Water-Dispersible Polymeric Structure Co-encapsulating a Novel

Oct 16, 2007 - We report the synthesis and characterization of a novel hexa-peri-hexabenzocoronene core (HBCC) containing chromophore with enhanced ...
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16846

J. Phys. Chem. C 2007, 111, 16846-16851

Water-Dispersible Polymeric Structure Co-encapsulating a Novel Hexa-peri-hexabenzocoronene Core Containing Chromophore with Enhanced Two-Photon Absorption and Magnetic Nanoparticles for Magnetically Guided Two-Photon Cellular Imaging Qingdong Zheng, Tymish Y. Ohulchanskyy, Yudhisthira Sahoo, and Paras N. Prasad* Department of Chemistry, Institute for Lasers, Photonics and Biophotonics, State UniVersity of New York at Buffalo, Buffalo, New York 14260 ReceiVed: June 18, 2007; In Final Form: August 30, 2007

We report the synthesis and characterization of a novel hexa-peri-hexabenzocoronene core (HBCC) containing chromophore with enhanced two-photon absorption (TPA) compared to that of a corresponding chromophore containing a hexaphenylbenzene core. With its planar core, the HBCC chromophore has a strong tendency to aggregate by π-stacking when entrapped into a biodegradable polymer, diacylphospholipid-polyethyleneglycol (PE-PEG). Both of these otherwise hydrophobic chromophores exhibit strong fluorescence in aqueous polymeric micellar solutions. The emission spectrum for the HBCC chromophore in water is strongly dependent on the concentration of the chromophore encapsulated in micelles. On co-entrapping the HBCC chromophore with magnetic Fe3O4 nanoparticles in the micelles, it was possible to achieve magnetically guided two-photon cellular imaging with ample bearing on targeted imaging and therapy.

1. Introduction During the past decade, increasing efforts have been put on the development of chromophores with enhanced two-photon absorption (TPA) because of their applications in photonics and biophotonics.1-8 For biological imaging by two-photon fluorescence microscopy, there is a need for a better signal-to-noise ratio that can be obtained by using a chromophore with a larger TPA cross-section value and a higher fluorescence quantum yield. As we know, one important molecular design strategy developed for obtaining organic chromophores with large twophoton absorption is to utilize a planar and extended π-conjugated core. Hexa-peri-hexabenzocoronene (HBC) has an ideal planar π-conjugated configuration. Its derivatives have attracted considerable attention in recent years because of their applications in field-effect transistors and photoconductive devices.9 By introducing HBC as a π-conjugated core, enhanced twophoton absorption may be expected, compared to the corresponding nonplanar π-conjugated core, hexaphenylbenzene. HBC derivatives have a strong tendency to aggregate by π-π stacking, which could also contribute to enhancement of their TPA. For biological applications, it is also necessary to design and synthesize materials that are water-soluble. Until now, there were only a few water-soluble materials with large two-photon absorption10-11 and the large majority of two-photon absorbing materials were nonpolar molecules that were barely suitable to form an aqueous dispersion. Recently, we developed a novel aqueous formulation by incorporating hydrophobic chromophores in a biodegradable polymeric micelle of diacylphospholipid-polyethyleneglycol (PE-PEG).12,13 These nanosized micelles are thermodynamically stable self-aggregates of amphiphilic molecules with a hydrophobic core that are capable of solubilizing the nonpolar molecules within them. One salient * Corresponding author. E-mail: [email protected].

