NANO LETTERS
Gd@C60[C(COOH)2]10 and Gd@C60(OH)x: Nanoscale Aggregation Studies of Two Metallofullerene MRI Contrast Agents in Aqueous Solution
2004 Vol. 4, No. 12 2373-2378
Balaji Sitharaman,† Robert D. Bolskar,‡ Irene Rusakova,§ and Lon J. Wilson*,† Department of Chemistry, the Center for Nanoscale Science and Technology, and the Center for Biological and EnVironmental Nanotechnology MS 60, Rice UniVersity, Houston, Texas 77251-1892, the Texas Center for SuperconductiVity, UniVersity of Houston, Houston, Texas 77204-5002, and TDA Research Inc., 12345 West 52nd AVenue, Wheat Ridge, Colorado 80033 Received September 1, 2004; Revised Manuscript Received September 28, 2004
ABSTRACT Derivatized water-soluble Gd-based metallofullerenes are excellent MRI contrast agents with unusually large proton relaxivities for agents with no direct Gd−OH2 bonding. In this study, dynamic light scattering (DLS), static light scattering (SLS), and transmission electron microscopy (TEM) have been used to characterize the propensity of two such species, Gd@C60[C(COOH)2]10 and Gd@C60(OH)x, to aggregate in aqueous solution, since aggregation is known to enhance proton relaxivities of MRI contrast agents by increasing their rotational correlation times (via more slowly tumbling aggregates). The present aggregation study has been conducted as a function of concentration, temperature, and pH and has revealed that both compounds aggregate at pH ) 9 to form spherical and irregular clusters having sizes between 30 and 90 nm, with little concentration or temperature dependency. Below pH ) 9, the aggregate sizes increase steadily and dramatically, reaching hydrodynamic diameters of 600−1000 nm by pH ) 5. Additionally, the intermolecular forces holding the aggregates together are weaker for Gd@C60[C(COOH)2] than for Gd@C60(OH)x. We conclude that the tendency of these metallofullerene species to self-assemble into nanoscale aggregates in aqueous solution likely produces their unusually large, outer-sphere, pH-sensitive proton relaxivities.
Derivatized water-soluble fullerenes1 are being developed in the field of medicine for a variety of potential applications such as photodynamic therapy agents,2 HIV-1 protease inhibitors,3,4 neuroprotective agents,5,6 and X-ray contrast agents.7 In addition, endohedral metallofullerenes also show promise for magnetic resonance imaging8,9 and nuclear medicine.10-13 Gd@C60 belongs to the most abundantly produced class of endohedral metallofullerenes generated by the carbonarc process. Previously, M@C60 materials in general (including Gd@C60) were thought to be unusable in practical applications due to their insolubility and air sensitivity. However, recent breakthroughs in derivatization, solubilization, and stabilization of these species have overcome these limitations.14 Gd-containing metallofullerenes are currently being pursued as a new generation of MRI contrast agent. These * Corresponding author. E-mail:
[email protected]. Tel: 713-348-3268. Fax: 713-348-5155. † Rice University. ‡ TDA Research Inc. § University of Houston. 10.1021/nl0485713 CCC: $27.50 Published on Web 11/17/2004
© 2004 American Chemical Society
molecules offer fundamental advantages over commerciallyavailable chelate compounds, with the most important being the complete lack of Gd(III) ion dissociation under physiological conditions. Recent studies have shown that watersoluble Gd-based metallofullerenes can produce proton relaxivities greater than commercially-available MRI contrast agents.14,15 In the case of metallofullerenes, the Gd(III) ion must interact electronically with bulk water protons through the fullerene cage walls and their functionalized groups since no direct Gd(III)-OH2 bonding is possible. Thus, their relaxivities are entirely outer-sphere in origin. We recently performed the first relaxometry studies16 of two water-soluble Gd@C60 metallofullerenes, Gd@C60[C(COOH)2]10 and Gd@C60(OH)x (Figure 1a and b, respectively), and discovered that both species displayed a maximum in the high-field (10-100 MHz) region of their nuclear magnetic resonance dispersion (NMRD) profile. This profile feature suggests that the relaxivity maxima might arise from an increase in the rotational correlation time, τR, of the contrast agent.17,18 An increase in τR reflects slower-tumbling contrast agent molecules in solution, potentially from an increase in molecular weight by aggregation. Indeed, ag-
Figure 1. Depiction of (a) Gd@C60[C(COOH)2]10 and (b) Gd@C60(OH)x.
