Multifunctional Dendrimer-Modified Multiwalled Carbon Nanotubes

May 21, 2009 - Elijah J. Petersen , D. Xanat Flores-Cervantes , Thomas D. Bucheli , Lindsay C. C. Elliott , Jeffrey A. Fagan , Alexander Gogos , Shann...
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Multifunctional Dendrimer-Modified Multiwalled Carbon Nanotubes: Synthesis, Characterization, and In Vitro Cancer Cell Targeting and Imaging Xiangyang Shi,*,† Su He Wang,*,‡ Mingwu Shen,† Mary E. Antwerp,‡ Xisui Chen,‡ Chang Li,‡ Elijah J. Petersen,§ Qingguo Huang,| Walter J. Weber, Jr.,§ and James R. Baker, Jr.*,‡ Nanobiotechnology Laboratory, College of Chemistry, Chemical Engineering, and Biotechnology, Donghua University, Shanghai 201620, People’s Republic of China, Michigan Nanotechnology Institute for Medicine and Biological Sciences, and Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan 48109, and Department of Crop and Soil Sciences, University of Georgia, Griffin, Georgia 30223 Received February 5, 2009; Revised Manuscript Received April 9, 2009

Carbon nanotubes hold great promise for their use as a platform in nanomedicine, especially in drug delivery, medical imaging, and cancer targeting and therapeutics. Herein, we present a facile approach to modifying carbon nanotubes with multifunctional poly(amidoamine) (PAMAM) dendrimers for cancer cell targeting and imaging. In this approach, fluorescein isothiocyanate (FI)- and folic acid (FA)-modified amine-terminated generation 5 (G5) PAMAM dendrimers (G5 · NH2-FI-FA) were covalently linked to acid-treated multiwalled carbon nanotubes (MWCNTs), followed by acetylation of the remaining primary amine groups of the dendrimers. The resulting MWCNT/G5.NHAc-FI-FA composites are water-dispersible, stable, and biocompatible. In vitro flow cytometry and confocal microscopy data show that the formed MWCNT/G5 · NHAcFI-FA composites can specifically target to cancer cells overexpressing high-affinity folic acid receptors. The results of this study suggest that, through modification with multifunctional dendrimers, complex carbon nanotube-based materials can be fabricated, thereby providing many possibilities for various applications in biomedical sensing, diagnosis, and therapeutics.

Introduction One-dimensional nanomaterials are of immense scientific and technological interest due to their unique structures and properties.1-4 Carbon nanotubes (CNTs) are well-ordered, allcarbon hollow graphitic nanomaterials with a high aspect ratio, high surface area, high mechanical strength, ultralight weight, unique electronic properties, and excellent chemical and thermal stability.5,6 These properties make them a promising nanoplatform for biomedical applications including, but not limited to, protein and peptide transporter,7-10 drug and gene delivery,1,9,11-16 medical imaging,2,17-20 and cancer targeting and therapeutics.2,21-26 Most of the applications involve nanotube surface functionalization with polymers or small molecules to improve the aqueous solubility of the CNTs. One of the most common methods of covalently functionalizing CNTs employs reactions with carboxylic acid (-COOH) residues on the CNTs,27 which are usually introduced by oxidation with strong acids at the more reactive (open) end or defect sites, instead of with their side walls.28 This approach has been proven to be simple and does not require multiple organic synthesis steps. Dendrimers are a novel class of highly branched, monodispersed, synthetic macromolecules with well-defined composition and architecture.29,30 The unique properties of dendrimers, especially poly(amidoamine) (PAMAM) dendrimers, make them * To whom correspondence should be addressed. E-mail: [email protected] (X.S.); [email protected] (S.H.W.); [email protected] (J.R.B.). † Donghua University. ‡ Michigan Nanotechnology Institute for Medicine and Biological Sciences. § Department of Chemical Engineering, University of Michigan. | University of Georgia.

