Fractal Behavior of Functionalized Fullerene Aggregates. I

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Fractal Behavior of Functionalized Fullerene Aggregates. I. Aggregation of Two-Handed Tetraaminofullerene with DNA† Qicong Ying,‡ Jun Zhang,‡ Dehai Liang,‡ Waka Nakanishi,§ Hiroyuki Isobe,§,| Eiichi Nakamura,§ and Benjamin Chu*,‡ Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, Department of Chemistry and ERATO (JST), The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and PRESTO, Japan Science and Technology Agency Received March 1, 2005 In tris-buffered saline (TBS) with a trace of dimethylformamide (DMF), the homoaggregation process of a functionalized fullerene, the two-handed tetraaminofullerene (TH), and the heteroaggregation process (complex formation) of TH with DNA (pGL3-control plasmid) were studied dynamically by using a combination of static and dynamic laser light scattering measurements. Fractal behavior was investigated in the aggregation process of both TH homoaggregates and TH-DNA heteroaggregates. The stability of aggregates in solution depends on the molar concentration ratio RM, defined as the molar ratio of moles of TH to moles of the DNA base pair. Higher RM values resulted in lower aggregate stability. The transition of the fractal dimension (Df) in TH homoaggregation by rapidly mixing 3.78 µM TH with an equal volume of the blank buffer was found to vary from a value of 1.46 to 2.02. Dynamic light scattering results revealed that, in the aggregation process, the change in the size distribution of aggregates with time could be related to a Df transition. In the Df transition region, the size distribution of homoaggregates displayed a drastic change from a single-mode distribution to a bimodal distribution, which clearly suggested a restructuring process with the formation of large aggregates. When the aggregation process finally reached equilibrium, Df ) 2.02, the size of the homoaggregates had a single mode but a broad distribution. However, TH-DNA heteroaggregation showed a Df transition from 1.58 to 1.7, but over a shorter time range of less than 5 min. Then, the Df value fluctuated in the range of 1.7 and finally reached an equilibrium value of Df ≈ 1.78, which was independent of molar concentration. There are two main action forces involved in the heteroaggregation process: van der Waals forces and attractive electrostatic forces, with the latter one being stronger and faster than that of the former. Therefore, a two-step action could occur in the heteroaggregation process. In the beginning of mixing, the attractive electrostatic forces dictated the aggregation process, and then van der Waals forces also got involved in the entire aggregation process. By using an initial concentration of 3.78 µM each and RM ) 1, TH-DNA heteroaggregates showed more stable solution behavior than the homoaggregates. The lower Df value of the heteroaggregates could be related to a looser compact structure. Results from scanning electron microscopy (SEM) also disclosed the different textures between TH homoaggregates and TH-DNA heteroaggregates; the former had a more dense packing than the latter one.

Introduction Fullerene (C60) has very limited solubility in organic solvents, such as benzene, toluene, pyridine, and so forth, and almost nondetectable solubility in water. Thus, the utilization of fullerene has been limited. Functionalized fullerenes containing different polar groups, such as amines or carboxylic acids, or polymer with polar groups, such as poly(ethylene oxide), have amphiphilic properties and become partially soluble. Their easier maneuverability has attracted a great deal of interest not only for their unusual molecular structure but also for their promising applications in biology and medicine.1 However, because of specific surface and charge interactions among modified fullerene and solvent molecules, the “dissolved” species exhibit a tendency to form aggregates or self-assembled supramolecular structures. Various interesting properties of fullerene and functionalized fullerene aggregates have been disclosed. The reversible aggregation behavior of C60 was revealed in benzene and later was also found in †

Part of the Bob Rowell Festschrift special issue. * Corresponding author. E-mail: [email protected]. ‡ Stony Brook University. § The University of Tokyo. | PRESTO, Japan Science and Technology Agency. (1) Nakamura, E.; Isobe, H. Acc. Chem. Res. 2003, 36, 807.