gregation has been reported to occur for C3, (a carboxyfullerene under development as a neuroprotective drug),6,19 as well as other water-soluble fullerene derivatives;20 however, no such information presently exists for a water-soluble metallofullerene. Due to the above considerations, we have conducted dynamic and static light scattering (DLS and SLS) experiments to examine Gd@C60[C(COOH)2]10 and Gd@C60(OH)x in aqueous solution in order to study their aggregation properties as a function of concentration, temperature, and pH. This has allowed the determination of their hydrodynamic diameters, size distributions, and average molecular weights. In addition, transmission electron microscopy (TEM) images have been obtained to provide visual structural information about the aggregate morphologies. The Gd@C60[C(COOH)2]10 and Gd@C60(OH)x samples used were prepared and characterized as described elsewhere.14,16 Both samples contained the metallofullerene of interest as well as similarly derivatized empty fullerenes of C60, C70, and C74 and Gd metallofullerenes of C70 and C74.14 The Gd@C60[C(COOH)2]10 sample was 2.8% Gd (m/m), while Gd@C60(OH)x was 0.64% Gd (m/m) by ICP. The sample solutions were prepared by the weighing method for all experiments (see Supporting Information). Simultaneous DLS and SLS experiments of the filtered solutions (0.45 mm syringe filter) were performed using a Malvern CGS-3 multiangle light scattering spectrophotometer with a HeNe laser (22 mW output power) operating at 632.8 nm. Each measurement was repeated at least three times. The deconvolution of the measured correlation curve to an intensitysize distribution was accomplished with a nonnegative leastsquares algorithm. The intensity-weighted mean hydrodynamic diameter (Dh) and the polydispersity index (PDI) were derived from cumulant fits of the intensity autocorrelation function. Figure 2a displays the hydrodynamic diameter of the two samples as a function of pH. The diameters were found to be highly pH dependent between pH ) 4-9, with an average diameter from 70 to 700 nm for Gd@C60[C(COOH)2]10 and 50-1200 nm for Gd@C60(OH)x. Figures 2b and c show the variation of average hydrodynamic diameter as a function of temperature and concentration. For the temperature range 15-55 °C, the average diameter varied from 58 to 70 nm for Gd@C60[C(COOH)2]10, while it varied from 45 to 50 nm for Gd@C60(OH)x. For Gd@C60[C(COOH)2]10, the average diameter was between 60 and 90 nm for concentrations greater than 2.5 mg/mL, while for Gd@C60(OH)x, it 2374
Figure 2. (a) Average hydrodynamic diameter of the aggregates formed by Gd@C60(OH)x and Gd@C60[C(COOH)2]10 in aqueous solution as a function of pH (conc. ) 5 mg/mL and temp ) 25 °C). (b) Average hydrodynamic diameter of the aggregates formed by Gd@C60(OH)x and Gd@C60[C(COOH)2]10 in aqueous solution as a function of temperature (conc. ) 5 mg/mL and pH ) 9.0). (c) Average hydrodynamic diameter of the aggregates formed by Gd@C60(OH)x and Gd@C60[C(COOH)2]10 in aqueous solution as a function of concentration (pH ) 9.0 and temp ) 25 °C).
was between 40 and 55 nm for concentrations greater than 1 mg/mL. The PDI for both samples in all experiments was found to be between 0.4 and 0.50. Also, the intensity of the light scattering increased with increasing concentration and temperature which can be attributed to an increase in the concentration of the aggregates without affecting the particle size. The autocorrelation curves were not well defined below 2.5 mg/mL for Gd@C60[C(COOH)2]10 and 1 mg/mL for Gd@C60(OH)x, indicating that the particle sizes were either too small or the solutions too dilute below these concentrations. Figures 3a and b show the intensity size distributions for Gd@C60[C(COOH)2]10 and Gd@C60(OH)x, respectively, at various concentrations. The data clearly display bimodal distributions for both compounds. For Gd@C60[C(COOH)2]10, Nano Lett., Vol. 4, No. 12, 2004
Figure 4. Debye plot for Gd@C60(OH)x and Gd@C60[C(COOH)2]10 in aqueous solution at 25 °C.