an ideal platform to covalently link dyes, targeting ligands, and drugs for multifunctional imaging, targeting, and therapeutic treatment of cancer cells.31-36 Furthermore, through various templating, stabilization, or assembly approaches, dendrimerentrapped,37-39 dendrimer-stabilized,40-42 or dendrimer-assembled inorganic nanoparticles (NPs)43-45 can be synthesized for biomedical applications, especially for targeted cancer imaging and therapeutics. Recent advances have revealed that dendrimers can be covalently functionalized on the surface of CNTs for subsequent metal or metal oxide nanoparticle synthesis46,47 and assembly,48 or for further electrical and biological applications.49-52 This implies that by combining the dendrimers’ surface functionality and unique molecular recognition ability with the electronic properties of CNTs, it may be possible to generate various complex composite nanodevices for a wide range of biomedical applications. In this present study, we directly functionalized multiwalled carbon nanotubes (MWCNTs) with generation 5 (G5) amineterminated PAMAM dendrimers that were covalently linked with fluorescein isothiocyanate (FI) and folic acid (FA). The carboxyl residues on the surface of MWCNTs treated with strong acids allow the conjugation with the amine groups of PAMAM dendrimers via EDC coupling chemistry. The remaining amine groups on the G5 dendrimers were then acetylated to neutralize their surface charge (Scheme 1). The resulting complex MWCNT/G5 · NHAc-FI-FA nanodevices and control MWCNT/G5.NHAc-FI composites without FA were characterized using UV-vis spectrometry, 1H NMR, transmission electron microscopy (TEM), zeta potential measurements, and thermogravimetric analysis (TGA). We show that multifunctional MWCNT/G5 · NHAc-FI-FA nanodevices are water dis-

10.1021/bm9001624 CCC: $40.75  2009 American Chemical Society Published on Web 05/21/2009

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Scheme 1. Schematic Representation of Reactions To Modify MWCNTs with Dendrimers for Cancer Cell Targeting

persible, stable, and biocompatible. In vitro flow cytometry and confocal microscopic imaging studies reveal that the fabricated nanodevices with FA modification can specifically target and be internalized by a cancer cell line (KB cells, a human epithelial carcinoma cell line) that overexpresses high-affinity folic acid receptors (FARs). To our knowledge, this is the first study related to the functionalization of CNTs with dendrimers for cancer cell targeting and imaging. The major advantage of our approach is that, unlike other methods, both targeting ligands and imaging molecules can be modified onto CNTs through a single step dendrimer-mediated reaction.

Experimental Section Materials. MWCNTs (diameter ) 30-70 nm, length ) 100 nm-2 µm) were synthesized and characterized in previous reports.53,54 The MWCNTs were treated with concentrated HNO3/H2SO4 (v/v ) 3:1) for 2 h, followed by filtration and drying to render the surface of MWCNTs with carboxylic acid residues.46 Ethylenediamine core amineterminated PAMAM dendrimers of generation 5 (G5 · NH2) with a polydispersity index less than 1.08 were purchased from Dendritech (Midland, MI). FA, FI, acetic anhydride, triethylamine, 1-ethyl-3-[3dimethylaminopropyl]carbodiimide hydrochloride (EDC), and all other chemicals and solvents were obtained from Aldrich and used as received. FI- and FA-functionalized generation 5 (G5 · NH2-FI-FA) PAMAM dendrimers were synthesized and characterized according to our previous work.45 FI-functionalized G5 dendrimers (G5 · NH2-FI) without FA conjugation were used as a control. KB cells were from American type Tissue Collection (ATCC, Rockville, Maryland). Penicillin, streptomycin, and fetal bovine calf serum (FBS) were purchased from Sigma (St. Louis, MO). Trypsin-EDTA, Dulbecco’s PBS, RPMI 1640 medium (with or without FA), and bovine serum albumin was obtained from GIBCO-BRL (Gaithersburg, MD). Water used in all experiments was purified using a Milli-Q Plus 185 water purification system (Millipore, Bedford, MA) with resistivity higher than 18 MΩ cm. Regenerated cellulose membranes with molecular weight cutoff (MWCO) of 50000 were acquired from Fisher. Fabrication of Multifunctional Dendrimer-Modified MWCNTs. The procedure used to fabricate multifunctional dendrimer-modified MWCNTs is shown in Scheme 1. In a typical synthesis, acid-treated MWCNTs (16.68 mg) were dispersed in 12 mL of water. EDC (12.82 mg) dissolved into DMSO (2 mL) was then mixed with the aqueous solution of MWCNTs. The mixture was allowed to react for 3 h under vigorous magnetic stirring, followed by addition of G5 · NH2-FI-FA solution (4.60 mg in 1 mL of water). The reaction was continued for 48 h under vigorous magnetic stirring, followed by 3 cycles of centrifugation/ redispersion (in water) to remove excess reactants. The MWCNT/ G5 · NH2-FI-FA composites were subjected to an acetylation reaction to neutralize the remaining amine groups of G5 · NH2-FI-FA dendrimers using a procedure described elsewhere.55,56 In brief, the composites

