Polyethyleneimine-Mediated Functionalization of Multiwalled Carbon

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Polyethyleneimine-Mediated Functionalization of Multiwalled Carbon Nanotubes: Synthesis, Characterization, and In Vitro Toxicity Assay Mingwu Shen,† Su He Wang,‡ Xiangyang Shi,*,† Xisui Chen,‡ Qingguo Huang,§ Elijah J. Petersen,| Roger A. Pinto,| James R. Baker, Jr.,‡ and Walter J. Weber, Jr.| College of Chemistry, Chemical Engineering, and Biotechnology, Donghua UniVersity, Shanghai 201620, People’s Republic of China, Michigan Nanotechnology Institute for Medicine and Biological Sciences, UniVersity of Michigan, Ann Arbor, Michigan 48109, Department of Crop and Soil Sciences, UniVersity of Georgia, Griffin, Georgia 30223, and Department of Chemical Engineering, UniVersity of Michigan, Ann Arbor, Michigan 48109 ReceiVed: October 21, 2008; ReVised Manuscript ReceiVed: December 27, 2008

Polymer-functionalized carbon nanotubes hold great promise for their use in environmental and biomedical applications. In this work, polyethyleneimine (PEI) was covalently bonded to acid-treated multiwalled carbon nanotubes (MWCNTs) through amide bond formation. The amine groups of PEI on the surface of MWCNTs were then reacted with acetic anhydride or succinic anhydride to form MWCNTs with neutral or negative surface charges, respectively. The structural transformation, surface potential, and morphology of the functionalized MWCNTs were characterized by nuclear magnetic resonance, thermogravimetric analysis, zeta potential, and transmission electron microscopy. The functionalized MWCNTs are water-soluble and stable. In vitro cytotoxicity assays using both FRO cells (a human thyroid cancer cell line) and KB cells (a human epithelial carcinoma cell line) reveal that the biocompatibility of these functionalized MWCNTs is largely dependent on their surface potential. Neutral and negatively charged MWCNTs are nontoxic to both cell lines at a concentration up to 100 µg/mL, whereas positively charged MWCNTs are toxic to FRO cells at 10 µg/mL. The results of this study demonstrate that PEI-modified MWCNTs can be chemically modified to alter their surface charges and cytotoxicity, thereby significantly improving the biocompatibility of the materials for a variety of biomedical applications. Introduction Recent advances in nanoscience and nanotechnology have revealed that carbon nanotubes (CNTs) can be used as a versatile platform for a variety of biomedical applications, including protein and peptide transporters,1-4 drug and gene delivery,1,5-14 medical imaging,15-20 and cancer targeting and therapeutics.15,21-25 These applications often involve improving the water solubility of the CNTs by modifying them through hydrophobic24,26,27 and covalent interactions with target molecules. For the covalent surface functionalization of CNTs, the most common method involves reactions with carboxyl (-COOH) functional groups on the CNTs, which are usually introduced by oxidation with strong acids at the more reactive (open) end or defect sites, instead of their side walls.28 In comparison to the side-wall functionalization of CNTs that usually includes nitrene cycloaddition, arylation using diazonium salts, or 1,3-dipolar cycloadditions,29 the former approach is simple and does not require multiple organic synthesis steps. Polyethyleneimine (PEI) polymers have been functionalized onto CNTs through the reaction of their amine groups with thionyl chloride-activated CNTs (with carboxyl groups),30-32 through the reaction of their amine groups with fluorinated single-walled carbon nanotubes (SWNTs),33 or through direct amination in organic solvents.30,34,35 These PEI-modified CNTs * To whom correspondence should be addressed. E-mail: [email protected]. † Donghua University. ‡ Michigan Nanotechnology Institute for Medicine and Biological Sciences, University of Michigan. § University of Georgia. | Department of Chemical Engineering, University of Michigan.

