Self-Assembled Photoresponsive Amphiphilic Diphenylaminofluorene

Mar 12, 2005 - ... Nanoscience and Engineering, University of Massachusetts, Lowell, .... Science and Technology of Advanced Materials 2008 9 (1), 014...
3 downloads 3 Views 410KB Size
Download PDF
Langmuir 2005, 21, 3267-3272

3267

Self-Assembled Photoresponsive Amphiphilic Diphenylaminofluorene-C60 Conjugate Vesicles in Aqueous Solution Sarika Verma,† Tanya Hauck,‡ Mohamed E. El-Khouly,§ Prashant A. Padmawar,† Taizoon Canteenwala,† Kenneth Pritzker,‡ Osamu Ito,§ and Long Y. Chiang*,† Department of Chemistry, Institute of Nanoscience and Engineering, University of Massachusetts, Lowell, Massachusetts 01854, Department of Pathology and Laboratory Medicine, Mount Sinai Hospital and University of Toronto, Toronto, Canada, and Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai 980-8577, Japan Received November 28, 2004. In Final Form: February 7, 2005 Water-soluble oligo(ethylene glycolated) derivatives of two-photon absorptive diphenylaminofluorenocarbonyl-methano[60]fullerene, denoted as C60(>DPAF-EG6), were synthesized with their molecular selfassembly characteristics in H2O studied. The formation of nano- to submicron-sized spherical hollow vesicles with a shell width of 15-20 nm was observed by transmission electron microscopy (TEM) micrographs. This shell width fits approximately with the length of a disordered bilayer-like molecular packing of C60(>DPAF-EG6), arising from strong intermolecular hydrophobic interactions of fullerene cages. Photoinduced intramolecular charge separation followed by charge recombination on the nanosecond time scale, from the DPAF moiety to the C60 cage in the vesicle structure, was detected via transient spectroscopic measurements.

Molecular assembly of amphiphilic fullerene derivatives in aqueous solution is associated primarily with the hydrophobic interaction of fullerene cages as the major attractive driving force for these types of molecules to aggregate in a self-organized shape. Several C60 monoadducts comprised of one or two hydrophilic heads, such as carboxylic acid or its anionic salt,1 poly(acrylic acid),2 cationic ammonium salts,3-7 and nonionic poly(ethylene glycol),8,9 serve well in molecular assembly, forming submicrospheres to nanospheres or nanorods. Significant intermolecular fullerenic interaction forces were revealed even with the highly water-soluble molecular micelle-like structure of hexa(sulfo-n-butyl)[60]fullerene (FC4S)10 and hexa(sulfo-n-butyloxypentylcarbonyl)[60]fullerene (FC10S).11 This self-assembly behavior resulted in the formation of FC4S nanospheres with a spherical radius of †

University of Massachusetts. Mount Sinai Hospital and University of Toronto. § Tohoku University. ‡

(1) Hao, J.; Li, H.; Liu, W.; Hirsch, A. Chem Commun. 2004, 602603. (2) Yang, J.; Li, L.; Wang, C. Macromolecules 2003, 36, 6060-6065. (3) Cassell, A. M.; Lee Asplund, C.; Tour, J. M. Angew. Chem., Int. Ed. 1999, 38, 2403-2405. (4) Oh-ishi, K.; Okamura, J.; Ishi-i, T.; Sano, M.; Shinkai, S. Langmuir 1999, 15, 2224-2226. (5) Sano, M.; Oishi, K.; Tshi-I, T.; Shinkai, S. Langmuir 2000, 16, 3773-3776. (6) Braun, M.; Hartnagel, U.; Ravanelli, E.; Schade, B.; Bo¨ttcher, C.; Vostrowsky, O.; Hirsch, A. Eur. J. Org. Chem. 2004, 1983-2001. (7) Georgakilas, V.; Pellarini, F.; Prato, M.; Guldi, D. M.; MelleFranco, M.; Zerbetto, F. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 50755080. (8) Felder, D.; Nava, M. G.; Carreon, M.; Eckert, J.-F.; Luccisano, M.; Schall, C.; Masson, P.; Gallani, J.-L.; Heinrich, B.; Guillon, D.; Nierengarten, J.-F. Helv. Chim. Acta 2002, 85, 288-319. (9) Song, T.; Dai, S.; Tam, K. C.; Lee, S. Y.; Goh, S. H. Langmuir 2003, 19, 4798-4803. (10) Bhonsle, J. B.; Chi, Y.; Huang, J. P.; Shiea, J.; Chen, B. J.; Chiang, L. Y. Chem. Lett. 1998, 465-466. (11) Jeng, U.; Lin, T.-L.; Liu, W.-J.; Tsao, C.-S.; Canteenwala, T.; Chiang, L. Y.; Sung, L. P.; Han, C. C. Physica A 2002, 304, 191-201.