(in 10 mL of water) were thoroughly mixed with triethylamine (9.8 µL). Then, a methanol solution (1 mL) containing 7.2 mg acetic anhydride was added dropwise into the composites/triethylamine solution. The reaction mixture was vigorously stirred for 24 h. The formed MWCNT/G5 · NHAc-FI-FA composites were purified by extensive dialysis against PBS buffer (3 times, 4 L) and water (3 times, 4 L) for 3 days using a membrane with MWCO of 50000, followed by lyophilization to obtain the MWCNT/G5 · NHAc-FI-FA composites. For biological testing, the composites were dispersed into a PBS buffer solution and stored at 4 °C before biological testing. The control composites (MWCNT/G5 · NHAc-FI) without FA conjugation were synthesized identically except that FI-modified amine-terminated G5 dendrimers (G5 · NH2-FI) were used. These dendrimers were prepared and characterized according to a previous report.45 General Characterization Methods. UV-vis spectra were collected using a Perkin-Elmer Lambda 20 UV-vis spectrometer. All MWCNT samples were dispersed in water at the concentration of 1 mg/mL. 1H NMR spectra of the functionalized MWCNTs were recorded on a Bruker DRX 500 nuclear magnetic resonance spectrometer. Samples were dissolved in D2O before NMR measurements. The surface potential of functionalized MWCNTs was measured by a Malvern Zetasizer Nano ZS model ZEN3600 (Worcestershire, U.K.) equipped with a standard 633 nm laser. The size and morphology of the MWCNT composites were characterized by a Philips CM-100 TEM equipped with a Hamamatsu Digital Camera ORCA-HR operated using AMT software (Advanced Microscopy Techniques Corp, Danver, MA). The operation voltage was kept at 60 kV. TEM samples were prepared by deposition of a diluted particle suspension (5 µL) onto a carbon-coated copper grid and were air-dried before the measurement. TGA measurements were performed using a Perkin-Elmer TGA-7 thermogravimetric analyzer with a heating rate of 48 °C/min in air. Cell Cultures. The KB cells were continuously grown in two 10cm culture dishes, one in FA-free media and the other in regular RPMI 1640 cell culture medium supplemented with penicillin (100 units/mL), streptomycin (100 µg/mL), 10% heat-inactivated FBS, and 2.5 µM FA. The cells grown in FA-free media express high-level FARs (for short, KB-HFAR cells), while the cells grown in FA-containing media express low-level FARs (for short, KB-LFAR cells). MTT Quantitation of Cell Viability. An MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to quantify the viability of the cells. Briefly, approximately 1 × 104 KB cells per well were seeded into a 96-well plate. After culturing overnight, functionalized MWCNTs at concentrations ranging from 0 to 100 µg/ mL in PBS were added to each well. After 24 h incubation, the metabolically active cells were then detected by adding MTT to a final concentration of 0.5 mg/mL for 4 h at 37 °C. Isopropanol with 0.04 N HCl was added, and the plates were read at 570 nm with a 630 nm reference. Mean and standard deviation for the triplicate wells were reported.