have many interesting applications such as gas adsorption,33-35 gene delivery,36 and as a neural growth substrate.32 They can also be further assembled and modified to form superhydrophobic multilayered surfaces30 and can be used to form hybrid inorganic/CNT materials.37,38 Thus, PEI-modified CNTs are an important intermediate material that can be further processed or functionalized for various applications. Although it has been shown that PEI-modified CNTs can be acylated by a mixed anhydride of octadecanoic acid to generate hydrophobic CNTs or CNT films,30 a systematic investigation of the acylation of PEI-CNTs to produce differently charged nanotubes is not reported in the literature. We anticipate that through judicious acylation of PEI-modified CNTs, neutralized or negatively charged CNTs could be created to improve the biocompatibility of the CNTs for various biological applications. In this present study, we first modified multiwalled CNTs (MWCNTs) with PEI through amide bond formation, and the amine groups of the attached PEI were further reacted with acetic anhydride or succinic anhydride to produce neutralized or negatively charged CNTs, respectively (Scheme 1). The functionalized MWCNTs were characterized using nuclear magnetic resonance (NMR) spectroscopy, thermogravimetric analysis (TGA), zeta potential measurements, and transmission electron microscopy (TEM). Biocompatibility of the differently functionalized MWCNTs was tested by an MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay using two different cell lines: FRO cells, a human thyroid cancer cell line, and KB cells, a human epithelial carcinoma cell line. To our knowledge, this is the first report regarding the systematic acylation of PEI-modified CNTs to generate CNTs with different

10.1021/jp809323e CCC: $40.75  2009 American Chemical Society Published on Web 02/04/2009

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SCHEME 1: Schematic Representation of Reactions To Modify MWCNTs through PEI-Mediated Functionalization

surface charges. Results from this study provide new insights in the polymer functionalization of CNTs for biomedical applications. Experimental Section Materials. MWCNTs (diameter ) 30-70 nm, length ) 100 nm-2 µm) were synthesized and characterized as described in previous reports.39,40 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 CNTs with carboxylic acid residues.41 Hyperbranched PEI (Mw ) 25000), acetic anhydride, succinic anhydride, and all other chemicals and solvents were obtained from Aldrich and used as received. Regenerated cellulose membranes (MWCO ) 50000) were acquired from Fisher. The water used in all experiments was passed through a Millipore Milli-Q Plus 185 purification system and had a resistivity exceeding 18.2 MΩ · cm. PEI-Mediated Functionalization of MWCNTs. As shown in Scheme 1, MWCNTs were treated with thionyl chloride to introduce acid chloride groups on the MWCNTs. Subsequent amidation with PEI in anhydrous N,N-dimethylformamide (DMF) yielded CNT/PEI, and these products were then treated with acetic anhydride or succinic anhydride to produce acetylated CNT/PEI.Ac (Ac denotes acetyl groups) or carboxylated CNT/PEI.SAH(SAHdenotessuccinamicacidgroups),respectively. In a typical synthesis, 99.8 mg of acid-treated MWCNTs was dispersed in 20 mL of SOCl2 and 1 mL of DMF and refluxed for 24 h. Then, the reaction mixture was centrifuged (5000 rpm, 10 min) and the solvent decanted. To fully remove the thionyl chloride, the mixture was centrifuged and redispersed in DMF for 5 times. Then, the SOCl2-treated MWCNTs were redispersed in dehydrated DMF (8 mL). PEI (93 mg) dissolved into 4 mL of DMF was added into the solution of SOCl2-treated MWCNTs under vigorous magnetic stirring. A total of 200 µL of triethylamine was then added to the reaction mixture. The reaction was carried out at 50 °C for 48 h. The DMF and the excess of reactants and byproduct were removed from the mixture by extensively dialysis against water (6 times, 4 L) for 3 days, followed by lyophilization to obtain the CNT/PEI. To yield modified CNT/PEI with a neutral surface charge, the amine groups of the PEI attached onto the MWCNTs were acetylated. Briefly, 500 µL of triethylamine was added to the solution of CNT/PEI (30 mg) dispersed in DMSO (5 mL), and the solution was thoroughly mixed. A DMSO solution (5 mL) containing acetic anhydride (390 µL) was added dropwise into the solution of CNT/PEI mixed with triethylamine while it was being stirred vigorously. The mixture was allowed to react for