advantage of these micelles is their capability to incorporate multiple functional constituents without compromising the functionalities and the overall stability of the micelles.13,14 For example, hydrophobic photosensitizer drugs and colloidally prepared magnetic particles have been co-loaded successfully in these micelles, which serve as nanocarriers, in order to achieve magnetically guided delivery of drugs in photodynamic therapy.13 In this paper, we report the design and preparation of an HBC core containing chromophore as well as its co-encapsulation with magnetic Fe3O4 nanoparticles in biodegradable polymeric micelles for magnetically guided two-photon cellular imaging. For comparison purposes, a hexaphenylbenzene core containing chromophore was also prepared. 2. Experimental Section 2.1. Materials and Instruments. All chemicals were purchased from Aldrich and were used without further purification. 1H NMR and 13C NMR spectra were recorded 500 and 75 MHz, respectively. MALDI-TOF MS spectra were recorded on a Bruker Biflex IV MS spectrometer with dithranol as a matrix. Elemental analysis was carried our by Atlantic Analysis Inc., Norcross, GA. Linear absorption spectra were recorded on a Shimadzu UV-3101 PC spectrophotometer. One-photon excited fluorescence was measured by using a Jobin-Yvon Fluorolog FL-311 spectrofluorometer. For linear fluorescence quantum efficiency measurements in solution, an external reference of Coumarin 152 (φf ) 0.21 in ethanol) was used.15 Size distribution of micelles was determined by dynamic light scattering (DLS) measurement with a Brockhaven instrument 90 Plus Particle Size Analyzer, with a scattering angle of 90° and particle size range measurements of 2 nm to 3 µm. The measurements were repeated three times, and each size data represents an average of 3 runs. 2.2. Synthesis. These star-shaped chromophores were prepared from the Pd-catalyzed Heck reaction between hexaiodo-

10.1021/jp074713g CCC: $37.00 © 2007 American Chemical Society Published on Web 10/16/2007

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SCHEME 1: Synthetic Routes for the Star-Shaped Two-Photon Absorbing Chromophores

peri-hexabenzocoronene or hexakis(4-iodophenyl)benzene and vinylated compound 3 (Scheme 1). Hexaiodo-hexaphenylbenzene was prepared according to literature procedures.9a The insoluble hexaiodo-peri-hexabenzocoronene (2) was prepared from hexakis(4-iodophenyl)benzene (1) via Lewis acid-mediated oxidative cyclodehydrogenation.9a The synthesis of the vinylated compound (3) has been described in a previous report.16 The Pd-catalyzed Heck reaction in the final step was proven to be quite successful. Compounds H1 and H2 were obtained in yields of 90% and 75%, respectively. All new compounds were characterized by 1H NMR, 13C NMR, MALDI-TOF mass spectrometry, and elemental analyses. These nonpolar chromophores are soluble in THF, toluene, chloroform, and so forth but barely soluble in water. Synthesis of Compound H1. A mixture of compound 117 (0.43 g, 0.33 mmol), vinylated compound 316 (1.15 g, 2.0 mmol), tetrabutylammonium bromide (1.75 g, 5.5 mmol), potassium carbonate (0.88 g, 6.25 mmol), palladium(II) acetate (25 mg, 113 µmol), and N,N-dimethylformamide (20 mL) was stirred at 150 °C (oil bath) for 48 h under an atmosphere of argon. After the mixture was cooled, 150 mL of methanol was added, and the precipitate was filtered and washed with methanol. The crude product was purified by column chromatography with hexane-toluene (4:1 to 1:1) as the eluent. We collected 1.18 g (0.297 mmol) of yellow crystalline solid (90% yield) after evaporating the eluent. 1H NMR (500 MHz, CDCl3, δ, ppm): 7.55-7.35 (complex multiplets, Ar-H, 60 H), 7.25-7.20 (complex multiplets, Ar-H, 36 H), 7.15-7.05 (complex multiplets, Ar-H, 48 H), 7.05-6.95 (complex multiplets, Ar-H, 24 H), 1.89-1.85 (m, CH2, 24 H), 1.10-1.04 (m, CH2, 24 H), 0.68-0.65 (m, CH2 + CH3, 60 H). 13C NMR (75 MHz, CDCl3, δ, ppm): 152.38, 151.10, 147.91, 147.12, 140.77, 140.33, 139.77, 137.14, 136.88, 136.41, 136.05, 135.89, 135.43, 131.99, 129.08, 128.00, 127.08, 126.79, 126.71, 126.49, 125.65, 125.14,