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Figure 1. (a) UV-vis spectra of the pristine acid-treated MWCNTs (curve 1), and the functionalized MWCNT/G5 · NHAc-FI (curve 2) and MWCNT/ G5 · NHAc-FI-FA (curve 3) composites. (b) Comparison of the stability of the pristine acid-treated MWCNTs (1), and the functionalized MWCNT/ G5.NHAc-FI (2) and MWCNT/G5.NHAc-FI-FA (3) composites in PBS buffer.

Flow Cytometry Analysis. Approximately 1 × 105 cells per well were seeded in 24-well plates the day before the experiments. An hour before initiating an experiment, the cells were rinsed four times with serum-free RPMI 1640 medium. Functionalized MWCNTs were added at final concentrations of 0-10 µg/mL. After 1 h incubation with the functionalized MWCNTs, the KB-HFAR and KB-LFAR cells were trypsinized and suspended in PBS containing 0.1% bovine serum albumin and analyzed using a Becton Dickinson FACScan analyzer. The FL1-fluorescence of 10000 cells was measured, and the mean fluorescence of the gated viable cells was quantified. Confocal Microscopy. Confocal microscopic analysis was performed using an Olympus FluoView 500 laser scanning confocal microscope (Melville, NY). The cells were plated on a plastic coverslip before measurements. The FI fluorescence was excited with a 488 nm argon blue laser, and the emission was measured through a 505-525 barrier filter. The optical section thickness was set at 5 µm. The cells were incubated with functionalized MWCNTs (10 µg/mL) for 2 h, followed by rinsing with PBS buffer. The nuclei were counterstained with 1 µg/mL of Hoescht33342 using a standard procedure. Samples were imaged using a 60× water-immersion objective lens and magnified with FluoView software.

Results and Discussion CNTs have been modified and bioconjugated with specific biomolecules for targeted cancer imaging and therapeutics. Most of the reported approaches involve multiple synthetic steps.2,57,58 The unique properties of dendrimer chemistry provide a straightforward strategy for modifying the surface of CNTs for various applications. For example, glycodendrimer-modified CNTs can significantly improve the biocompatibility of the CNTs;52 dendrimer-modified CNTs can be used as an intermediate material for complex hierarchical materials synthesis,46,47 and can be used for chemical and biochemical sensing.50 None of the literature reports are related to the modification of dendrimers onto CNTs for cancer targeting and imaging studies, which is the subject of our present research. Figure 1a shows the UV-vis spectra of the functionalized MWCNTs. The pristine acid-treated MWCNTs display a peak at 255 nm (Curve 1), which is typical for MWCNT materials.59 The characteristic FI absorption peak at 500 nm in both curves 2 and 3 confirms the modification of both G5 · NHAc-FI-FA and G5 · NHAc-FI dendrimers on the MWCNTs. The prominent absorption peak at 260 nm for MWCNT/G5 · NHAc-FI-FA composites is related to the overlap of the FA absorbance (280 nm) and the typical MWCNT peak at 255 nm. In contrast, at the same wavelength, MWCNT/G5 · NHAc-FI composites only show a weak band, which is solely related to the absorbance of the modified MWCNTs. Unlike the pristine acid-treated