gyration (Rg) of ∼19 Å or an estimated sphere long-axis diameter of 60 Å for the aggregates, where the radius equals (5/3)1/2Rg, as determined by small-angle X-ray scattering and neutron scattering techniques.11,12 The aggregation size and shape of FC4S nanospheres remain relatively unchanged with the variation of concentrations from 0.35 to 25.6 mM. Hydrophilic FC4S nanospheres exhibit effective biological activities against several disease-related indications13 and photodynamic cytotoxicity against fibrosarcoma tumor cells.14 Unlike small amphiphilic surfactant molecules in forming micelle structures in water, C60-derived amphiphilics, such as pentamethyl[60]fullerene salt Me5C60K and Ph5C60K, have a tendency to form closed submicrospheres with a bilayer shell below the critical aggregation concentration and multi-bilayer vesicles above the critical aggregation concentration.15,16 In another example of amphiphilic C60 adducts, the attachment of longer alkyl (12) Jeng, U.; Lin, T. L.; Tsao, C. S.; Lee, C. H.; Canteenwala, T.; Wang, L. Y.; Chiang, L. Y.; Han, C. C. J. Phys. Chem. B 1999, 103, 1059-1063. (13) Lee, Y. T.; Chiang, L. Y.; Chen, W. J.; Hsu, H. C. J. Soc. Exp. Biol. Med. 2000, 224, 69-75. Hsu, H. C.; Chiang, L. Y.; Chen, W. J.; Lee, Y. T. J. Cardiovasc. Pharmacol. 2000, 36, 423. Huang, S. S.; Tsai, S. K.; Chih, C. L.; Chiang, L Y.; Hsieh, H. M.; Teng, C. M.; Tsai, M. C. Free Radical Biol. Med. 2001, 30, 643-649. Huang, S. S.; Chih, L. H.; Lin, C. H.; Chiang, L. Y.; Mashino, T.; Mochizuki, M.; Okuda, K.; Hirota, T.; Tsai, M. C. Fullerene Sci. Technol. 2001, 9, 375. Lai, Y. L.; Chiou, W. Y.; Lu, F. J.; Chiang, L. Y. Br. J. Pharmacol. 1999, 126, 778-784. Chueh, S. C.; Lai, M. K.; Lee, M. S.; Chiang, L. Y.; Ho, T. I.; Chen, S. C. Transplant. Proc. 1999, 31, 1976-1977. Chen, M. J.; Chiang, L. Y.; Lai, Y. L. Toxicol. Appl. Pharmacol. 2001, 171, 165. (14) Chi, Y.; Canteenwala, T.; Chen, H. H. C.; Chen, B. J.; Canteenwala, M.; Chiang, L. Y. Proc. Electrochem. Soc. 1999, 99-12, 234249. (15) Burger, C.; Hao, J.; Ying, Q.; Isobe, H.; Sawamura, M.; Nakamura, E.; Chu, B. J. Colloid Interface Sci. 2004, 275, 632-641. (16) Zhou, S.; Burger, C.; Chu, B.; Sawamura, M.; Nagahama, N.; Toganoh, M.; Hackler, U. E.; Isobe, H.; Nakamura, E. Science 2001, 291, 1944-1947.

