyldichloride was 2:2:1. The solution was stirred at room temper-ature under N2 protection for 3 h, concentrated and droppedinto a large amount of ethyl ether, and then fractionated by asilica gel column.

Gd3N@C80, DiPEG, DBU, and CBr4 were dissolved inchlorobenzene in molar ratio 1:20:20:67. After deaeration withbubbling argon for 30 min, the solution was stirred overnightat room temperature. The resultant mixture was evaporated, andthe residue was dissolved in toluene. To this solution, 18-crown-6 and 50% sodium hydroxide solution were added, andthe resultant solution was stirred for 4 h. 1.5 mL hydrogenperoxide was added drop by drop, and more water was added.The mixture was stirred overnight at room temperature. Theresultant solution was concentrated and separated on a SephadexG25 (Pharmacia) size-exclusion gel column with distilled wateras eluent to afford a narrow golden band (first fraction, pH e7). (See Supporting Information Figure 1.)The yield was up to95%.

Relaxivity Measurements. The concentrations of Gd3+ ionswere determined by inductively coupled plasma optical emissionspectroscopy (ICP-OES, Perkin-Elmer Optima 5300DV) at342.247 nm. The instrument was calibrated by a 10 ppmICP standard solution. Each molecule was assumed toencapsulate three Gd3+ ions, so the concentration ofGd3N@C80[DiPEG(OH)x] was assumed to be one-third of theGd3+ ion concentration.

The proton relaxation times were measured at three differentmagnetic field strengths: 0.35 T (TEACH SPIN PS1-B), 2.4 T(Bruker/Biospec), and 9.4 T (Varian Inova 400). The inversion-recovery method was used to measure T1 and the Carr-Pucell-Meiboom-Gill method was used for measurement of T2. Therelaxivities were extracted from linear fits of plots of relaxationrates (1/T1 and 1/T2) vs the concentrations of the paramagneticspecies.

Dynamic Light Scattering. Dynamic light scattering (DLS)measurements were carried out with an ALV/CGS-3 compactgoniometer system and ALV/LSE-5003 multi-τ digital correlatorat 25 °C. He-Ne laser producing vertically polarized light of

λ0 ) 632.8 nm was used as a light source. Each sample wasfiltered with a 0.5 µm cellulose acetate membrane filter.

Animal Studies. We used a convection-enhanced deliverymethod (7) to infuse 0.0235 mM Gd3N@C80[DiPEG350(OH)x]into a live rat brain tumor and followed the functionalized EMFredistribution over the course of several days. Infusion wasapplied for 180 min at a rate of 0.2 µL/min with a total infusionvolume of 36 µL.

RESULTS AND DISCUSSION1H NMR. 1H NMR spectra of Gd3N@C80[DiPEG(OH)x] was

performed on an Inova 400. Figure 2a displays the 1H NMRspectra of Gd3N@C80[DiPEG350(OH)x] and malonyl DiPEG350in D2O. In Gd3N@C80[DiPEG350(OH)x] the -OCH3 and-OCH2CH2- signals appeared at δ3.35 ppm (s, 6H) and 3.67ppm (m, 52H), respectively; the signals for -COOCH2- andCOOCH2CH2- centered at 4.32 ppm (t, 4H) and 3.78 ppm (t,4H) in the malonate disappeared in the TNT EMFs derivativeapparently due to the strong paramagnetic effect of the Gd3+

ions. The NMR spectra of other PEG derivatives gave similarresults.

UV-Vis. The UV-Vis spectra of Gd3N@C80 andGd3N@C80[DiPEG2000(OH)x] are shown in Figure 2b. ForGd3N@C80, there is an absorption peak at about 710 nm and asecond absorption occurs at 410 nm (20). These absorptionsare attributed to the ΠfΠ* transitions (21). For the function-alized metallofullerenes, however, these characteristic featuresare significantly reduced. This indicates that the conjugated πstructure of the carbon cage has been significantly altered.Similar results were obtained with other samples with differentmolecular weights (Gd3N@C80[DiPEG5000, 750, 350(OH)x]).