24 h. The DMSO, excess reactants, and byproduct were removed from the mixture by extensive dialysis against PBS buffer (3 times, 4 L) and water (3 times, 4 L) for 3 days, followed by lyophilization to obtain the CNT/PEI · Ac. To yield modified CNT/PEI with a negative surface charge, the amine groups of PEI attached onto MWCNTs were carboxylated using succinic anhydride. In brief, CNT/PEI (30 mg) dispersed in DMSO (5 mL) was mixed with succinic anhydride (410 mg) dissolved in 5 mL of DMSO under vigorous magnetic stirring. The mixture was allowed to react for 24 h. The DMSO and the excess reactants and byproduct were removed from the mixture by extensive dialysis against water (6 times, 4 L) for 3 days, followed by lyophilization to obtain the CNT/PEI · SAH. Characterization Techniques. 1H NMR spectra of functionalized MWCNTs were recorded on a Bruker DRX 500 nuclear magnetic resonance spectrometer. Samples were dispersed in D2O before NMR measurements. TGA measurements were performed using a Perkin-Elmer TGA-7 thermogravimetric analyzer with a heating rate of 48 °C/min in air. Zeta potential measurements were performed using a Malvern Zetasizer Nano ZS model ZEN3600 (Worcestershire, U.K.) equipped with a standard 633 nm laser. TEM measurements were performed at 60 kV on a Philips CM-100 microscope equipped with a Hamamatsu Digital Camera ORCA-HR operated using AMT software (Advanced Microscopy Techniques Corp, Danver, MA). A 5 µL aqueous solution of a MWCNT sample (3 mg/ mL) was dropped onto a carbon-coated copper grid and airdried before TEM analysis. Cell Biological Evaluation. FRO cells (a human thyroid cancer cell line, ATCC, Rockville, MD) were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1× nonessential amino acids, and 1.0 nM sodium pyruvate. KB cells (a human epithelial carcinoma cell line, ATCC, CLL17, Rockville, MD) were continuously grown in RPMI 1640 medium supplemented with 10% heat-inactivated FBS and 2.5 µM folic acid. An MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay was used to quantify the viability of both cell lines. Briefly, 8 × 103 FRO or KB cells per well were seeded into a 96-well plate. After overnight incubation, functionalized MWCNTs at concentrations ranging from 0-100 µg/mL in PBS buffer (pH 7.4) was added. After 24 h incubation with MWCNTs at 37 °C, MTT reagent in PBS solution was added to detect the metabolically active cells in each well. Then, the plates were read at 570 nm. Mean and standard deviation for the triplicate wells were reported. The comparison of the modified MWCNTs with the pristine acid-

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Figure 1.

Shen et al.

H NMR spectra of PEI, MWCNTs, CNT/PEI, CNT/PEI · Ac, and CNT/PEI · SAH.

1

treated MWCNTs and the comparison of all MWCNT materials at different concentrations with the PBS control were tested using ANOVA statistical method (SAS 9.0, SAS Institute Inc., Cary, North Carolina). In parallel, after treatment with functionalized MWCNTs for 24 h, the cell morphology was observed by Leica DMIRB fluorescent inverted microscope. The magnification is set at 200× for all samples. Results and Discussion The covalent linkages between MWCNTs and PEI were qualitatively confirmed by 1H NMR spectroscopy (Figure 1). Acid-treated MWCNTs do not display any salient features related to the proton signals. However, after linked with PEI, the -CH2- proton signals of 1-3 related to PEI are clearly observed in the spectrum of the PEI-modified MWCNTs (CNT/ PEI). Further acetylation of the amines of PEI that was modified onto MWCNTs (CNT/PEI.Ac) introduces a new proton signal 4 (1.87 ppm), which is related to the -COCH3 protons. For the carboxylated MWCNTs (CNT/PEI.SAH), signals 5 (2.6 ppm) and 6 (2.9 ppm) related to the -CH2- protons of succinamic acid end groups are present. The somewhat more isolated proton signals of 1-3 for both CNT/PEI · Ac and CNT/PEI · SAH when compared with those of PEI and CNT/PEI also indicate the transformation of the PEI amine groups to acetyl and succinamic acid groups, respectively. TGA analysis (Figure 2) was also used to characterize the surface modification of the MWCNTs. For pristine acid-treated MWCNTs, there is no significant weight loss after increasing the temperature to 500 °C (4.2%). However, at the same temperature, CNT/PEI shows 30.6% weight loss, which is ascribed to the presence of PEI grafted onto the surface of MWCNTs. After further modification of the PEI amine groups with acetic anhydride and succinic anhydride, respectively, the formed CNT/PEI · Ac and CNT/PEI · SAH exhibit respective 40.7 and 43.6% weight losses after heating to 500 °C. The increased weight loss of CNT/PEI · Ac and CNT/PEI · SAH (when compared with CNT/PEI) verifies the successful transformation of the PEI amine groups to acetyl and succinamic acid end groups, respectively. The carboxylation of CNT/PEI gives rise to end groups of -NHCOCH2CH2COOH, which have

Figure 2. TGA curves of MWCNT, CNT/PEI, CNT/PEI · Ac, and CNT/PEI · SAH.