MWCNTs which precipitate out of PBS buffer after 3 days, the dendrimer-functionalized MWCNTs are water-dispersible and stable for at least 6 months in water, PBS buffer, and serum at 4 °C (Figure 1b and Figure S2 in Supporting Information). The stability of the functionalized MWCNTs in both water and PBS buffer was also occasionally confirmed using UV-vis spectrometry within 6 months. We did not see any appreciable changes in the absorption features of these nanocomposites. This implies that the dendrimer functionalization on MWCNTs significantly increases the water solubility and stability of MWCNTs, which is essential for them to be used for biological applications. The modification of MWCNTs with dendrimers was also qualitatively confirmed by 1H NMR spectroscopy (Figure S1, Supporting Information). After the acetylation reaction, the appearance of the peak at 1.85 ppm related to the -CH3 protons of the acetyl groups clearly indicates the formation of acetamide groups on the dendrimer surface for both MWCNT/G5 · NHAcFI-FA and MWCNT/G5 · NHAc-FI composites.55 However, the aromatic proton peaks related to both FI and FA did not show any significant changes compared to the 1H NMR spectra of the G5 · NH2-FI and G5 · NH2-FI-FA dendrimers, respectively.45 Zeta potential measurements show that after the acetylation reaction, the surface potentials of the MWCNT/G5 · NH2-FIFA (+28.3 mV) and MWCNT/G5 · NH2-FI (+31.6 mV) composites significantly decreased when the MWCNT/G5 · NHAcFI-FA (+3.1 mV) and the MWCNT/G5 · NHAc-FI (+4.5 mV) composites were formed, further confirming the successful transformation of the dendrimer terminal amines to acetamide groups. TEM images of the pristine acid-treated MWCNTs, MWCNT/ G5 · NHAc-FI, and MWCNT/G5 · NHAc-FI-FA are shown in Figure 2. It is clear that after modification with dendrimers, the morphology of the MWCNTs does not change significantly. No aggregation of MWCNTs was observed in the images, suggesting that the dendrimers were uniformly attached to the -COOH groups of the acid-treated MWCNTs. The lower contrast of dendrimers does not allow the visualization of the dendrimer coating onto carbon nanotubes. The variable wall thicknesses of the MWCNTs originate from the nature of the chemical vapor deposition approach used to produce them and are not indicative of changes to the MWCNTs after reaction with the dendrimers.53,54 TGA analysis (Figure 3) was used to characterize the surface modifications of the MWCNTs. For pristine acid-treated MWCNTs, there is no significant weight loss up to 500 °C (6.6%). However, at the same temperature, MWCNT/G5 · NHAcFI and MWCNT/G5 · NHAc-FI-FA show 21.0 and 24.2% weight

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Figure 2. TEM images of (a) pristine acid-treated MWCNTs, (b) MWCNT/G5 · NHAc-FI, and (c) MWCNT/G5 · NHAc-FI-FA.

Figure 3. TGA curves of pristine acid-treated MWCNTs, MWCNT/ G5 · NHAc-FI, and MWCNT/G5 · NHAc-FI-FA.

loss, respectively, which is ascribed to the presence of the corresponding G5 · NHAc-FI and G5 · NHAc-FI-FA dendrimers grafted onto the surface of MWCNTs. The higher weight loss of MWCNT/G5 · NHAc-FI-FA compared to that of the MWCNT/ G5 · NHAc-FI is attributed to the higher molecular weight of the G5 · NHAc-FI-FA dendrimers (when compared with the G5 · NHAc-FI dendrimers). The cytotoxicity of the functionalized MWCNTs was evaluated by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay. The cell viability data (Figure S3, Supporting Information) show that the KB cells treated with both MWCNT/G5 · NHAc-FI and MWCNT/ G5 · NHAc-FI-FA composites display more or less similar OD (570 nm) values at a concentration range of 0-100 µg/mL. This implies that the formed functionalized MWCNTs are biocompatible at a concentration of up to 100 µg/mL. Literature data show that single-walled CNTs interfere with the results of MTT assay when testing the cell viability.60,61 However, this does not occur for the MWCNTs investigated in this study. To confirm this, we mixed MTT with the dendrimer-functionalized MWCNTs in the absence of cells for 24 h at 37 °C. MTT assay data show that there were no appreciable differences in the absorbances for either MWCNT/G5 · NHAc-FI or MWCNT/ G5 · NHAc-FI-FA composite nanotubes at different concentrations (Figure S4, Supporting Information). This result is consistent with our previous work.27 The toxicity of the functionalized MWCNTs modified with dendrimers was also evaluated by visualizing the morphologies of KB cells (Figures S5 and S6). The morphology of KB cells treated with MWCNT/ G5 · NHAc-FI and MWCNT/G5 · NHAc-FI-FA at a concentra-