benzonitrile.2,3 Extensive studies on the aggregation of C60 or C70 in benzene mixture solvents, in carbon disulfide solution, and in aqueous colloidal solution have been reported.4-6 Recently, the self-assembled behavior of C60Ph5K and of C60 (CH3)5K in aqueous solution was investigated by laser light scattering (LLS). The results exhibited that the Ph5C60- (or (CH3)5C60-) anions associated and formed spherical bilayer vesicles. In addition, a more complex form of aggregation exists for C60(CH3)5K at relatively high concentrations.7,8 The flowerlike core-shell multimolecular micelles of modified fullerenes armed with different numbers of polymer chains were investigated in different organic solvents. The polymer chains included poly(ethylene oxide), a diblock copolymer of polystyrene-poly(p-vinylphenol), and poly(alkyl methacrylate).9-11 (2) Ying, Q.; Marecek, J.; Chu, B. J. Chem. Phys. 1994, 101, 2665. (3) Nath, S.; Pal, H.; Plit, D. K.; Sapre, A. V.; Mittal, J. P. J. Phys. Chem. B 1998, 102, 10158. (4) Bokare, A. D.; Patnaik, A. J. Chem. Phys. 2003, 119, 4529. (5) Ghosh, H. N.; Sapre, A. V.; Mittal, J. P. J. Chem. Phys. 1996, 100, 9439. (6) Andrievsky, G. V.; Klochkov, V. K.; Karyakina, E. L.; MchedlovPetrossyan, N. O. Chem. Phys. Lett. 1999, 300, 392. (7) Zhou, S.; Burger C.; Chu, B.; Sawamura, M.; Nagahama, N.; Toganoh, N.; Hackler, U. E.; Isobe, H.; Nakamura, E. Science 2001, 291, 1944. (8) Burger, C.; Hao, J.; Ying, Q.; Isobe, H.; Sawamura, M.; Nakamura, E.; Chu, B. J. Colloid Interface Sci. 2004, 275, 632.

10.1021/la050557y CCC: $30.25 © 2005 American Chemical Society Published on Web 08/13/2005

Fractal Behavior of Fullerene Aggregates

Globule aggregates of C60-poly(ethylene oxide) in tetrahydrofuran (THF), in dimethyl formamide (DMF), and in water were reported.12 Aggregation is a complex random process without apparent regular features as often revealed by selfsimilarity and universality. The mathematic concept of fractals in aggregation is a quantitative way to describe the nature of aggregates. Computer simulations have established models to reveal the regular features in random aggregation processes. Two universal aggregation regimessdiffusion-limited cluster aggregation (DLCA) and reaction-limited cluster aggregation (RLCA)shave been identified in many different colloid systems (e.g., colloidal gold, colloidal silica, and polymer latex suspensions) and also used as a template to characterize the structure and kinetics of aggregates.13-16 In DLCA, the aggregation is a relatively fast process, as limited by cluster diffusion, which shows a fractal dimension in the range of Df ≈ 1.7-1.8 in irreversible aggregation and Df ≈ 2.03 ( 0.05 in reversible aggregation.17 A power law with Rh ∝ t1/Df describes the kinetics, where Rh is the hydrodynamic radius of the cluster. However, RLCA is a slow aggregation process, as limited by the collision times for two clusters to overcome a repulsive energy barrier between them. It shows a fractal dimension in the range of Df ≈ 2.0-2.1 and exponential kinetics with Rh ∝ eCt, where C is a constant depending on the experimental conditions. A transition region between DLCA and RLCA was also reported.18 The fractal behavior of fullerene aggregation was revealed in benzene solution with Df ) 2.1 and exponential kinetics. It characterized the aggregation of C60 in benzene to be in the RLCA regime.2 The aggregation behavior of a functionalized fullerene with two ammonium headgroups in water showed that the vesicles also aggregated to form a fractal structure with Df ) 1.4.19 With the C60 aqueous colloid solution being prepared by transferring fullerene from toluene into water and dispersed by sonication,6,20 the C60 clusters had Df ≈ 1.95. Recently, the cationic C60 surfactant of two-handed tetraaminofullerene (TH) was synthesized. It could be dissolved in DMF and an aqueous solution with a small amount of DMF.21-23 The cationic C60 surfactant formed a complex with DNA (pGL3-control plasmid) in buffer solution. The transfection efficiency of the TH-DNA complex was reported to be comparable to that of a commercially available lipofection reagent.23 It is impor(9) Song, T.; Dai, S.; Tam, K. C.; Lee, S. Y.; Goh, S. H. Langmuir 2003, 19, 4798. (10) Okamura, H.; Ide, N.; Minoda, M.; Komatsu, K.; Fukuda, T. Macromolecules 1998, 31, 1859. (11) Wang, X.; Goh, S. H.; Lu, Z. H.; Lee, S. Y.; Wu, C. Macromolecules 1999, 32, 2786. (12) Song, T.; Dai, S.; Tam, K. C.; Lee, S. Y.; Goh, S. H. Polymer 2003, 44, 2529. (13) Weitz, D. A.; Oliveria, M. Phys. Rev. Lett. 1984, 52, 1433. (14) Weitz, D. A.; Huang, J. S.; Lin, M. Y.; Sung, J. Phys. Rev. Lett. 1985, 54, 1416. (15) Zhou, Z.; Wu, P.; Chu, B. J. Colloid Interface Sci. 1991, 146, 541. (16) Poon, W. C. K.; Haw, M. D. Adv. Colloid. Interface Sci. 1997, 73, 71. (17) Kolb, M. J. Phys. A: Math. Gen. 1986, 19, L263. (18) Odriozola, G.; Tirado-Miranda, M.; Schmitt, A.; Lo´pez, F. M.; Callejas-Ferna´ndez, J.; Martinez-Garcia, R.; Hidalgo-A Ä lvarez, R. J. Colloid Interface Sci. 2001, 90, 240. (19) Sano, M.; Oishi, K.; Ishi-I, T.; Shinkai, S. Langmuir 2000, 16, 3773. (20) Bulavin, L.; Adamenko, I.; Prylutskyy, Y.; Durov, S.; Graja, A.; Bogucki, A.; Scharff, P. Phys. Chem. Chem. Phys. 2000, 2, 1627. (21) Nakamura, E.; Isobe, H.; Tokuyama, H.; Sawamura, M. Chem. Commun. 1996, 1747. (22) Isobe, H.; Tomita, N.; Jinno, S.; Okayama, H.; Nakamura, E. Chem. Lett. 2001, 1214. (23) Nakamura, E.; Isobe, H.; Tomita, N.; Sawamura, M.; Jinno, S.; Okayama, H. Angew. Chem., Int. Ed. 2000, 39, 4254.