123.80, 123.35, 122.46, 120.59, 120.32, 119.20 (sp2 carbons); 54.84, 39.98, 25.98, 22.92, 13.77(sp3 carbons). Elemental analysis Calcd: C, 90.87; H, 7.02; N, 2.12. Found: C 90.71, H 7.12, N 1.95. MALDI-TOF MS Calcd for M+Na+ C300H276N6Na: 3988.18. Found: 3988.40. Synthesis of Compound H2. The same procedure as described for the preparation of compound H1 was used. Yellow crystalline solid (75% yield). 1H NMR (500 MHz, CDCl3, δ, ppm): 7.55-7.38 (complex multiplets, Ar-H, 36 H), 7.25-7.20 (complex multiplets, Ar-H, 30 H), 7.15-7.06 (complex multiplets, Ar-H, 48 H), 7.04-6.85 (complex multiplets, Ar-H, 42 H), 1.90-1.85 (m, CH2, 24 H), 1.09-1.10 (m, CH2, 24 H), 0.72-0.65 (m, CH2 + CH3, 60 H). 13C NMR (75 MHz, CDCl3, δ, ppm): 152.43, 151.17, 147.99, 147.18, 140.79, 140.29, 135.99, 135.55, 131.69, 129.15, 127.24, 126.65, 123.85, 122.52, 120.62, 120.32, 119.30 (sp2 carbons); 54.88, 40.04, 26.04, 22.98, 13.83 (sp3 carbons). Elemental analysis Calcd: C, 91.14; H, 6.73; N, 2.13. Found: C, 91.02; H, 6.77; N, 2.02. MALDITOF MS Calcd for M+Na+ C300H264N6Na: 3976.08. Found: 3976.00. Preparation and Characterization of Aqueous Micelles. Polymeric diacyllipid micelles were prepared from 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (ammonium salt) (PE-PEG) according to the methodology described previously by Torchilin.18 Briefly, 5.0 mg of PE-PEG was dissolved in chloroform and different volumes of chromophore solution (10 µM in THF) were added. Each resulting dispersion was sonicated for 5 min. After the removal of the organic solvent, the lipidic film, deposited on the vial walls, was rehydrated in 10 mL of water and subjected to further ultrasonication for 10 min using a bath sonicator. The resulting micellar solution was filtered through a 0.2 µm cut off SFCA membrane filter (Corning Co., NY), before any use.

