NANO LETTERS
Nanoscale Aggregation Properties of Neuroprotective Carboxyfullerene (C3) in Aqueous Solution
2004 Vol. 4, No. 9 1759-1762
Balaji Sitharaman,† Subashini Asokan,‡ Irene Rusakova,§ Michael S. Wong,*,‡ and Lon J. Wilson*,† Department of Chemistry, Department of Chemical Engineering, Center for Nanoscale Science and Technology, and Center for Biological and EnVironmental Nanotechnology, Rice UniVersity, Houston, Texas 77251-1892, and Texas Center for SuperconductiVity and AdVanced Materials, UniVersity of Houston, Houston, Texas 77204-5002 Received May 7, 2004
ABSTRACT Water-soluble malonic acid derivatives of C60 are known to have potent antioxidant activity with potential medical applications as neuroprotective agents. It is commonly assumed that e,e,e tris-malonic acid-C60 (or C3) exists as discrete molecules solubilized in aqueous solution. In this work, C3 is revealed to aggregate in water. The aggregation properties have been studied as a function of concentration, temperature, and pH by dynamic light scattering (DLS). The C3 aggregates are polydisperse under physiological conditions, do not vary much in size as a function of concentration or temperature, and tend to larger sizes at low pH values. Transmission electron microscopy (TEM) and cryo-TEM have been used to visualize the morphology of the nanocrystalline aggregates. The results suggest that 40−80 nm aggregates of C3, not individual C3 molecules, are responsible for their neuroprotective action in cells.
Water-soluble fullerene derivatives hold promise for medicinal applications.1,2 Increased availability of these watersoluble fullerene derivatives has enabled many biologically relevant studies of these compounds which have potential uses for photodynamic therapy,3 inhibitors of the HIV-1 protease,4,5 and possibly X-ray contrast agents.6 They have also been shown to be potent radical scanvengers7 and, hence, highly effective antioxidants. Stress or pathological conditions in a cell or organism generate free radicals that act as mediators in cell damage and even death. In the central nervous system, overstimulation of the glutamate receptor produces superoxide and nitric oxide radicals which form the genesis of many neurogenerative diseases.8-11 In a landmark paper, Dugan and co-workers harnessed the potent radical-scavenging property of C60 and demonstrated its potential therapeutic use as a neuroprotective agent both in vitro and in vivo.12 They used the water-soluble malonic acid trisadduct, C60[C(COOH)2]3, of C3 symmetry (Figure 1), which is highly water soluble, with a well-defined structure that has good stability.13 Termed C3, this fullerene is actively being pursued * Corresponding authors:
[email protected] (LJW); Voice: (713) 3483268; Fax: (713) 348-5155.
[email protected]; Voice: (713) 348-3511; Fax: (713) 348-5478. † Department of Chemistry, Rice University. ‡ Department of Chemical Engineering, Rice University. § University of Houston. 10.1021/nl049315t CCC: $27.50 Published on Web 07/28/2004
© 2004 American Chemical Society
Figure 1. Structure of C3.
as a prime drug candidate for various neurological diseases, as well as for possible lifespan extension.14,15 Complete characterization of any new drug candidate is fundamental to its success, and a key component of any characterization is an accurate assessment of its aggregation state. Compounds can aggregate as a function of temperature, pH, ionic strength, and concentration. Even small degrees of aggregation can be significant, since they cause conformational shifts in molecular structure which can alter the function of a therapeutic agent. Indeed, biological tests
Figure 2. (a) Average hydrodynamic diameter of the aggregates formed by C3 as a function of pH (concentration ) 5 mg/mL and temp ) 25 °C). (b) Average hydrodynamic diameter of the aggregates formed by C3 as a function of concentration (pH ) 7.4 and temp ) 25 °C). (c) Average hydrodynamic diameter of the aggregates formed by C3 as a function of temperature (concentration ) 10 mg/mL and pH ) 7.4).