tion range of 1-100 µg/mL is similar to that of untreated KB cells, suggesting that the nanotubes are very biocompatible. Even when the formed aggregated carbon nanotubes significantly covered the cell surfaces at the highest nanotube concentrations (100 µg/mL), the cells remained healthy. It is worthwhile to note that the aggregated carbon nanotubes on the surface of cells might be due to the cellular uptake after the long time exposure to cells via a nonspecific mechanism (e.g., lipid raftmediated uptake), and these aggregated nanotubes on the cell surfaces should not be related to the colloidal stability of the functionalized MWCNTs. The vitamin FA and the dye FI conjugated onto the G5 dendrimer surface were used as a targeting ligand and an imaging molecule, respectively, providing the functionalized MWCNTs with both targeting and imaging capabilities. The FAR is widely known to be overexpressed in several human carcinomas including breast, ovary, endometrium, kidney, lung, head and neck, brain, and myeloid cancers.62-64 The high-affinity of FAR for FA (Kd ) 0.1-1 nM) affords specific binding and internalization of FAmodified particles to cancer cells in the presence of normal cells through receptor-mediated endocytosis. In this study, both KBHFAR and KB-LFAR cells were selected for the intracellular uptake of functionalized MWCNTs. Figure 4 shows the flow cytometric analyses of KB-HFAR and KB-LFAR cells after exposure to functionalized MWCNTs (1 µg/mL) for 1 h. Treatment of KB-HFAR cells with MWCNT/G5 · NHAc-FI-FA but not MWCNT/G5 · NHAc-FI results in a significant increase in the fluorescence signal within the cells (Figure 4a), suggesting binding of only MWCNT/G5 · NHAc-FI-FA composites. Conversely, KBLFAR cells treated with either MWCNT/G5 · NHAc-FI-FA or MWCNT/G5 · NHAc-FI-FA show a fluorescence intensity similar to the PBS control (Figure 4b). The cellular uptake of the FAfunctionalized MWCNTs by KB-HFAR cells shows a dosedependent response (Figure 4c). For KB-LFAR cells, neither MWCNT/G5 · NHAc-FI-FA nor MWCNT/G5 · NHAc-FI show significant binding, even at a concentration of up to 10 µg/mL (Figure 4d). The conjugation of the FI-modified G5 dendrimers onto MWCNTs also enables confocal microscopic imaging of the cellular uptake of the functionalized MWCNTs. Figure 5 shows that only KB-HFAR cells treated with FA-modified MWCNT/ G5 · NHAc-FI-FA display prominent fluorescence signals, which is associated with the specific uptake of MWCNT/G5 · NHAcFI-FA into the cytoplasm and also onto the membrane of the cells (Figure 5c,f). In contrast, the same KB cells treated with MWCNT/G5 · NHAc-FI do not show any salient fluorescence signals (Figure 5b,e), which is similar to KB cells treated with PBS buffer (Figure 5a,d). The weak fluorescence signals in both

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Figure 4. Flow cytometric studies of the binding of functionalized MWCNTs with KB cells. (a and b) Binding of MWCNT/G5 · NHAc-FI and MWCNT/G5 · NHAc-FI-FA composites (1 µg/mL) with KB-HFAR cells and KB-LFAR cells, respectively: 1, PBS control; 2, MWCNT/G5 · NHAcFI; 3, MWCNT/G5 · NHAc-FI-FA. (c and d) Dose-dependent binding of MWCNT/G5 · NHAc-FI and MWCNT/G5 · NHAc-FI-FA with KB-HFAR and KB-LFAR cells, respectively.