Mass Spectra. To confirm the functionalization, MALDI-TOF mass spectra were obtained for DiPEG750, Gd3N@C80[DiPEG750], and Gd3N@C80[DiPEG750(OH)x] (see Sup-porting Information Figure 2). The spectrum for DiPEG 750was a series of peaks centered at about m/z 1388. The peakswere separated by m/z 44, which is the mass of the (CH2CH2O)unit. The spectrum of Gd3N@C80[DiPEG750] was a series ofpeaks centered at about m/z 2907. The separation of m/z 1 isattributed to the isotope effect of Gd3N@C80. We can clearlysee that Gd3N@C80 was successfully functionalized withDiPEG750. However, in the mass spectrum of Gd3N@C80[DiPEG750(OH)x] we only observed peaks for metallo-fullerene (m/z 1447) and PEG fragments (centered at m/z 759and separated by 44). Even though the molecular ion peak wasnot observed, the metallofullerene and PEG fragments indicatesuccessful functionalization.

MRI Relaxivity. The ability of paramagnetic compounds toenhance the relaxation rate can be described by the followingequation (1, 2):

1/T1 and 1/T2 are the longitudinal and transverse relaxationrates. 1/Ti,obs is the observed value of the solution and 1/Ti,para

is the contribution of the paramagnetic compound. [M] is theconcentration of the paramagnetic species. r1 and r2 are definedas longitudinal and transverse relaxivities, respectively, whichare expressed in (mM · s)-1.

A major contribution to 1/Ti,para arises from the simultaneousrelaxation of multiple water molecules. This is the result of thedipolar electron-nuclear spin interaction between the largefluctuating Gd3+ electron magnetic moment and the waterhydrogen nucleus. These relaxed water molecules undergosubsequent rapid exchange with bulk water. Dipole-dipoleinteractions are described by the Solomon-Bloembergen-Morgan

1Ti,obs

) 1Ti,H2O

+ 1Ti,para

) 1Ti,H2O

+ ri[M] i ) 1,2 (1)

High Relaxivity Trimetallic Fullerene MRI Contrast Agents Bioconjugate Chem., Vol. 21, No. 4, 2010 611

(SBM) equations. In the high-field region (above 10 MHz), theseequations reduce to (1, 2)

If a water exchange process exists, then the relaxation ratesare (1, 2):

[M] is in units of mM, τR is the rotational correlation time,τM is the residence time of the coordinated water molecules,ωH is the Larmor frequency of proton, τ1e is the longitudinalrelaxation time of electron, and q is the number of the watermolecules that coordinate to the paramagnetic molecules. τC isdominated by the shortest correlation time among τR, τM, andτ1e. If τR is the dominant time, the relaxivity as a function offrequency shows a broad maximum peak that increases withincreasing τR (1).

In the present investigation, the relaxivities of all theGd3N@C80[DiPEG(OH)x] samples were measured. The re-sults are summarized in Table 1. Our previously reportedrelaxivities for Gd3N@C80[DiPEG5000(OH)x] (7) are alsolisted, and they agree with the current data. All theGd3N@C80[DiPEG(OH)x] samples have high r1 and r2 valuesat all three magnetic field strengths. The r1 values range from

Figure 2. (a) 400 MHz 1H NMR spectra of Gd3N@C80[DiPEG350(OH)x] (top) in comparison with DiPEGM350 malonate (bottom) in D2O; (b) UV-Visspectra of Gd3N@C80 and Gd3N@C80[DiPEG2000(OH)x]. Note the disappearance of the characteristic peaks at 330 and 410 nm in the derivative.

1T1M

) 615

γI2g2µB

2 S(S + 1)

rH6 ( τC

1 + ωH2 τc

2) (2)

1T2M

) 115

γI2g2µB

2 S(S + 1)

rH6 (4τc +

3τC

1 + ωH2 τc

2) (3)

τC-1 ) τM

-1 + τR-1 + τ1e

-1 (4)

1T1,para

) q[M]/55600T1M + τM

(5)

1T2,para

) q[M]/55600T2M + τM

(6)

612 Bioconjugate Chem., Vol. 21, No. 4, 2010 Zhang et al.

139 mM-1 s-1 (Gd3+ based relaxivity 46.3 mM-1 s-1) to 237mM-1 s-1 (Gd3+-based relaxivity 79.0 mM-1 s-1) for thedifferent PEG chains at 2.4 T compared to the value of about4 mM-1 s-1 for commercially available Gd3+ ion chelatedcontrast agents. Several other groups have also reported r1

values of mono gadofullerene derivatives, that is, one Gd3+

ion per fullerene cage. These r1 values are typically higherthan those of commercial MRI contrast agents. In our work,the relaxivities of all the Gd3N@C80[DiPEG(OH)x] samplesare significantly higher on a per molecule basis. A summaryof these other studies and the relaxivities of Gd3N@C80-[DiPEG350(OH)x] in the current study are presented in Table2 (6, 8, 9, 11, 12, 22).