10.1021/la047082f CCC: $30.25 © 2005 American Chemical Society Published on Web 03/12/2005

3268

Langmuir, Vol. 21, No. 8, 2005

chains onto C60, making the molecule more surfactantlike, was proposed to form bilayer vesicles readily in water.17 Recently, we demonstrated high two-photon absorptivity of acceptor-donor type fullerene-chromophore dyads including 7-(1,2-dihydro-1,2-methanofullerene[60]61-carbonyl)-9,9-diethyl-2-diphenylaminofluorene, C60(>DPAF-C2) 1, with efficient intramolecular energy transfer between the C60 cage moiety and the diphenylaminofluorene (DPAF) donor moiety upon two-photon pumping at 800 nm.18 This analogue of photoresponsive materials also exhibits intramolecular electron transfer from the DPAF moiety to the fullerene cage upon onephoton excitation in polar media.19 The observed phenomena may allow their potential utilities in photodynamic therapy, fluorescence bioimaging, and optical power limiting. Thus, it is of our interest to incorporate either energy- or electron-transfer activity, associated with multiphoton absorptivity in the membrane, on fullerenyl bilayer vesicles in aqueous solution to encompass its potential biological applications. Experimental Section General. Reagents of 4-(dimethylamino)pyridine (DMAP), 1,3dicyclohexylcarbodiimide (DCC), poly(ethylene glycol) bis(carboxymethyl) ether (molecular weight 600), and anhydrous dichloroethane were purchased from Aldrich. A C60 sample with a purity of 99.5% was used. Further purification of C60 was made by thin-layer chromatography (TLC, SiO2, toluene). The precursor compound 9,9-di(ethanol)-2-diphenylaminofluorenated [60]fullerene as C60(>DPAF-OH) was prepared according to the reported procedure18 with chemical modification at the C-9 group to incorporate alcohol functionality. Toluene and benzene were dried and distilled over sodium. Infrared spectra were recorded as KBR pellets on a Thermo Nicolet 370 FT-IR spectrometer. 1H NMR and 13C NMR spectra were recorded on either a Bruker Spectrospin-400 or Bruker AC-300 spectrometer. Fluorescence spectra were recorded on a FLUOROLOG (ISA Instruments) spectrofluorometer. Transmission electron microscopy (TEM) images were taken on a Philips Tecnai 20 transmission electron microscope at 200 kV. Synthesis of Amphiphilic 9,9-Di[oligo(ethylene glycol)]2-diphenylaminofluorenocarbonyl-methano[60]fullerene 3. Poly(ethylene glycol) bis(carboxymethyl) ether (molecular weight 600, 463 mg, 0.08 mmol) was taken in a round-bottom flask (100 mL) and stirred under reduced pressure in an oil bath maintained at 80 °C for 5.0 h. The reaction flask was cooled to room temperature, and 1,3-dicyclohexylcarbodiimide (DCC, 159 mg, 0.08 mmol), 4-(dimethylamino)pyridine (DMAP, 94 mg, 0.08 mmol), and 9,9-di(ethanol)-2-diphenylaminofluorenocarbonylmethano[60]fullerene C60(>DPAF-OH) (4, 150 mg, 0.013 mmol) were added in sequence. To the reaction mixture was then added anhydrous dichloroethane (50 mL), and this solution was stirred in an oil bath maintained at 65 °C for an additional 2.0 h until the complete disappearance of the starting material on a thinlayer chromatography (TLC) plate. The resulting reaction mixture was filtered and the filtrate dried on a rotary evaporator. The crude semisolid product was then dissolved in a solvent mixture of THF-H2O (1:1, 10 mL) and dialyzed against distilled water using a dialysis membrane, with the molecular weight cutoff at 1000, for effective removal of residual impurities and unreacted poly(ethylene glycol) bis(carboxymethyl) ether. After completion of the dialyses and solvent evaporation by the freeze-dry technique, the product of 9,9-di[oligo(ethylene glycol)]-2-diphe(17) Sano, M.; Oishi, K.; Ishi-i, T.; Shinkai, S. Langmuir 2000, 16, 3773-3776. (18) Chiang L. Y.; Padmawar, P. A.; Canteenwala, T.; Tan, L.-S.; He, G. S.; Kannan, R.; Vaia, R.; Lin, T.-C.; Zheng, Q.; Prasad, P. N. Chem. Commun. 2002, 17, 1854-1855. Padmawar, P. A.; Canteenwala, T.; Verma, S.; Tan, L.-S.; Chiang, L. Y. J. Macromol. Sci., Pure Appl. Chem. 2004, 41, 1387-1400. (19) Luo, H.; Fujitsuka, M.; Araki, Y.; Ito, O.; Padmawar, P.; Chiang, L. Y. J. Phys. Chem. B 2003, 107, 9312-9318.