performed on water-soluble fullerene derivatives have shown the results to be strongly affected by aggregation.16 Specifically for C3, Guldi and co-workers17,18 earlier used UVvis spectrophotometry and pulse radiolysis on very dilute aqueous samples to characterize the compound as being monomeric, but no data were given for higher concentrations. Using dynamic light scattering (DLS) and transmission electron microscopy (TEM), the present work demonstrates that C3 exhibits strong aggregation behavior in aqueous solution. Furthermore, this aggregation has been investigated as a function of pH, temperature, and concentration by DLS, while TEM images have also been obtained to provide more detailed structural information about the aggregates. The C3 sample used was prepared by a literature method as described elsewhere.13 The purified product was characterized by 1H and 13C NMR spectroscopy. The C3 solutions were prepared by the weighing method. In all the experiments, HPLC grade water was used and was filtered through a 0.22 µm syringe filter (Millpore, MILLEX-GP). The solutions were prepared by ultrasonication, followed by centrifugation and decantation. The decanted solutions were then filtered through a 0.45 mm syringe filter (Whatman). Prior to sample loading, appropriate quartz cuvettes were cleaned with soap solution followed by rinsing with ethanol, acetone, and finally distilled water, before being dried in a vacuum oven. DLS measurements were performed on the samples using a Brookhaven 90Plus submicron particle size analyzer with HeNe laser (30 mW) operating at 656 nm wavelength. The slightly colored C3 samples had an absorbance (measured at 656 nm) below 0.1. The cumulant method was used to derive information about the aggregate size distribution in the form of the polydispersity index, and a Laplace inverse 1760
program called NNLS was used to determine the intensityweighted aggregate particle size in the form of hydrodynamic diameter (Dh). NNLS was chosen over the more common CONTIN inversion method in this study because it can account for broad and multimodal size distributions.19,20 Measurements were repeated at least three times. The aggregate Dh’s were measured as a function of pH, concentration, and temperature (Figure 2a-c). The average Dh’s were found to be essentially invariant with concentration or temperature, but they were found to have a strong dependence on solution pH. The aggregates increased in size with decreased pH, which can be explained by considering the effect of proton dissociation of the C3 malonic acid groups. At pH above their pKa (assumed to be close to the pKa values of diethylmalonic acid, 2.99 and 5.83), the acid groups are deprotonated, thereby maximizing C3 solubility in water. Any aggregation would be due to hydrophobic interactions between the exposed C3 fullerene surface (Figure 1). At lower pH, the acid groups are less dissociated and therefore less charged; particles with a reduced surface charge would be more susceptible to aggregation than those with high surface charges. Thus, the pH effect on C3 aggregation is consistent with the DLVO (Derjaguin-Landau-VerveyOverbeek) theory of colloidal stability.21 At pH below 4, the C3 molecules rapidly aggregated to form a precipitate. The precipitation may be aided by hydrogen bonding between the COOH groups of adjacent C3 molecules. Under all solution conditions, the aggregate sizes were broadly distributed. The polydispersity index (PDI) provides a metric for describing the size distribution. PDI values between 0 and 0.05 indicate very monodisperse particles, and values above 0.05 indicate broadened size distributions.22 Nano Lett., Vol. 4, No. 9, 2004
Figure 3. (a) Cryo-TEM micrograph of the aggregates formed by a C3 solution microfilm (concentrated ) 0.1 mg/mL, pH ) 7.4). (b) Microdiffraction pattern of the same microfilm taken under cryogenic conditions. (c) TEM image of the same solution microfilm at room temperature. (d) TEM image of the same solution microfilm at higher resolution.