As previously indicated, a primary reason for the high molarrelaxivity of the Gd3N@C80[DiPEG(OH)x] samples in compari-son with other endohedral metallofullerenes derivatives origi-nates from the presence of three Gd3+ ions within the cage: thecharge transfer between the (Gd3N)6+ cluster and the (C80)6-

cage leads to a very stable nanoparticle. Previous studies haveconfirmed that ferromagnetic coupling in this cluster gives riseto a large magnetic moment, 21 µB (23). The dipolarelectron-nuclear spin interaction between the fluctuating Gd3+

electron magnetic moment and the large number of exchange-able water hydrogen atoms induces efficient proton relaxation.This is in contrast with the conventional Gd-DTPA complex,which has a single water molecule bound in the first coordinationsphere of the metal ion, exchanges rather slowly with the bulkwater, and the entire complex tumbles very rapidly.

Further factors contributing to the relaxation process relateto the unique property of the fullerenes to aggregate and formlarge nanoclusters. Aggregation occurs frequently for water-soluble metallofullerenes and affects the relaxivity significantlyas previous studies have shown (5, 9). For the low molar massGd3N@C80[DiPEG(OH)x] samples, the aggregates are formedby hydrogen bonding between -OH groups; however, thehydrophobic fullerene-fullerene interactions are also strong.Hence, fullerene aggregates can be formed. According to theDebye-Stokes equation for rigid spherical particles

the rotational correlation time τR increases as the particle radiusincreases, which is an important parameter influencing relaxivity

as a function of magnetic field strength. It has also beenpostulated that the presence of these aggregates might lead toa “confined” pool of water molecules in the interstitial spacesbetween individual gadofullerenes, which exchanges rapidlywith the bulk water increasing again the relaxivity (24).Depending on the PEG length and aggregate size, differentrestricted pools of water would be created as mentioned aboveinfluencing the measured overall relaxivities.

In the limit of τM , T1M, T2M for eqs 2 and 3, we obtain

At low field, ωH2τC

2 , 1 and the ratio approaches the value7/6 ) 1.17. Our measured ratios of r2/r1 are listed in Table 3.On the basis of eq 7, the ratios at 0.35 T are consistent with thepredicted value. Another result of the SBM equations is ωHτC

≈ 1 at maximum r1. According to eq 7, this leads to r2/r1 ≈11/6 ) 1.83 for the peak. The ratios for the 750 and 350 PEGsat 2.4 T are close to 11/6 ) 1.83 and hence the maximum r1

values should occur near 100 MHz (2.4 T). We can also seethat at 2.4 T the 750/350 Da species has the maximum r1 of allthe samples. Hence, at 2.4 T for the 750/350 samples we obtainτC ) 1/ωH ) 1.59 ns, which is significantly longer than thecommercial contrast agent, e.g., τR ) 66 ps for the [Gd(DTPA-BMA)] (1). This assumes that τC is dominated by a rotationalcomponent.

The aggregation behavior was studied by DLS experiments.The DLS results for all Gd3N@C80[DiPEG(OH)x] samplesare shown in Figure 3. The mean peak positions are 75 nm,76 nm, 58 nm, and 37 nm for Gd3N@C80[DiPEG350(OH)x],Gd3N@C80[DiPEG750(OH)x], Gd3N@C80[DiPEG2000(OH)x],and Gd3N@C80[DiPEG5000(OH)x], respectively. The 350PEGand 750PEG samples have the largest average aggregation size(∼75 nm), while the 5000PEG sample is much smaller (∼37nm). The trend of decreasing aggregation size with increasingmolecular weight is consistent with the relaxivity trend. It should

Table 1. Relaxivity Data of Gd3N@C80[DiPEG(OH)x] Seriesa

mol. weightof PEG

conc. range(µM)

r1 (0.35 T)(mM-1 s-1)

r2 (0.35 T)(mM-1 s-1)

r1 (2.4 T)(mM-1 s-1)

r2 (2.4 T)(mM-1 s-1)

r1 (9.4 T)(mM-1 s-1)

r2 (9.4 T)(mM-1 s-1)

5000 0.4-6.5 107 ( 8 127 ( 36 139 ( 6 221 ( 11 52.5 ( 2.4 186 ( 125000 (7) 1.6-12.6 102 144 143 222 32 1372000 1.0-15.2 130 ( 4 148 ( 8 158 ( 6 249 ( 12 41.9 ( 3.0 218 ( 11750 1.1-17.4 152 ( 5 169 ( 20 232 ( 10 398 ( 22 63.3 ( 1.8 274 ( 9350 1.5-23.5 227 ( 31 268 ( 19 237 ( 9 460 ( 23 68.2 ( 3.3 438 ( 5

a The relaxivities were measured at room temperature in water.