Figure 3. Intensity size distributions for (a) Gd@C60[C(COOH)2]10 and (b) Gd@C60(OH)x in aqueous solution at different concentrations (pH ) 9.0 and temp ) 25 °C).
as the concentration is decreased, the apparent size of both the faster and slower diffusing modes show a shift to lower values, while no such shift is observed for Gd@C60(OH)x. This result implies a shift in the population of larger aggregates to smaller ones for Gd@C60[C(COOH)2]10 but not for Gd@C60(OH)x. This difference in aggregation behavior may be due to a difference in the intermolecular and intramolecular interactions between aggregated species. A similar trend was observed, but to a lesser extent, in their intensity size distribution at various temperatures as seen in Figure S3c and d (Supporting Information). To examine the possible intermolecular/intramolecular interactions in the two samples, SLS experiments were also performed. Practically no angular dependence of the scattered light intensity was observed. This may be due to a majority of the aggregates in solution being smaller than the wavelength of incident light, thus reducing Mie scattering. Figure 4 shows the plot of KC/Rθ vs concentration for both solutions. The plot allows for the determination of the molecular weight, Mw, and second osmotic virial coefficient, A2, using the relationship between the Rayleigh ratio Rθ and the mass concentration of the compounds, C21 KCMw/Rθ ) 1 + 2 A2 Mw C
(1)
where K ) (2π n0/λ2)2 (dn/dC)2/NA. In eq 1, NA is Avogadro’s number, n0 is refractive index (RI) of the solvent, and λ is wavelength of scattered light. The presentation of the static light scattering results in terms of eq 1 is often called a Debye plot. The refractive Nano Lett., Vol. 4, No. 12, 2004
index increments, dn/dC, needed for interpretation of the SLS data were determined by a Wyatt Technologies optilab device and were found to be 0.1 mg/mL for both the samples. All measurements were carried out at 25 ( 0.1 °C. The measured average molecular weight, 2nd virial coefficient, and the calculated average aggregation number are presented in Table 1. The results show that the aggregates in both solutions to have a high average molecular weight (∼105) and hence a high average aggregation number (∼102). Gd@C60(OH)x (assuming x ∼ 27) displays a higher average molecular weight and a greater aggregation number (by about a factor of 2) than Gd@C60[C(COOH)2]10. Also, for Gd@C60[C(COOH)2]10, the average molecular weight decreases with a decrease in concentration, indicating that the aggregates are breaking up into smaller fragments. This trend is consistent with that observed in the intensity-size distribution in Figure 2. The second virial coefficient represents particle interaction strength and has been correlated with sample solubility.22 The negative slope value measured for Gd@C60(OH)x indicates that it has a slight preference for aggregation as opposed to solvation. Gd@C60[C(COOH)2]10 shows a positive slope and also a clear discontinuity in the plot with two distinct data series. The slope of the series below 8 mg/mL is more positive than the series above 8 mg/mL indicating that the positive value increases with dilution. This trend indicates that repulsive forces between aggregates dominate so that aggregate-solvent interactions are favored over those between the aggregates alone. Thus, the individual molecules within a Gd@C60[C(COOH)2]10 aggregate seem to be held together by weaker forces when compared to Gd@C60(OH)x. Biodistribution data for water-soluble fullerene compounds in small animals has been reported in several recent studies. In vivo radiotracing13,23 and MRI9 studies using polyhydroxylated fullerene compounds, each differing in terms of carbon cage structure, degree of hydroxylation, and endohedral metal content (or lack thereof), revealed significant uptake of the polyhydroxylated fullerenes by the organs and tissues of the reticuloendothelial system (RES). Conversely, the carboxylated Gd metallofullerenes, Gd@C60[C(COOH)2]10, displayed different biodistribution characteristics.14 This 2375