16848 J. Phys. Chem. C, Vol. 111, No. 45, 2007 For in vitro interactions with cells and stability studies, the micelles were resuspended in phosphate buffer saline PBS at pH 7.4. For the magnetically guided imaging studies, the magnetic nanoparticles co-encapsulated sample was prepared by adding chromophore H2 and Fe3O4 nanoparticles19 with a weight ratio of 10:2. The monodiperse Fe3O4 nanoparticles, with an average diameter of 8 nm, were prepared in accordance with the recipe adopted in an earlier work.13 In short, 1 mmol iron acetylacetonate, 3 mmol oleic acid, 5 mmol 1,2-hexadecanediol, and 20 mL phenylether were taken together in a three-necked flask and the mixture was heated at 265 °C for 90 min. The particles were first centrifuged from the reaction mixture by adding a small amount of toluene and requisite ethanol indicated by the brownish cloudy appearance. The precipitate could be readily redispersed with chloroform. 2.3. Two-Photon Absorption Measurements. The TPA spectra were determined by using laser pulses (∼120 fs duration, 76 MHz repetition rate, ∼2 nJ/pulse) from 715 to 940 nm generated by a mode-locked Ti:sapphire laser (Mira from Coherrent). A spectrum analyzer (IST-rees) was used to monitor the excitation wavelengths. The ηδ values at 775 nm was determined by using the pulses (∼775 nm, ∼150 fs duration, 1 kHz repetition rate) from a Ti:sapphire laser oscillator/amplifier system (CPA-2010 from Clark-MXR). All data were taken by two-photon excited fluorescence method with Rhodamine 6G (110 µM solution in methanol) as a reference.20 2.4. In Vitro Studies Imaging with Tumor Cells. HeLa (human cervix epitheloid carcinoma) cells (American Type Culture Collection, Manassas, VA) were cultured in a minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), according to instructions supplied by the vendor. For studying magnetically controlled dye uptake and imaging, we trypsinized and resuspended the cells in a MEM alpha medium with 10% fetal bovine serum (FBS) at a concentration of 7.5 × 105 cells/mL and plated in 35-mm culture plates, using 2.5 mL of the medium containing 0.10 mL of the cell suspension. The plates were incubated overnight at 37 °C with 5% CO2. The next day, the cells (50% confluency) were rinsed carefully with phosphate buffered saline (PBS), and 2.5 mL of the medium containing 50 µL of the aqueous dispersion of polymeric micelles was added to plate and mixed gently. The treated cells were returned to the incubator, and the treated culture dish was placed on the top of a 0.5 T magnet (20 mm diameter) and incubated overnight. On the following day, the plates were rinsed with sterile PBS, and fresh media was added. The cells were then directly imaged using two-photon laser scanning fluorescence microscopy. Two-photon excitation for the two-photon laser scanning fluorescence microscopy was performed at 800 nm using a Ti:sapphire laser (Tsunami, Spectra-Physics) pumped by a frequency-doubled diode-pumped solid-state laser (Millennia, Spectra-Physics). It provided 90-fs pulses at an 82 MHz repetition rate. A spectrum analyzer (ISTREES, Germany) was used to monitor the wavelength and the bandwidth of the excitation light. A confocal laser scanning microscope (Bio-Rad, model MRC-1024) with an upright microscope (Nikon, model Eclipse E800) along with a 10 X objective lens (Nikon, Plan-10X, NA ) 0.25) was used for cell imaging. A long-pass filter (460 LP) was used as the emission filter for imaging. 3. Results and Discussion Table 1 summarizes the linear and two-photon absorption properties for compounds H1 and H2 in THF and H2O,

Zheng et al. TABLE 1: Linear and Two-Photon Absorption Properties for Compounds H1 and H2 samples and solvents

λ1max (nm)

λem (nm)

ηa

H1 in THF H1 in H2Od H2 in THF H2 in H2Od

401 398 404 400

499 499 501 541

0.85 0.37 0.58 0.15

λ2max (nm)

δ (GM)b

ηδ at 775 nm (GM)c

760

881

820

2022

545 250 916 336

a Fluorescence quantum yield. b Maximum two-photon absorption cross section, 1 GM ) 10-50 cm4 s photon-1, experimental uncertainty (15%. c Experimental uncertainty (15%. d The concentrations for H1 and H2 are 5.0 µM, and the concentration for PE-PEG solution was fixed at 0.6 mg/mL; the micelles sizes for H1 and H2 are 148.6 ( 6.7 nm and 185.8 ( 4.2 nm, respectively.

Figure 1. Linear absorption and emission spectra for H1 and H2 in THF and micelles: (a) absorption, (b) emission. (The concentration for H1 and H2 in THF and water was fixed at 5 µM, and the concentration for PE-PEG in water was fixed at 0.6 mg/mL.)

including linear absorption maxima, emission maxima, fluorescence quantum yields, two-photon absorption peak, peak TPA cross-section, and the ηδ (quantum yield × TPA cross section) value at 775 nm. Figure 1 depicts the linear absorption and emission spectra for compounds H1 and H2 in THF as well as in H2O. As shown in the figure, the linear absorption and emission spectra for H1 and H2 are quite similar in their dilute THF solutions, except that H2 exhibits a red-shift in the linear absorption and emission bands. However, H1 has a higher fluorescence quantum yield (85%) compared to compound H2 (58%). This is reasonable considering the fact that compound H2 has a planar π-conjugated core, HBC, which is prone to form face-to-face π-interactions, when two molecules are in close proximity to each other.17 As shown in Figure 1b, the aqueous solution of H2 (5 µM) exhibits a significantly redshifted emission, which can be accounted for by a strong tendency for π-π stacking aggregation in the dimer structures by virtue of their planar cores within the confined environment of the micelles.17 In contrast, the hexaphenylbenzene cored