Table 2. Relaxivity Data of Gd3N@C80[DiPEG(OH)x] Seriesa

metallofullerene derivatives r1 (mM-1 s-1) magnetic field strength (T)

Gd@C82O6(OH)16(NH2CH2CH2COOH)8 (6) 9.1 1.5Gadofullerenol (22) 47 9.4Gd@C82(OH)40 (12) 67 0.47

81 1.031 4.7

Gd@C60(OH)x (26) 83.2 1.4Gd@C60[C(COOH)2]10 (11, 26) 24.0 1.4

4.6 0.47Gd3N@C80[DiPEG350(OH)x] 227 (75.7 Gd3+ based) 0.35

237 (79.0 Gd3+ based) 2.468.2 (22.7 Gd3+ based) 9.4

a The relaxivities were measured at room temperature in water.

τR ) 4πηreff3 /3kBT

Table 3. r2/r1 Ratios of the Pegylated/Hydroxylated Samples

sample r2/r1 (0.35 T) r2/r1 (2.4 T) r2/r1 (9.4 T)

5000 1.19 1.59 3.542000 1.14 1.58 5.20750 1.11 1.72 4.33350 1.18 1.94 6.42

r2/r1 ) [3 + 4(1 + ωH2 τC

2 )]/6 (7)

High Relaxivity Trimetallic Fullerene MRI Contrast Agents Bioconjugate Chem., Vol. 21, No. 4, 2010 613

also be noted that these pegylated particles clearly deviate fromspherical shape and the ones with longer PEG chains mightexhibit internal flexibility. This could be an important additionalfactor for the observed reduced relaxivities for these largermolecules due to increased anisotropic rotation described by afast local motion component that controls relaxivity and a slowglobal motional component (25).

Animal Infusion Study. Pure T1 computed images of threeconsecutive brain slices of a live rat brain following infusionof 0.0235 mM Gd3N@C80[DiPEG350(OH)x] into the braintumor are shown in Figure 4. The middle column indicates theslice with the tumor, while the left and the right columns areslices without tumor used as controls. Immediately afterthe infusion is terminated (“3.5 h”), the Gd3N@C80

[DiPEG350(OH)x] nanoparticles have been distributed through-out the tumor, reducing the tissue T1 values. The tumor appearsas a dark area, as the white circle indicates. As time progresses,the brain tumor grows and the Gd3N@C80[DiPEG350(OH)x]nanoparticles appear to be pushed out into the tumor periphery.This is illustrated at day 7 in the T1 map, which showsaggregates on the surface, and the T1-weighted image showsan enhancement ring around the tumor. These results illustratethe enhanced in ViVo relaxivity of this optimized Gd3N@C80-[DiPEG350(OH)x] system at low molar concentrations. It shouldalso be noted that after 7 days the nanoparticles can still enhancethe MRI contrast, which indicates that these metallofullereneagents can be used for long-term imaging diagnostic applica-tions. In addition, these metallofullerene agents provide aplatform for effective brachytherapy applications when Gd isreplaced and/or combined with other radioactive agents (177Lu).

CONCLUSIONS

We synthesized hydroxylated and pegylated Gd-containingTNT EMFs, Gd3N@C80[DiPEG(OH)x], with different chainlengths of PEG. The relaxivities were measured at threemagnetic field strengths. All the samples show significantlyhigher r1 and r2 compared to commercially available Gd-chelatesand show considerable promise as a new class of contrast agents.They represent some of the highest relaxivities reported in theliterature for commercial and investigational MRI contrastagents. Analyzing the field dependence of r1, r2, and r2/r1

indicates reasonable agreement with the predictions of the SBMtheory under the assumption that the rotational correlation timeτR dominates the relaxation process. Direct infusion of these

particles into the live tumor-bearing rat brain demonstrates aninitial uniform distribution and hence the potential for effectivebrachytherapy applications when Gd is replaced with radioactive177Lu.

ACKNOWLEDGMENT

We thank the National Science Foundation [DMR-0507083]and the National Institute of Health [1R01-CA119371-01] forsupport. The authors thank Dr. William Keith Ray and Kim C.Harich for the help with the mass spectra.

Supporting Information Available: Additional informationas described in the text. This material is available free of chargevia the Internet at http://pubs.acs.org.

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