The C3 solutions were determined to have PDI values in the range of 0.25-0.55 and 0.35-0.54 in temperature and concentration ranges studied, respectively. The PDI increased with decreasing pH, from 0.22 to 0.51, indicating that the C3 aggregates became more nonuniform as they grew in size. To visualize the actual aggregate size and morphology, cryo-TEM and TEM experiments were performed. An important aim of these experiments was to check for aggregation in the very dilute concentration range of 0.050.5 mg/mL. These very dilute solutions did not scatter sufficiently for DLS measurements, and hydrodynamic diameters could not be obtained. Cryo-TEM and TEM analyses of C3 were carried out on a JEOL 2000 FX electron microscope equipped with a cryogenic and heating sample holder operated at 130 kV. TEM analysis included conventional imaging and selected area electron diffraction (SAED). Samples for cryo-TEM studies were prepared by dipping a copper grid coated with amorphous carbon-holey film into the sample solution. The grid was then frozen in liquid nitrogen and immediately moved into the TEM cold stage, inserted into the column of the microscope, and kept at -153 °C. Precautions were taken to minimize the heating and irradiation influence of the electron beam on the samples. The cryo-TEM technique is well suited to the observation of these materials since the chance of perturbing the actual structure in solution is minimized. Nano Lett., Vol. 4, No. 9, 2004
Images were taken in the concentration range of 0.05 to 5 mg/mL. Figure 3a displays a cryo-TEM image at 0.1 mg/ mL. The image shows spherical and ellipsoidal clusters homogeneously distributed, with an average aggregate size of 40-80 nm. These aggregates are estimated to contain ∼6 × 104 to ∼5 × 105 C3 molecules. The SAED displays a ring diffracting pattern (Figure 3b), which indicates the polycrystalline nature of C3 aggregates for the first time, similar to the recently observed polycrystallinity of unfunctionalized C60 aggregates suspended in water.23 Electron microscopy studies of higher concentration samples indicated no significant difference in the observed size or structure of the C3 aggregates. After completing the cryo-TEM studies, the same TEM samples were lyophilized inside the TEM column, which allowed for in situ observation of possible structural changes. The final stage of the TEM study was performed at room temperature and in the concentration range 0.05 to 1 mg/ mL. Figures 3c,d show low- and high-magnification TEM images of the aggregates at room temperature and at 0.1 mg/ mL. The aggregates were mostly spherical in shape, along with a few ellipsoidal and nanotubular clusters. Interestingly, electron diffraction measurements taken in the same area as the images showed diffuse rings rather than discrete spots. It appears that C3 aggregates are crystalline while in 1761
suspension, and that they lose their internal ordering upon drying. The observed aggregation behavior of C3 in this work may have ramifications about ongoing studies of C3 as an antioxidant against biologically reactive oxygen species (such as superoxide anion, hydrogen peroxide, and hydroxyl radical). Until recently, it was thought that C3 was stoichiometrically involved in its radical-scavenging reaction,12,24 but new data on C3 as a superoxide dismutase mimic suggest that it may be involved as a catalyst rather than as a stoichiometric reactant.25 In this scenario, the superoxide anions would be catalytically converted to oxygen and hydrogen peroxide by crystalline aggregates containing ∼104 to 105 C3 molecules, possibly working in some concerted fashion. The influence of aggregate size and structure on the catalytic properties of C3 must now be considered. In conclusion, DLS and TEM studies on the fullerene derivative C3 have shown that the compound exists as aggregates in water at various concentrations (0.05-10 mg/ mL). The aggregates are polydisperse and polycrystalline. Their average size is independent of concentration and temperature, but is strongly dependent on pH between pH 4-8. The aggregates, rather than individual C3 molecules, are possibly interacting with biologically reactive oxygen species during their neuroprotective action within cell membranes.24 Acknowledgment. L.J.W. thanks the Robert A. Welch Foundation (Grant C-0627) for support of this work. We also thank C Sixty, Inc. for the sample of C3, and the Texas Center for Superconductivity and Advanced Materials, University of Houston, for the TEM images. M.S.W. gratefully acknowledges financial support from Rice University, Oak Ridge Associated Universities, and the National Science Foundation (EEC-0118007). References (1) Wilson, L. J. Interface 1999, 8 (4, winter), 24.