Langmuir, Vol. 21, No. 22, 2005 9825 Scheme 1. Structure of Two-handed Tetraaminofullerene (TH)

tant to elucidate the mechanistic features of C60-surfactant-DNA complex formation. In this work, the time evolution of the aggregation process of TH itself and of the TH-DNA complex formation in TBS buffer solution with a trace amount of DMF was investigated by alternatively conducting static and dynamic laser light scattering measurements. The transition in the fractal dimension as a function of structural evolution existed in both TH homoaggregate and TH-DNA heteroaggregate formation processes, with the latter one proceeding faster than the former one. The stability of aggregates in solution depended on the concentration of aggregates, with the higher concentration resulting in a lower stability of aggregates in solution. TH-DNA heteroaggregates were more stable in solution than TH homoaggregates, which had a higher fractal dimension and also a denser texture than the heteroaggregates. Experimental Section Materials. The synthesis and purification processes of twohanded tetraminefullerene (TH) were the same as described elsewhere.21,23 After TH was protonated by trifluoroacetic acid, it could be dissolved in DMF or a DMF/water mixture. Each TH molecule combined with four others.23 Afterwards, TH was protonated by trifluoroacetic acid molecules, which carried four positive charges here defined as a cationic C60 surfactant, TH. The chemical structure of TH is shown in Scheme 1. The molecular weight of TH, when combined with four trifluoroacetic acid molecules, has a value of 1683 g/mol. The DNA sample used in the present study was pGL3-control plasmid. Sample Preparation and Mixing Process. TH could be dissolved in DMF. The TH/DMF stock solution used in the mixing process with DNA had a concentration of 2 mM, which was a dark-brown color. The DNA/TBS stock solution was prepared by dissolving 3.78 µM DNA (here M is moles of DNA base pairs, 1 M ) 660 g/mol L) in TBS buffer solution, in which the salt content was 165 mM at pH 7.4. The concentration of the DNA/TBS solution was kept at a constant value of 3.78 µM in the mixing process. The variation in concentration of the TH-DNA mixed solution depended only on the amount of TH/DMF stock solution added to the DNA/TBS solution. Thus, in the present work, the concentration of the mixed solution was denoted in terms of the molar ratio RM, defined as the ratio of the number of moles of TH molecules to the number of moles of DNA base pairs. The conversion of RM to concentration in µM of TH and of DNA is listed in Table 1. In the homoaggregation process, the mixing experiment was conducted by adding a desired amount of TH/ DMF stock solution to the TBS buffer solution, with the molar concentration of TH being the same as the one used for the Table 1. Correlation of Molar Concentration Ratio RM with Concentration in µM of TH and of DNA Used DNA (µM)a