Verma et al. nylaminofluorenocarbonyl-methano[60]fullerene 3 as C60(>DPAFEG6) was obtained as a semisolid in 40% yield. Spectroscopic Data of 3. 1H NMR (200 MHz, CDCl3, ppm) δ 8.58 (dd, J ) 8 Hz, J ) 1.6 Hz, 1H), 8.43 (d, J ) 1.6 Hz, 1H), 7.88 (d, J ) 8 Hz, 1H), 7.69 (d, J ) 8 Hz, 1H), 7.40-7.10 (m, 12H), 5.77 (s, 1H), 4.33 (t, J ) 4 Hz, 4H), 4.18 (s, 4H), 3.88 (s, 4H), 3.68 (broad, 80H), 2.26-2.61 (m, 4H). FT-IR (KBr) νmax: 3423, 2872, 1746, 1671, 1592,1489, 1466, 1425, 1349, 1277, 1247, 1200, 1109, 950, 844, 756, 695, 526 cm-1. Preparation of Spherical Vesicles. The preparation of C60(>DPAF-EG6)-derived vesicles was done by dissolving 3 (1.4 mg) in THF-DMSO (1:1, 0.1 mL) with ultrasonication for ∼5.0 min, followed by the addition of H2O (1.9 mL) with vigorous stirring to form a solution of 3.0 × 10-4 M in concentration. A portion of this solution was diluted to solutions with concentrations of 1.0 × 10-5 and 5.0 × 10-6 M for subsequent transmission electron microscopy measurements. Optical Absorption Measurements. Optical absorption spectra (250-800 nm) were determined in a quartz cuvette with either a UV-vis spectrophotometer (model UV160U, Shimadzu, Kyoto, Japan) or a computer-controlled Perkin-Elmer UV-visNIR Lambda 9 series spectrophotometer with C60(>DPAF-EG6)derived vesicles in H2O. Dynamic Light Scattering Measurements. Light scattering experiments were performed on a Brookhaven light scattering instrument equipped with a light scattering system, a BI9000 AT digital correlator, and a photon counter with a BI200SM research goniometer. A Uniphase ´ıBlue laser source with an output power of 50 mW was used that supplies vertically polarized light with a wavelength of 514 nm. The data were collected at 25 °C by monitoring the scattered light intensity at a 90° detection angle. Each light scattering measurement was performed at least three times. Transient Absorption Measurements. Collection and analysis of nanosecond transient absorption spectra in the nearIR region (600-1600 nm) were made using third-harmonic generation (HG, 355 nm) of a Nd:YAG laser (532 nm, SpectraPhysics, Quanta-Ray GCR-130, fwhm 6 ns) as an excitation light source. Light intensity from a pulsed Xe lamp was monitored and detected with a Ge-APD device (Hamamatsu Photonics, B2834). For measurement of the spectrum in the visible region 400-1000 nm, a Si-PIN photodiode (Hamamatsu Photonics, S1722-02) was used as the detector. Triplet lifetime was estimated by using a photomultiplier. All the samples in a quartz cell (1.0 × 1.0 cm2) were deaerated by bubbling argon through the solution for 10 min.

Results and Discussion Chemical modification of the multiphoton absorptive compound 1 was made by attaching nonionic poly(ethylene glycol) (PEG) groups to enhance its solubility in water. We took the size balance of the PEG chain width to a relatively larger molecular width of the hydrophobic C60DPAF moiety into account in the construction of the amphiphilic analogue of 1. Accordingly, the use of double PEG chain tails with an extended chain length was proposed to match with the overall molecular radius and to balance the hydrophilicity and hydrophobicity between different moieties. However, a high solubility of PEG chains may be expected to induce dynamic mobility in water that gives rise to the poor orientation and disorder of the intermolecular close-packing of C60-DPAF chromophore moieties. Attachment of a highly fluorescent DPAF dye donor moiety onto the C60 cage was made by a R-cyclopropylketo linker that places the DPAF chromophore into close vicinity of the fullerenyl π-electron system. The precursor compound 9,9-di(methoxyethyl)-2-diphenylaminofluorenated fullerene C60(>DPAF-OMe) 2 was used for the preparation of 9,9-di[oligo(ethylene glycol)]-2-diphenylaminofluorenocarbonyl-methano[60]fullerene 3, C60(>DPAF-EG6), as shown in Scheme 1. Compound 2 was synthesized from the key intermediate

Diphenylaminofluorene-C60 Conjugate Vesicles Scheme 1a

a Reagents and synthetic conditions: (i) BBr3, NaI, 15-crown5, CH2Cl2, -30 °C; (ii) PEG bis(carboxymethyl) ether (MW 600), DCC, DMAP, ClCH2CH2Cl.