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(2) Wilson, S. R. In Fullerenes Chemistry, Physics, and Technology; Kadish K. M., Ruoff, R. S., Eds; John Wiley & Sons: New York, 2000; p 437. (3) Tokuyama, H.; Yamago, S.; Nakamura, E.; Shiraki, T.; Sugira, Y. J. Am. Chem. Soc. 1993, 115, 6506. (4) Friedman, S. H.; DeCamp, D. L.; Sijbesma, R. P.; Srdanov, G.; Wudl, F.; Kenyon, G. L. J. Am. Chem. Soc. 1993, 115, 7918. (5) Sijbesma, R.; Srdanov, G.; Wudl, F.; Castoro, A.; Wilkins, C.; Friedman, S. H.; Decamp, D. L.; Kenyon, G. L. J. Am. Chem. Soc. 1993, 115, 6510. (6) Wharton, T.; Wilson L. J. Bioorg. Med. Chem. 2002, 3545-3554. (7) Krusic, P. J.; Keizer, J.; Morton, J. R.; Preston, K. F. Science 1991, 254, 1183. Hirsch, A. The Chemistry of the Fullerenes; Thieme: Stuttgart 1994. (8) McGeer, E. G.; McGeer, P. L. Nature 1976, 263, 517-524. (9) Rothman, S. M.; Olney, J. W. Ann. Neurol. 1986, 19, 105-111. (10) Choi, D. W. Neuron 1988, 1, 623-634. (11) McIntosh, T.; Soares, H.; Hayes, R.; Simon, R. In Frontiers in Excitatory Amino Acid Research; Cavallo, E. A.; el al., Eds.; Liss: New York, 1988; pp 653-656. (12) Dugan, L. L.; Turetsky, D. M.; Du, C.; Lobner, D.; Wheeler, M.; Almli, C. R.; Shen, C. K.; Luh, T. Y.; Choi, D. W.; Lin, T. S. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 9434. (13) Lamparth, I.; Hirsch, A. J. Chem. Soc. Chem. Commun. 1994, 1727. (14) Dugan, L. L.; Lovett, E. G.; Quick, K. L.; Hardt, J. I. 2004, U.S. Patent Application, Pub. No. US 20040034100A1. (15) Dugan, Laura L.; Lovett, Eva G.; Quick, Kevin L.; Hardt, Joshua I. 2003, U.S. Patent Application, Pub. No. 20030162837A1. (16) Da Ros, T.; Prato, M. Chem. Commun. 1999, 663-669. (17) Guldi, D. M.; Hungerbu¨hler, H.; Asmus, K. J. Phys. Chem. B 1999, 103, 1444-1453. (18) Guldi, D. M. Res. Chem. Intermed. 1997, 23, 653-673. (19) Pecora, R. J. Nanopart. Res. 2000, 2, 123-131. (20) Morrison, I. D.; Grabowski, E. F.; Herb, C. A. Langmuir 1985, 4, 496-501. (21) Hunter, R. J. Foundations of Colloid Science, 3rd ed.; Oxford University Press: New York, 2001. (22) Pecora, R. Dynamic Light Scattering; Plenum Press: New York, 1985. (23) Deguchi, S.; Alargova, R. G.; Tsujii, K. Langmuir 2001, 17, 60136017. (24) Wang, C. I.; Tai, L. A.; Lee, D. D.; Kanakamma, P. P.; Shen, C. K.; Luh, T.; Cheng, C. H.; Hwang, K. C. J. Med. Chem. 1999, 42, 46144620. (25) Ali, S. S.; Hardt, J. I.; Quick, K. L.; Kim-Han, J. S.; Erlanger, B. F.; Huang, T.; Epstein, C. J.; Dugan, L. L. The Electrochemical Society 2004, 205th meeting, Abs. 590.
NL049315T
Nano Lett., Vol. 4, No. 9, 2004