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TABLE 2: Average Size of PEG-PE Nanocarriers with Different Amounts of Chromophore Loadinga H2/PE-PEG (w/w)

1/24

1/12

1/6

1/3

2/3

H2 concentration (µM) size (nm) polydispersity

0.625 76.4 ( 3.0 0.303

1.25 99.8 ( 2.4 0.190

2.5 113.0 ( 1.2 0.159

5.0 148.6 ( 6.7 0.143

10 191.7 ( 2.0 0.167

a The concentration of PE-PEG in water was fixed at 0.6 mg/mL; all of the samples were filtered through a 0.2 µm microfilter, and some particles with larger sizes were removed after filtration.

Figure 3. Two-photon absorption spectra for compounds H1 and H2.

Figure 2. Linear absorption (a) and emission (b) spectra for H2 in micelles at different concentrations. (The concentration for PE-PEG was fixed at 0.6 mg/mL.)

chromophore H1 in the same concentration (5 µM) does not form the π-π stacking aggregation because of the nonplanar stereo configuration and thus exhibits no shift in the emission peak. As shown in Figure 1a, the linear absorption band for H2 did not exhibit red-shift in going from the THF solution to the micelles solution. Therefore, self-absorption cannot be used for the explanation of the red-shifted emission found for H2 in micelles. Micellar formulations with different ratios of H2 to PE-PEG were prepared in order to investigate the aggregation behavior of the former. The sizes (hydrodynamic radii) of these micelles with different ratios of H2 to PE-PEG were measured by dynamic light scattering (DLS), and the results are shown in Table 2. As can be noted from Table 2, the average size of the micelles increased monotonically with increasing load of H2, which is an expected trend.19 The linear absorption and the emission spectra of the same micelles are shown in Figure 2a and b. The linear absorption spectra retain the same shape, except that the optical density increases with the increasing load of H2 (Figure 2a). However, a gradual change in the emission spectra evolves. There is almost no aggregation found for H2 micelles when its concentration is considerably low (0.625 µM). With increasing concentration, more molecules form the π-π stacking aggregation, which emit red-shifted fluorescence.

Therefore, there is a gradual red shift in emission with increasing concentration of H2 in the micelle (Figure 2b). TPA spectra for compounds H1 and H2 were shown in Figure 3. H2 has a TPA peak value of 2022 GM at 820 nm, whereas H1 has a TPA peak value of 881 GM at 760 nm. The TPA cross-section value of H1 was measured to be 641 GM at 775 nm by using an independent nonlinear transmission method with AF 350 as a reference (δ ) 206 GM at 775 nm).21 This value is in good agreement with the value (δ ) 656 GM at 775 nm for H1 as shown in Figure 3) measured by using fluorescence method in this work. Compound H2 exhibited a red-shifted TPA band compared to H1. At the same time, the TPA peak value for H2 is 230% as large as that for H1. Because the only difference between H1 and H2 is the core, their remarkable difference in TPA substantiates the fact that the planarity of a molecule has a strong effect on its two-photon absorptivity. At the same time, the formation of π-π stacking aggregation for H2 may help to extend its π-conjugation, which in turn enhances its two-photon absorption.22 For obtaining any biological applications, it is vital to make these strong two-photon absorbing chromophores water-soluble. Thus, these chromophores have been evaluated for their quantitative fluorescence behavior within the micelles and it was found that they still have substantial two-photon excited fluorescence, although with reduced fluorescence quantum yields when compared to their organic solutions. For example, H1 has a fluorescence quantum yield of 0.37 in water, against 0.85 in THF, a reduction to ∼44% from its original value. Similarly, H2 has a fluorescence quantum yield of 0.15 in water, compared to 0.58 in THF, a fall to ∼26% from its original value. The greater reduction in the quantum yield for H2 can be attributed to the π-π interactions for H2 as we mentioned before. Also as shown in Table 1, both H1 and H2 have decreased ηδ values in water, compared to those in THF at 775 nm. The ηδ value for H1 decreases from 545 GM in THF to 250 GM in water, and in case of H2, from 916 GM in THF to 336 GM in water, both measured at 775 nm. Despite the decrease of fluorescence quantum yield for H2 induced by the π-π interactions, the ηδ value for H2 is still larger than that for H1 because the TPA