7-bromoacetyl-9,9-di(methoxyethyl)-2-diphenylaminofluorene with C60 using the Bingel cyclopropanation reaction in toluene in the presence of 1,8-diazabicyclo[5.4.0]undec7-ene (DBU, 1.0 equiv).20 Conversion of 2 to 3 was carried out by the demethylation reaction of 2 first with BBr3 in the presence of NaI and 15-crown-5 in CH2Cl2 at -30 °C for a period of 15 h to afford the corresponding 2-diphenylaminofluorenocarbonyl-methano[60]fullerene-9,9-diethanol 4. Esterification of diethanol 4, C60(>DPAF-OH), was performed by the treatment of 4 with oligo(ethylene glycol) bis(carboxymethyl) ether (molecular weight 600, excess) using 1,3-dicyclohexylcarbodiimide (DCC) and 4-(dimethylamino)pyridine (DMAP) as the reagents in ClCH2CH2Cl. The resulting fullerene monoadduct product C60(>DPAF-EG6) 3 was obtained as a semisolid in 40% yield after dialysis. Removal of the residual starting oligo(ethylene glycol) bis(carboxymethyl) ether was made by dissolving the crude product in water and subsequently subjecting the solution to dialysis purification against distilled water using a dialytic membrane having a molecular weight cutoff (MWCO) of 1000. The precursor 2 exhibits a close spectroscopic resemblance to C60(>DPAF-C2) 1. The structure of 1 was substantiated by X-ray single-crystal structural analyses18 and correlated to the structural characterization of the product C60(>DPAF-EG6) 3 using various spectroscopic analysis methods. The molecular weight of the intermediate C60(>DPAF-OH) 4 was confirmed by its mass spectroscopic (MS) measurement using the matrix assisted laser desorption ionization (MALDI) technique in R-cyano4-hydroxycinnamic acid (CHCA). It displayed a group of mass ion peaks at m/z 1181-1184 corresponding to the molecular ion mass of 4 and its isotopic masses. Linkage (20) Bingel, C. Chem. Ber. 1993, 126, 1957.

Langmuir, Vol. 21, No. 8, 2005 3269

of DPAF to the C60 cage was substantiated by the chemical shift of unique R-proton H1 to the cyclopropylketo group of 3, showing the downshift to δ 5.77 (s, 1H), presumably under the influence of the fullerenic current through the space by a strong deshielding effect. It is accompanied by ethylene glycolic protons at δ 3.68 (broad, 80H) in the 1H NMR spectrum of 3, consistent with its composition and the structure. Chemical conversion of hydroxyl groups of C60(>DPAF-OH) 4 to the corresponding oligo(ethylene glycolated) ester moieties of 3 was verified by a strong symmetrical ether stretching absorption band (sCsOs Cs) at 1109 cm-1 and a strong infrared band centered at 1746 cm-1 corresponding to the absorption of CdO functional groups, indicating the presence of ester and acid moieties. This is in addition to the stretching absorption of a carbonyl group, which bridges the fluorene and fullerene moieties of 3, at 1670 cm-1. Characteristics of the oligo(ethylene glycol) moiety were also observed in the MALDI-MS measurements of 4 in CHCA by many consecutive mass ion peaks above m/z 1200 each separated apart from the adjacent peak by a mass of 44, which fits well with the mass of an ethylene glycol subunit. The related 13C NMR spectrum showed a peak at δ 189.7 corresponding to the chemical shift of the carbonyl carbon, two peaks at δ 172.2 and 170.4 corresponding to the chemical shift of ester and acid carbons, and a total of 29 carbon signals distributed in a range of δ 137-149, indicating a C2 symmetry for 58 sp2 carbons of the fullerene cage as evidence for the monoadduct structure of 3. Dissolution of the product C60(>DPAF-EG6) in water is in slow kinetics. It took 2-3 days to dissolve 10 mg of the compound in 1.0 mL of water. However, its solubility in H2O can be highly enhanced by dissolving 3 first in a minimum amount of THF-DMSO (1:1) under ultrasonication for ∼5.0 min. The wetted sample showed good compatibility with H2O when stirred vigorously. Accordingly, a solution of 3.0 × 10-4 M in concentration was prepared for the formation of vesicles. A portion of this solution was then diluted to concentrations of 2.0 × 10-5 and 5.0 × 10-6 M for subsequent dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements. These solutions were filtered directly into the scintillation vials of 1.5 cm diameter through the filter of 0.45 µm pore size. The topography of the molecular assembly structures of C60(>DPAF-EG6) was investigated by TEM images. Both Formvar and carbon-copper grids in a variety of mesh sizes were used. Samples for microscopic measurements were prepared by the freeze-dry technique under vacuum. It was performed by coating the grid with a solution of 3 in various concentrations followed by placing the coated grid on a metal dish that was in thermal equilibrium with liquid nitrogen. This apparatus was then placed inside a vacuum chamber overnight to remove all solvents completely prior to the measurement. Consistent TEM microimages of C60(>DPAF-EG6)derived vesicles, prepared at a concentration of 3.0 × 10-4 M in H2O, were obtained. The micrograph illustrated many regular spheres in a size ranging from 50 nm to greater than 400 nm in diameter that made up roughly over 90% of the particle-like subjects on the grid, as shown in Figure 1a. The majority of the spheres were larger than the typical micelle size of 20-30 nm in diameter derived from small molecular amphiphilics. As the concentration of the solution decreased to DPAF-EG6) 3-derived vesicles prepared at concentrations of (a) 3.0 × 10-4 M and (b) 2.0 × 10-5 M, with the view of defective vesicles in the insets showing the edge of the bilayer shell.