Figure 5. Confocal microscopic images of KB-HFAR cells treated with PBS buffer (a, d), MWCNT/G5 · NHAc-FI (10 µg/mL; b, e), and MWCNT/ G5 · NHAc-FI-FA (10 µg/mL; c, f) for 2 h, respectively. Images were collected under similar instrumental conditions.

Figure 5a and b originate from the intrinsic green fluorescence of the cells. These results suggest that the binding and

intracellular uptake do not occur significantly in the cells treated with non-FA modified MWCNTs.

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Conclusion In summary, we have developed a facile approach to covalently conjugate functionalized dendrimers onto MWCNTs for cancer cell targeting and imaging. Both targeting ligands and imaging molecules can be modified onto the MWCNTs through a one-step dendrimer-mediated reaction. The formed multifunctional MWCNTs are water dispersible, stable, and biocompatible. In vitro cell biological assay data show that the FA-modified MWCNTs can specifically target to cancer cells overexpressing FARs. Because carbon nanotubes are known to strongly bind to hydrophobic cancer drugs,24,65 we anticipated that the developed dendrimer-functionalized MWCNTs could be used as a nanoplatform for targeted cancer chemotherapy. In addition, recent advances show that carbon nanotubes can release heat in a radiofrequency (RF) field,25 which may be used to produce thermal cytotoxicity in malignant cells. Our study clearly shows that the afforded targeting ability of MWCNTs to cancer cells through dendrimer functionalization may be very useful for future development of MWCNT-based platforms for targeted cancer chemotherapy and physicotherapy. When compared to the dendrimer platform alone, the MWCNT/dendrimer composite should allow delivery of a higher payload of drug molecules as well as the intrinsic heating generation property of MWCNTs, thereby offering many advantages over conventional therapy. Furthermore, this approach to functionalizing MWCNTs via dendrimer chemistry may be applied to various other biological ligands (e.g., sugars, peptides, proteins, and antibodies) for targeting and imaging various biological systems. We are actively exploring this area of research. Acknowledgment. X.S. and S.H.W. contributed equally to this work. This work was financially supported by a subcontract from the University of Michigan Graham Environmental Sustainability Institute. X.S. thanks the support from the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning and the National Basic Research Program of China (973 Program, 2007CB936000). Q.H., E.J.P., and W.J.W., Jr. recognize support from EPA Grants RD833321 and R834094. We thank Sasha Meshinchi for his assistance with TEM experiments and Dr. Nicholas Kotov for use of his Malvern Zetasizer instrument. Supporting Information Available. Additional NMR characterization data for dendrimer-functionalized MWCNTs, MTT assay of KB cell viability, and KB cell morphology after treatment with differently functionalized MWCNTs. This material is available free of charge via the Internet at http:// pubs.acs.org.

References and Notes (1) Kostarelos, K.; Lacerda, L.; Pastorin, G.; Wu, W.; Wieckowski, S.; Luangsivilay, J.; Godefroy, S.; Pantarotto, D.; Briand, J. P.; Muller, S.; Prato, M.; Bianco, A. Cellular uptake of functionalized carbon nanotubes is independent of functional group and cell type. Nat. Nanotechnol. 2007, 2, 108–113. (2) Liu, Z.; Cai, W. B.; He, L. N.; Nakayama, N.; Chen, K.; Sun, X. M.; Chen, X. Y.; Dai, H. J. In vivo biodistribution and highly efficient tumour targeting of carbon nanotubes in mice. Nat. Nanotechnol. 2007, 2, 47–52. (3) Yan, X.; He, Q.; Wang, K.; Duan, L.; Cui, Y.; Li, J. Transition of cationic dipeptide nanotubes into vesicles and oligonucleotide delivery. Angew. Chem., Int. Ed. 2007, 46, 2431–2434. (4) Yang, Y.; He, Q.; Duan, L.; Cui, Y.; Li, J. Assembled alginate/chitosan nanotubes for biological application. Biomaterials 2007, 28, 3083– 3090. (5) Ajayan, P. M. Nanotubes from carbon. Chem. ReV. 1999, 99, 1787– 1800.

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