TH (µM)

1b

3.78

1c 10.6b 10.6c

3.72

3.78 3.78 39.2 39.2

RM

DMF vol % 0.2 0.2 2 2

a Moles of DNA base-pair, 1 M ) 660 g/L. b Heteroaggregation process. c Homoaggregation process.

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Ying et al.

heteroaggregation process. In the heteroaggregation process, the mixing experiment was conducted by adding the desired amount of TH/DMF stock solution to the DNA/TBS buffer solution. Thus, the DMF content in the final mixed solution was also varied with the amount of TH/DMF stock solution added, but the variation of DMF was only a small percentage ( 1 region, the value Df could be estimated from the slope in the linear region of q > ∼0.01 nm-1. The change in the fractal dimension Df of TH-DNA heteroaggregates as a function of time after mixing is shown in Figure 8 with the Rh,app distributions for RM ) 1 in different time zones after mixing (marked as A-C in Figure 8) also being shown in panels A-C, respectively. A comparison of the behavior of TH homoaggregates (as shown in Figure 5) with the heteroaggregates showed that the Df values of TH-DNA

heteroaggregates also underwent a transition from 1.58 to 1.7, but only over a very short time period of less than 5 min. Then the Df value fluctuated in the range of 1.7 and finally reached an equilibrium value of Df ≈ 1.78, which was independent of the initial molar concentration. A faster Df transition could be attributed to the strong attractive electrostatic forces. In the meantime, the average Rh,app of TH-DNA heteroaggregates varied very little from or remained essentially the same as a value of 109-119 nm for the entire 2 h mixing process after the formation of the TH-DNA heteroaggregates, as shown in panels A-C of Figure 8. The Rh,app distribution of THDNA heteroaggregates showed a single peak without large clusters formed. To make a comparison between the Rh,app distributions measured 11.5 min after mixing, as shown in panel A, and that measured at 122 min, corresponding to an equilibrium time, as shown in panel C, the two Rh,app distributions had a similar shape in the distribution curve and the same variance value of ∼0.5. This revealed that the TH-DNA heteroaggregates’ formation was a faster process and quickly reached a steady size distribution because the aggregates showed only slight restructuring (as shown in panel B). This result is consistent with the AFM observation in the study of condensation of plasmid DNA with functionalized fullerene.29 The SLS results also showed that the average Rg,app of TH-DNA heteroaggregates held a steady value of around 200 nm in the 2 h period after mixing. However, in the same time period the average Rg,app value of TH homoaggregates increased from ∼250 to ∼600 nm, as shown in Figure 9. In TH-DNA TBS solution at pH 7.4, the theoretical neutralization point of DNA phosphate charges with TH is RM ≈ 0.5. Generally, the DNA molecular size could become smaller or collapsed when the DNA phosphate charges were all neutralized. However, we found that the average size of TH-DNA heteroaggregates was even slightly expanded at RM ) 1 and the Rh,app value of DNA was about 71 nm whereas that of TH-DNA heteroaggregates was ∼120 nm, again in agreement with the observations by AFM.29 This finding could be illustrated as an intercalation effect. Because of attractive electro(29) Isobe, H.; Sugiyama, S.; Fukui, K.; Iwasawa, Y.; Nakamura, E. Angew. Chem., Int. Ed. 2001, 40, 3364.

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Figure 9. Apparent radius of gyration as a function of the mixing time of TH homoaggregates and TH-DNA heteroaggregates at a molar concentration ratio of RM ) 1.

Ying et al.