Figure 3. Distribution of effective sphere diameters of C60(>DPAF-EG6)-derived vesicles in H2O formed at concentrations (a) between 3.0 × 10-4 and 1.0 × 10-6 M and (b) between 3.0 × 10-5 and 1.0 × 10-6 M, where the marks b and [ represent the data points of three repeat trial runs (except more than six trial runs at 3.0 × 10-4 M).

Figure 2. TEM micrographs of (a) C60(>DPAF-EG6) 3 aggregates derived from the heat treatment (60 °C) of the vesicles prepared at a concentration of 3.0 × 10-4 M and (b) membranelike aggregates of 3 formed at a high dilution of 5 × 10-6 M.

the location of defective sites at the surface, and the morphology of holes among different spheres, we suggest that these holes do not result from the density gradient image of the spherical vesicles. Therefore, the edge of these holes should be measurable to reveal the thickness of the vesicle shell. To facilitate this possibility and substantiate these vesicles being hollow in nature, the perfect spherical vesicles (Figure 1a) prepared at a 3.0 × 10-4 M concentration were subjected to the heat treatment for a period of a few hours. At 45 °C treatment, the spheres were relatively stable. However, the heat treatment above 60 °C for a period of 2.0-3.0 h resulted in many disrupted spheres with a broken morphology in irregularly shaped membranelike structures. One example of broken vesicles is shown in Figure 2a. The morphology of this slightly “tilted” shell membrane at the top was revealed by a dark ring area with an irregular wall width and shape around the ring image. The morphology can be compared with the membranelike aggregates of 3 formed at a high dilution

of 5 × 10-6 M (Figure 2b). Measurement of the membrane wall thickness at the tilted membrane site perpendicular to the view, as shown in Figure 2a marked by the arrows, gave an edge width of roughly 15-20 nm. This distance across the membrane wall fits approximately with the linear bilayer molecular dimension of 16 nm, estimated by the 3D molecular structural modeling of 3 with a fully stretched oligo(ethylene glycol) chain having a repeating EG unit of 13. A slighly larger width of 20 nm observed in TEM images may imply a possible disordered bilayer head-head packing of C60(>DPAF) chromophore moieties in the membrane. These morphology analyses led us to propose a hollow spherical vesicle structure for the majority of molecularly assembled C60(>DPAF-EG6) in H2O, consisting of a shell-like bilayer membrane at the vesicle surface. In this instance, possible formation of spheres in a complex multilayer structure resembling that of a liposome cannot be ruled out. The distribution of average effective diameters of C60(>DPAF-EG6)-derived vesicle spheres, prepared at various concentrations between 3.0 × 10-4 and 1.0 × 10-6 M, was determined by dynamic light scattering measurements and summarized in Figure 3. A distinct relationship between the concentration and the sphere size was found at higher concentrations above 4.0 × 10-5 M, where the effective diameter of the sphere increases with the increase of concentration. A maximum average effective sphere diameter of 300 ( 50 nm based on more than three repeat trial runs was obtained, as shown in Figure 3a, perhaps, due to the effect of membrane filtration (a pore size of 0.45 µm). In the low concentration region below 4.0 × 10-5 M, the average effective sphere diameter remains relatively constant at 150-200 nm independent of the concentration (Figure 3b). Slight scattering of the DLS data among different repeated experiments indicated