Table 1. Average Molecular Weight, 2nd Virial Coefficient, and the Calculated Average Aggregation Number Obtained from SLS Data for Gd@C60(OH)x and Gd@C60[C(COOH)2]10 sample Gd@C60(OH)x Gd@C60[C(COOH)2]10
conc. C (mg/mL)
average molecular weight Mw (g/mol)
monomeric molecular weight M (g/mol)
average aggregation number Mw/M
2nd virial coefficient A2 (mL mol/g2)
1-5
3.26 × 105
1269
257
-0.01 × 10-5
5-8 8-10
1.27 × 105 2.57 × 105
2458 2458
51 105
0.04 × 10-5 0.03 × 10-5
carboxylated metallofullerene did not localize in the RES, but instead was rapidly excreted renally. These results raise an important question: why is RES uptake notably reduced for the carboxylated fullerene relative to the hydroxylated ones? The answer likely has important implications for fullerene-based drug development, as excessive or rapid RES uptake can mitigate the prospects for new drug candidates. The current DLS and SLS results, as well as the TEM imaging (see below) seemingly provide a first step toward answering this question. RES uptake is governed by many factors, one of which is particle size. Estimates of the size threshold that can induce RES uptake vary widely, but there is general agreement that particles larger than ca. 50 to 100 nm in diameter will not evade the RES. In the present study, aggregates of both Gd@C60[C(COOH)2]10 and Gd@C60(OH)x were found to be larger than this range at pH 7, but aggregates of the carboxylated derivative were also found to be held together by weaker forces. Possibly, as a result, the RES does not recognize the carboxylated materials as large aggregates, while the hydroxylated fullerene aggregates are too large and cohesive to evade RES uptake. The difference in surface charge between the two derivatives may also play a role in both aggregate cohesion and charge-based RES response. Finally, it should be mentioned that different derivatization procedures (of which there are several for fullerene polyhydroxylation) might produce compounds of varied structure and composition that have different aggregation characteristics. To further characterize the nature of the aggregates found by light scattering, TEM imaging was performed using a JEOL 2000 FX electron microscope operated at 130 kV and equipped with a cryogenic and heating sample holder. TEM analyses included conventional and high-resolution TEM imaging and selected-area electron diffraction (SAED). The samples were prepared and loaded as described in the Supporting Information. Cryo and RT TEM studies were performed with caution to minimize the effect of heating and irradiation influence by the e-beam. No solvent dissipation process was observed. Figures 5a and b show cryo and RT TEM images of Gd@C60(OH)x at 1.25 mg/mL and pH ) 9. The aggregates are diffuse spherical and irregularly shaped clusters. The SAED patterns, shown as insets and taken in the same area as the images, display ring diffracting patterns at low temperature as well as at RT, indicating a nanocrystalline structure for Gd@C60(OH)x under both conditions. A C60(OH)x control did not show any crystallinity at RT, while for the Gd@C60(OH)x sample, crystallinity was observed only from the dark areas in Figure 5b. Thus, the 2376
observed crystallinity seems due to only the metallofullerenes in 5b (dark areas) which aggregate preferentially with themselves and not with empty fullerene species (light areas). Figures 5c and d show the cryo and RT TEM images of Gd@C60[C(COOH)2]10 at 3.4 mg/mL and pH ) 9. The images display a large number of small spherical clusters and irregular-shaped clusters. The SAED pattern (shown in the inset) for the cryo temperature was an impressive spot diffraction pattern demonstrating a single-crystal structure for the Gd@C60[C(COOH)2]10 aggregates with the size of crystalline areas being rather large. Gd@C60[C(COOH)2]10 clusters lose their crystallinity when warmed, as evidenced by the SAED diffraction pattern at RT. The size of the clusters for both samples was found to be between 30 and 90 nm, in good agreement with the average hydrodynamic diameter obtained by DLS. Comparing these images to those taken at higher concentrations, there is no major change in size or structure of