Figure 3. Comparison of the stability of MWCNTs (1), CNT/PEI (2), CNT/PEI · Ac (3), and CNT/PEI · SAH (4) in PBS buffer.

a higher molecular weight, compared to the -NHCOCH3 end groups generated from acetylation of CNT/PEI. Thus, the slightly higher weight loss for the CNT/PEI · SAH compared to the CNT/PEI · Ac follows the expected trend. MWCNTs grafted with PEI or PEI derivatives are fairly stable in aqueous solution. A uniform black color of the MWCNTs grafted with PEI or PEI derivatives (Figure 3) indicates the colloidal stability of the functionalized MWCNTs. It should be noted that the purification of CNT/PEI through a dialysis process using a membrane with MWCO ) 50000 ensures that both the acetylation and carboxylation reactions occur on the PEI molecules that were covalently attached on MWCNTs. Both acylation reactions do not alter the hydrophilicity of the functionalized MWCNTs. We assume that both acetylation and carboxylation of PEI molecules do not change the hydrophilicity

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Figure 4. TEM images of (a) pristine acid-treated MWCNTs, (b) CNT/PEI, (c) CNT/PEI · Ac and (d) CNT/PEI · SAH.

TABLE 1: Zeta Potential Values of Functionalized MWCNTs materials zeta-potential (mV)

MWCNTs

CNT/PEI

CNT/PEI · Ac

CNT/PEI · SAH

-45.6

+34.6

-0.756

-20.6

of PEI, which is quite similar to the acetylation and carboxylation of poly(amidoamine) dendrimers.42 As compared with pristine acid-treated MWCNTs, which precipitate after 3 days’ storage in PBS buffer solution, the PEI-modified MWCNTs are stable for at least a month under similar conditions. Further transformation of the amine groups of the grafted PEI does not seem to alter the stability of the MWCNTs. No precipitation occurred for all CNT/PEI, CNT/PEI · Ac, and CNT/PEI · SAH solutions for at least a month (Figure 3). The precipitated MWCNTs without PEI modification in aqueous solution are probably related to the aggregated bundles of MWCNTs. TEM images of the pristine acid-treated MWCNTs, CNT/PEI, CNT/PEI · Ac, and CNT/PEI · SAH are shown in Figure 4. The variable wall thicknesses of the MWCNTs originate from the nature of the chemical vapor deposition approach used to produce MWCNTs.39,40 It is clear that after grafting with PEI and PEI derivatives, the morphology of the MWCNTs does not change significantly. No aggregation of MWCNTs was observed in the images, suggesting that the PEI was uniformly coated onto each nanotube. The lower contrast of PEI polymers does not allow for visualization of the polymer coating on the carbon nanotubes. The surface modification of MWCNTs was assessed by zeta potential measurements (Table 1). The surface potential of the acid-treated MWNCTs (-45.6 mV) became positive (34.6 mV) after modification with PEI. Subsequent acetylation and carboxylation of the amines of PEI generated neutral (-0.756 mV)

and negatively charged (-20.6 mV) nanotubes, respectively. The zeta potential changes reflect the successful surface modification of MWCNTs, suggesting that the surface potentials of MWCNTs can be manipulated through PEI-mediated reactions. The cytotoxicity of MWCNTs before and after the grafting of PEI or PEI derivatives was assessed by an MTT assay of two different cell lines (FRO cells and KB cells). After incubation of the pristine MWCNTs, CNT/PEI, CNT/PEI · Ac, and CNT/PEI · SAH with cells for 24 h, an MTT assay was performed to evaluate the viability of both FRO and KB cells (Figure 5). There was not a statistically significant difference in the cell viability compared to the controls for both FRO and KB cells treated with pristine MWCNTs, CNT/PEI · Ac, and CNT/PEI · SAH at concentrations ranging from 0-100 µg/mL (p > 0.05). However, CNT/PEI starts to exhibit cytotoxicity at 10 µg/mL and 50 µg/mL to FRO cells (Figure 5a) and KB cells (Figure 5b), respectively (p < 0.0001 for both treatments). In general, the cytotoxicity of amine-terminated PEI or dendrimers stem from strong electrostatic interaction between the positively charged polymers or nanoparticles and the negatively charged cell membranes.43-45 This study underlines the fact that pristine acid-treated MWCNTs can be modified using PEI and then exhibit cytotoxicity, but the nanotubes can be made nontoxic after additional acylation of the amine groups of the grafted PEI to form neutrally or negatively charged MWCNTs thus making the nanotubes suitable for various biomedical applica-

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Figure 5. MTT assay of FRO (a) and KB (b) cell viability after treatment with differently functionalized MWCNTs for 24 h. Mean and standard deviation for the triplicate wells were reported. The data are expressed as mean ( S. D. Statistical significance was calculated using ANOVA method and is indicated with (*) for p < 0.05, (**) for p < 0.001, and (***) for p < 0.0001.