Diphenylaminofluorene-C60 Conjugate Vesicles

Figure 4. Steady-state visible absorbance spectra of C60(>DPAF-EG6)-derived vesicles in H2O prepared at concentrations of (a) 1.0 × 10-4 M, (b) 2.0 × 10-4 M, and (c) 3.0 × 10-4 M. Inset profile: absorption spectrum of C60(>DPAF-EG6) in a dilute solution of CHCl3 at a concentration of 2.0 × 10-5 M.

that the sphere size may depend on the procedure of sample preparation and subsequent treatments of the solution. However, the sphere-size distributions at all concentrations agree well with those observed in TEM micrographs. The photophysical characteristics of C60(>DPAF-EG6)derived submicrospherical vesicles in H2O were investigated via steady-state absorption, fluorescence, and nanosecond transient absorption spectroscopic measurements to demonstrate the effect of its molecular assembly on the intramolecular electron- and/or energy-transfer efficiency. Accordingly, concentration-dependent steadystate visible absorption spectra are shown in Figure 4. A very weak broad absorption band centered at 700 nm is correlated to characteristics of π-electron systems of the C60 cage moiety. The absorption band in the region of 400470 nm is attributed to the DPAF moiety. In a dilute solution of CHCl3 at a concentration of 2.0 × 10-5 M where good solubility of both the fullerene cage and DPAF moiety makes C60(>DPAF-EG6) in a lesser degree of aggregation, the absorption maximum of DPAF appeared at 405 nm, as shown in the inset profile of Figure 4. The intensity of this band increased as the concentration increased from 1.0 × 10-4 to 3.0 × 10-4 M. The progressive shift of the peak maximum from 410 nm of Figure 4a to 460 nm of Figure 4c was interpreted as characteristics of increasing the tight molecular packing of diphenylaminofluorenyl rings during the aggregation of C60(>DPAF-EG6), that is dominated by strong intermolecular hydrophobic interaction forces of the fullerene cages in aqueous solution. The behavior is consistent with the vesicle formation at the latter concentration. The effect of molecular assembly was clearly observed in steady-state fluorescence spectra of C60(>DPAF-EG6) in H2O (Figure 5) at the same concentration range as that for the absorption spectra, DLS, and TEM measurements. It exhibits a main peak centered at 500 nm which is attributed from the fluorescence of the DPAF moiety upon photoexcitation at 400 nm. In contrast to the increase of optical absorption upon the increase of concentration in Figure 4, a systematic decrease in the fluorescence intensity was detected as the concentration of 3 in H2O increased from 1.0 × 10-4 to 3.0 × 10-4 M. This intensity decrease was accounted for the effect of fluorescence quenching of the DPAF moiety due to the increasing degree of molecular aggregation at a concentration of 3.0 × 10-4 M. The results clearly support the observed vesicle formation at the same concentration.

Langmuir, Vol. 21, No. 8, 2005 3271

Figure 5. Steady-state fluorescence spectra of C60(>DPAFEG6) vesicles formed at (a) 1.0 × 10-4 M, (b) 2.0 × 10-4 M, and (c) 3.0 × 10-4 M in H2O under excitation at λex ) 400 nm.

Figure 6. Transient absorption spectra obtained by nanosecond 532 nm laser photolysis of C60(>DPAF-EG6) at 3.0 × 10-4 M in Ar-saturated H2O.