the aggregates. The fact that only the Gd@C60(OH)x aggregates maintain their crystallinity when warmed to RT (while those of Gd@C60[C(COOH)2]10 do not) suggests that Gd@C60(OH)x molecules within the aggregates are more strongly associated with one another when compared to Gd@C60[C(COOH)2]10. Previously, we reported preliminary light scattering measurements for Gd@C60[C(COOH)2]10, Gd@C60(OH)x, and C60(OH)x by DLS at pH ∼ 7.14 These measurements showed aggregates >100 nm diameter for Gd@C60(OH)x and C60(OH)x, but no aggregation was detected for Gd@C60[C(COOH)2]10. These earlier results, obtained with less sensitive instrumentation, differ from the current results, which were obtained with more sophisticated DLS and SLS instrumentation under systematically varied conditions (temperature, pH, concentration), as well as being independently verified by cryo-TEM. For these reasons, we now believe the results stated here are correct. The emerging picture from the above results leads us to conclude that 30-90 nm aggregates of Gd@C60(OH)x and Gd@C60[C(COOH)2]10 at pH ) 9 are large enough to tumble slowly and thus increase the molecular rotational correlation time sufficiently (ps f ns) to generate the unusually large proton relaxivities observed for these metallofullerene species in aqueous solution.16 The marked pH dependency on proton relaxivity exhibited by both compounds can be interpreted as resulting from the pH-dependent molecular aggregation, since the increased aggregate size at low pH (Figure 2a) undoubtly leads to slower molecular tumbling. This suggests that gadofullerenes could serve as pH-sensitive MRI contrast agents for diagnosing abnormal tissue such as tumors and Nano Lett., Vol. 4, No. 12, 2004
Figure 5. (a) Cryo- and (b) room-temperature TEM micrographs of the aggregate formed by an Gd@C60(OH)x aqueous solution microfilm (conc. ) 1.25 mg/mL, pH 9.0). Insets are the microdiffraction patterns of the microfilm. (c) Cryo- and (d) room-temperature TEM micrographs of the aggregate formed by an Gd@C60[C(COOH)2]10 aqueous solution microfilm (conc. ) 3.4 mg/mL, pH 9.0). Insets are the microdiffraction patterns of the microfilm.
arterial plaques, which are known to possess lower pH values (e 0.5 pH units) than healthy tissue.24,25 In conclusion, both Gd@C60(OH)x and Gd@C60[C(COOH)2]10 aggregate in aqueous solution with sizes ranging from 30 to 90 nm at pH ) 9. From the variable-pH (pH ) 4-9) DLS studies, aggregation for both species was found to be highly pH dependent, with Gd@C60(OH)x showing a greater degree of aggregation than Gd@C60[C(COOH)2]10. From variable-concentration and variable-temperature DLS studies at pH ) 9, only Gd@C60[C(COOH)2]10 showed a measurable dependency. SLS and TEM results indicate that the intermolecular forces between Gd@C60(OH)x molecules within aggregates are stronger than those between Gd@C60[C(COOH)2]10 molecules within its aggregates. Thus, Gd@C60(OH)x aggregates are less affected by changes in parameters such as concentration and temperature. Nevertheless, slowly tumbling, rigid aggregates of Gd@C60[C(COOH)2]10 and Gd@C60(OH)x undoubtedly reduce the rotational correlation time of both species sufficiently to produce the unusually large proton relaxivities for these atypical MRI contrast Nano Lett., Vol. 4, No. 12, 2004
agents which have only an outer-sphere contribution to relaxivity. In fact, the outer-sphere relaxivities for the gadofullerenols are apparently the largest outer-sphere relaxivities ever observed for any MRI contrast agent. Acknowledgment. The work was supported by the NIH (Grant 1-R01-EB000703) and the Robert A. Welch Foundation (Grant C-0627). We thank Prof. Andre´ E. Merbach, Dr. EÄ va To´th and Dr. Lothar Helm for helpful discussions about relaxivities and NMRD profiles. We also thank Prof. Peter G. Vekilov and Mr. Weichun Pan for access to the laser light scattering instrumentation. Supporting Information Available: Experimental details. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Nakamura, E.; Isobe, H. Acc. Chem. Res. 2003, 36, 807-815 and references therein. 2377
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