Figure 6. Phase-contrast photomicrographs of untreated FRO cells (a) and FRO cells treated with 10 µg/mL pristine acid-treated MWCNTs (b), CNT/PEI (c), CNT/PEI · Ac (d), and CNT/PEI · SAH (e).

tions. Earlier research has shown that MTT assays may give false positive results when testing the cytotoxicy of single-walled carbon nanotubes.46 It is possible that MTT may interact differently with MWCNTs based on their surface modifications, which could lead to false interpretations. After mixing MTT with the pristine acid-treated and different functionalized MWCNTs in the absence of cells, however, there was not a significant difference in the MTT assay results between unmodified and functionalized MWCNTs at different concentrations (Figure S1, Supporting Information). Cytotoxicity results from cell treatments with the variously modified nanotubes thus cannot be attributed to artifacts from nanotube interactions during the MTT assay. The toxicity of the pristine acid-treated MWCNTs and MWCNTs modified with PEI or PEI derivatives was also evaluated by microscopically examining the FRO and KB cells

(Figures 6 and 7). The microscopic results closely matched those obtained from the MTT assay. The morphology of FRO cells treated with the pristine acid-treated MWCNTs, CNT/PEI · Ac, and CNT/PEI · SAH at a concentration of 10 µg/mL is similar to the morphology of untreated FRO cells, suggesting that after acylation of the amine groups of PEI grafted onto MWCNTs to form neutralized and negatively charged materials, the nanotubes exhibited very good biocompatibility (Figure 6). In contrast, without further functionalization of the PEI amines, the CNT/PEI is toxic to FRO cells at the same concentration. Similarly, when treated with the pristine acid-treated MWCNTs, CNT/PEI · Ac, and CNT/PEI · SAH at the concentration of 50 µg/mL, KB cells appear healthy when compared with the untreated control cells (Figure 7). However, under similar conditions, cytotoxicity to KB cells was visually evident when treated with CNT/PEI without the functionalization of PEI

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Figure 7. Phase-contrast photomicrographs of untreated KB cells (a) and KB cells treated with 50 µg/mL pristine acid-treated MWCNTs (b), CNT/PEI (c), CNT/PEI · Ac (d), and CNT/PEI · SAH (e).

amines (Figure 7c). The KB cells became rounded and nonadherent, indicating that there is a significant percentage of cell death (Figure 7c). It should be noted that both FRO and KB cells studied are cancer derived cell lines. It is likely that the CNT/PEI would also be toxic to normal cells. Our group previously showed that amine-terminated dendrimers at sufficiently high concentrations are toxic to both cancer cells and normal cells,43 and that the toxicity is solely related to the positive charge of the used polymers.43,44 However, further experiments are necessary to assess if these findings would also pertain to the CNT/PEI. Conclusion In summary, the present study shows that pristine acid-treated MWCNTs covalently modified by PEI can be further functionalized through PEI-mediated acetylation and carboxylation reactions. In all cases, the MWCNTs display enhanced water solubility after functionalization with PEI or PEI derivatives. More importantly, the surface charge of the MWCNTs can be modulated to be positive, negative, or neutral. We show that the pristine acid-treated MWCNTs, the neutral (CNT/PEI · Ac), and the negatively charged MWCNTs (CNT/PEI · SAH) do not display cytotoxicity at a concentration up to 100 µg/mL. However, without further modification, the PEI-modified MWCNTs are cytotoxic. It implies that through PEI modification, MWCNTs can be further functionalized to significantly improve their water solubility and biocompatibility. We anticipate that the approach may be extended to conjugate various biomolecules onto carbon nanotube surfaces with enhanced water solubility and biocompatibility, providing many opportunities for the applications of carbon nanotubes in biomedical sciences. Acknowledgment. We thank Sasha Meshinchi for his assistance with TEM experiments and Dr. Nicholas Kotov for use of his Malvern Zetasizer instrument. 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. Supporting Information Available: The interaction of MTT with pristine acid-treated MWCNTs and different surface-

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