Nanosecond transient absorption measurements were carried out by nanosecond laser photolysis at 532 nm excitation with the transient absorption spectrum data collection in the visible and near-IR regions (400-1000 nm). All the samples of C60(>DPAF-EG6) at a concentration of 3.0 × 10-4 M were deaerated by bubbling argon through the aqueous solution for 10 min. Immediately after the laser pulse, no significant transient absorption of the triplet 3C60* state at 740 nm was observed,19 indicating little or no involvement of the fullerenic triplet state during the photoexcitation process. On the contrary, two new bands at 840 and 1000 nm, which were attributed to absorptions of the DPAF radical cation (DPAF•+) and the radical anion of the C60 moiety (C60•-), respectively,19 were detected, as shown in Figure 6. In the inset time profiles of Figure 6, both 840 and 1000 nm bands ascended immediately after nanosecond laser irradiation on a 10 ns time scale, indicating clearly the intramolecular electron transfer and charge-separation process taking place via the singlet excited state of the C60 cage moiety. The decay rate of the 840 nm band as the charge recombination value (kCR) was evaluated to be 1.5 × 108, 6.3 × 107, 5.0 × 107, and 5.0 × 107 s-1 for C60(>DPAF-EG6) vesicles prepared at concentrations of 1.0 × 10-4, 2.0 × 10-4, 3.0 × 10-4, and 4.0 × 10-4 M, respectively. The results correspond to a relatively longer lifetime (20 ns) of the charge-separated state of C60-DPAF moieties in the vesicles formed at a higher concentration of 3.0 × 10-4 M compared to that (7.0 ns) of the vesicles prepared at a 1.0 × 10-4 M concentration. In the region of higher concen-

3272

Langmuir, Vol. 21, No. 8, 2005

trations than 3.0 × 10-4 M, a relatively steady charge recombination rate of 5.0 × 107 s-1 might reveal close resemblance in the molecular packing of 3 in the vesicles formed. Furthermore, in a 100 ns time scale, a slower decay rate with a longer kCR value was calculated to be from 7.6 × 106 s-1 (lifetime 130 ns) to 5.0 × 106 s-1 (lifetime 200 ns) with increasing concentration from 1.0 × 10-4 to 4.0 × 10-4 M, respectively. This implies the existence of longer-lived charge-separated states as minor components in self-assembled systems. Conclusion We developed a facile synthetic method for the preparation of novel oligo(ethylene glycolated) derivatives of highly two-photon absorptive conjugate18 C60(>DPAF-C2) using the corresponding 9,9-di(methoxyethyl)-2-diphenylaminofluoreno [60]fullerene derivative 2 as the precursor molecule. The synthesis led to the water-soluble analogue C60(>DPAF-EG6). Systematic investigation of the molecular self-assembly characteristics of C60(>DPAFEG6) in H2O resulted in observation of many nano- to submicron-sized spherical hollow vesicle formations with a shell width of 15-20 nm, estimated by TEM micrographs. This shell width fits approximately with the length of a disordered bilayer-like molecular packing of C60(>DPAF-EG6). That allowed us to propose that these spherical hollow vesicles are likely made of the bilayer shell-membrane organization with a head (C60-DPAF)to-head conformation. The arrangement of a bilayer structure is reasonable based on geometric packing considerations and strong intermolecular hydrophobic

Verma et al.

interactions of C60-DPAF heads that form a long-range, coherent size distribution of the vesicles. These vesicles based on bilayer-membrane structures may serve as an alternative to lipid membranes and liposome vesicles. Photoinduced intramolecular charge separation followed by charge recombination on the nanosecond time scale, from the DPAF moiety to the C60 cage in the vesicle structure, was detected via transient spectroscopic measurements. As the utility of the photodynamic cytotoxicity of amphiphilic C60 derivatives against the growth of fibrosarcoma tumor cells becomes a crucial approach and viable mechanism for cancer therapy,14 the photogeneration of charge carriers in the bilayer membrane comprised of multiphoton absorptive C60(>DPAF-EG6) conjugates may allow us to explore its potential in biomedical treatments. Acknowledgment. The authors thank AOARD/ AFOSR for partial financial support of this work under the contract FA520904P0540. This research was also supported by a Grant-in-Aid for the COE project, Giant Molecules and Complex Systems, 2002 (to M.E.E.-K.), and for Scientific Research on Primary Area (417) from the Ministry of Education, Science, Sport and Culture of Japan (to O.I.). Supporting Information Available: 1H NMR spectra and 13C NMR spectra of 2, 3, and 4; infrared spectra of 3 and 4; and TGA profile and UV-vis spectrum of 3. This material is available free of charge via the Internet at http://pubs.acs.org. LA047082F