TH-DNA aggregates, which is then followed by a further restructuring process. Both the SLS and DLS results indicated that at RM ) 1 the solution behavior of TH-DNA heteroaggregates was more stable than that of the TH homoaggregates. However, for the aggregation process of TH-DNA at RM ) 10.6, the mixed solution did not show Df transition behavior, and precipitation appeared after ∼50 min. The DNA condensation dependence on the concentration of TH-DNA and the enormous TH-DNA aggregates were also observed by AFM even at RM ) 2.6.29 The equilibrium Df value of 1.78 for the heteroaggregates was lower than the Df value of 2.02 for the TH homoaggregates. The TH-DNA complex could have a looser aggregate structure than that of TH homoaggregates. Panels a and b of Figure 10 are SEM pictures of TH homoaggregates and TH-DNA heteroaggregates, respectively. A comparison of the two SEM pictures clearly shows that the structure of TH-DNA heteroaggregates (Figure 10b) with a low Df value ∼1.78 is less compact than that of TH homoaggregates with a higher Df value of 2.02 (Figure 10a). The SLS and DLS results are in agreement with the observations from AFM.29 However, a combination of SLS and DLS results could provide more detailed information on the fractal dimension and its transition as related to the restructuring in the aggregation process. Conclusions

Figure 10. SEM pictures of (a) TH aggregates and (b) THDNA heteroaggregates. Scheme 2. Complexation of DNA and Two-handed Tetraaminofullerene (TH)

Molecular model shows a possible structure of the complex (Blue and green ) DNA strand, purple ) TH).

static forces, the C60 cores bind the DNA molecules that could cause the TH-DNA aggregates to behave as though they have a relatively sparse structure and also slow the aggregation rate. The TH molecules could bind DNA in the major groove of the DNA duplex to maintain the structural integrity of the helix (a possible representation is shown in Scheme 2). By combining the results of SLS and DLS, it is reasonable to understand that in the heteroaggregation process a two-step action could be involved: in the beginning period immediately after the mixing process, TH molecules bind DNA chains to form

Light scattering results revealed that the structural evolution of both TH and TH-DNA aggregates (homo- and heteroaggregates, respectively) could be presented in terms of fractal dimensions. The stability of aggregates in solution depended on molar concentration ratio RM with the higher RM value resulting in a lower stability of the aggregates. For RM ) 1, the Df as a function of time after mixing changed from a value of 1.46 to 2.02 for the TH homoaggregation, and a very fast transition of Df from a value of 1.58 to 1.77 in the TH-DNA heteroaggregation process occurred. Fractal dimension Df ) 2.02 ( 0.04 found in TH homoaggregation was higher than the value of Df ≈ 1.7-1.8 for an irreversible DLCA but was in the range of 2.03 ( 0.05 for a reversible DLCA. The TH homoaggregates could be grouped with the reversible DLCA. The DLS results revealed an aggregate restructuring process that occurred in the Df transition region. In the TH homoaggregation, the Rh,app distribution started from a single peak with a (large) variance of 0.6, progressed to two broad peaks appearing in the middle of the Df transition region with the larger peak exhibiting an even larger tail, and then finally reached an equilibrium state with one broad peak. However, in the TH-DNA heteroaggregation process, two different processes could be detected. The transition of Df from a value of 1.58 to 1.7 in a very short time range could be attributed to strong attractive electrostatic interactions in the beginning of the heteroaggregation process. The formation of heteroaggregates proceeded quickly, but the TH-DNA heteroaggregates had a steady size distribution with the average size being only slightly larger than (or comparable to) that of the DNA chains before complex formation. The LLS results found in this study agreed with the AFM observations. In comparison with TH homoaggregates, TH-DNA heteroaggregates had a low Df value corresponding to a looser packing structure. Acknowledgment is made to the donors of the American Chemical Society Petroleum Research Fund for partial support of this research as well as to the National Science Foundation (DMR-0454887) for their support. This study was also financially supported by Monbukagakusho,

Fractal Behavior of Fullerene Aggregates

Japan (the 21st Century COE Program for Frontiers in Fundamental Chemistry to E.N.), and SUNBOR (to H.I.). W.N. thanks the Japan Society for Promotion of Science for a predoctoral fellowship. This article is dedicated to Robert Rowell to honor him on the occasion of his 20th

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anniversary as an Editor for Langmuir and to honor his distinguished career and contributions to colloid and surface chemistry LA050557Y