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Zheng et al. magnetic particles. This was enabled because the micelles preserved their integrity in spite of the application of an external magnetic field. Magnetic control on an otherwise nonmagnetic substance such as this chromophore would be impossible. It is thus unambiguously demonstrated that the magnetic control subjected on the micelles results in a greater cellular uptake than would be possible by a simple diffusion-controlled process. 4. Conclusions We have designed and prepared a novel HBC core containing chromophore with enhanced two-photon absorption. The chromophore could be incorporated into a polymeric micelle successfully to ensure a biocompatible aqueous formulation. Furthermore, magnetic Fe3O4 nanoparticles were co-encapsulated with the chromophore within the micelles and magnetically guided in vitro cellular imaging was demonstrated by twophoton excitation. The efficacy of this formulation is derived from the combined advantages of the retention of a good twophoton fluorescence quantum yield of the novel chromophore and the feasibility of homogeneous co-encapsulation of multiple constituents within the polymeric micelles.

Figure 4. Magnetically guided two-photon excited fluorescence microscopic images of cells stained with PEG-PE micelles coentrapping H2 and magnetic nanoparticles. A magnet was applied on the topright triangular area of the sample. (a) A schematic illustration of the magnetic field applied area; (b) fluorescence from H2 labeled micelles; (c) transmission; (d) combined images for fluorescence and transmission.

cross section for H2 is significantly enhanced by the π-π interactions. This behavior has been observed in porphyrin derivatives, where the self-assembled porphyrin dimers are shown to have a large TPA cross-section value because of the expansion of porphyrin-porphyrin π-conjugation.22 As we mentioned previously, one of the important advantages for micelles is their colloidal stability in spite of multicomponent loading and thus, in principle, all nonpolar materials can be coloaded within micelles. In this work, Fe3O4 nanoparticles are co-encapsulated into micelles successfully, together with the two-photon absorbing chromophore H2. The sizes for these magnetic micelles were found to be 171.0 ( 3.7 nm with a polydispersity of 0.280. The ηδ value at 775 nm for these micelles was measured to be 280 GM, close to the value of the micelles without magnetic particles. This indicated its potential applications for the imaging of biological membranes. As an extension of this experiment, the possibility of magnetically guiding the polymeric micelles to enhance the uptake in a targeted area was tested using cell cultures selectively exposed to an external magnetic field. H2 containing polymeric magnetic micellar dispersion was added to a cell dish and properly mixed. Then the dish was located on the top of a magnet and kept overnight. Then two-photon excited confocal fluorescence images were taken from the locations inside the area where the magnetic field was applied, as well as from the area outside it. As is demonstrated in Figure 4a-d, there is a clear difference between the top-right of the images, where magnetic field was applied, and the bottom-left of the images, where no magnetic field was applied. In Figure 4b and d, one can easily notice that the cellular uptake is much higher in the top-right area where magnetic filed is applied, in contrast to that in the bottom-left area where cellular uptake is not assisted by magnetic field. This demonstrates that the cellular uptake of the chromophore can be magnetically controlled in the present formulation because on application of the magnetic field the chromophore molecules were co-guided along with the co-encapsulated

Acknowledgment. This work was supported in part by a grant from the Chemistry and Life Sciences Directorate of the Air Force Office of Scientific Research and in part by the John R. Oishei Foundation. Partial support from the center of Excellence in Bioinformatics and Life Sciences at University at Buffalo is also acknowledged. References and Notes (1) Spangler, C. W. J. Mater. Chem. 1999, 9, 2013. (2) Prasad, P. N. Introduction to Biophotonics; John Wiley & Sons: Hoboken, NJ, 2003. (3) Ogawa, K.; Hasegawa, H.; Inaba, Y.; Kobuke, Y.; Inouye, H.; Kanemitsu, Y.; Kohno, E.; Hirano, T.; Ogura, S.-i.; Okura, I. J. Med. Chem. 2006, 49, 2276. (4) (a) Parthenopoulos, D. A.; and Rentzepis, P. M. Science 1989, 245, 843. (b) Lee, K.-S.; Yang, D.-Y.; Park, S. H.; Kim, R. H. Polym. AdV. Technol. 2006, 17, 72. (c) Cumpston, B. H.; Ananthavel, S. P.; Barlow, S.; Dyer, D. L.; Ehrlich, J. E.; Erskine, L. L.; Heikal, A. A.; Kuebler, S. M.; Lee, I.-Y. S.; McCord-Maughon, D.; Qin, J.; Rockel, H.; Rumi, M.; Wu, X.-L.; Marder, S. R.; Perry, J. W. Nature 1999, 398, 51. (5) (a) Frederiksen, P. K.; Jørgensen, M.; Ogilby, P. R. J. Am. Chem. Soc. 2001, 123, 1215. (b) Frederiksen, P. K.; McIlroy, S. P.; Nielsen, C. B.; Nikolajsen, L.; Skovsen, E.; Jørgensen, M.; Mikkelsen, K. V.; Ogilby, P. R. J. Am. Chem. Soc. 2005, 127, 255. (6) (a) Abbotto, A.; Beverina, L.; Bozio, R.; Facchetti, A.; Ferrante, C.; Pagani, G. A.; Pedron, D.; Signorini, R. Chem. Commun. 2003, 2144. (b) Cho, B. R.; Son, K. H.; Lee, S. H.; Song, Y. S.; Lee, Y. K.; Jeon, S. J.; Choi, J. H.; Lee, H.; Cho, M. J. Am. Chem. Soc. 2001, 123, 10039. (7) Samoc, M.; Morrall, J. P.; Dalton, G. T.; Cifuentes, M. P.; Humphrey, M. G. Angew. Chem., Int. Ed. 2007, 46, 731. (8) Kannan, R.; He, G. S.; Yuan, L.; Xu, F.; Prasad, P. N.; Dombroskie, A. G.; Reinhardt, B. A.; Baur, J. W.; Vaia, R. A.; Tan, L. S. Chem. Mater. 2001, 13, 1896. (9) (a) Wu, J.; Watson, M. D.; Zhang, L.; Wang, Z.; Mu¨llen, K. J. Am. Chem. Soc. 2004, 126, 177. (b) Wu, J.; Watson, M. D.; Tchebotareva, N.; Wang, Z.; Mu¨llen, K. J. Org. Chem. 2004, 69, 8194. (c) Grimsdale, A. C.; Mu¨llen, K. Angew. Chem., Int. Ed. 2005, 44, 5592. (10) Woo, H. Y.; Hong, J. W.; Liu, B.; Mikhailovsky, A.; Korystov, D.; Bazan, G. C. J. Am. Chem. Soc. 2005, 127, 820. (11) Krishna, T. R.; Parent, M.; Werts, M. H. V.; Moreaux, L.; Gmouh, S.; Charpak, S.; Caminade, A.-M.; Majoral, J.-P.; Blanchard-Desce, M. Angew. Chem., Int. Ed. 2006, 45, 4645. (12) Baba, K.; Ohulchanskyy, T. Y.; Zheng, Q.; Lin, T.-C.; Bergey, E. J.; Prasad, P. N. Mater. Res. Soc. Sym. Proc. 2005, 845, 209. (13) Cinteza, L. O.; Ohulchanskyy, T. Y.; Sahoo, Y.; Bergey, E. J.; Pandey, R. K.; Prasad, P. N. Mol. Pharmaceutics 2006, 3, 415. (14) (a) Xu, H.; Yan, F.; Monson, E. E.; and Kopelman, R. J. Biomed. Mater. Res. 2003, 66A, 870. (b) Kopelman, R.; Koo, Y.-E. L.; Philbert, M. M.; Bradford, A.; Ramachandra, Reddy, G.; McConville, P.; Hall, D. E.;

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