SWCNT PEG-eggs: Single-walled carbon nanotubes in biocompatible shell-crosslinked micelles

Carbon 45 (2007) 2388–2393 www.elsevier.com/locate/carbon

SWCNT PEG-eggs: Single-walled carbon nanotubes in biocompatible shell-crosslinked micelles Runtang Wang a, Paul Cherukuri a, Juan G. Duque b, Tonya K. Leeuw a, Melinda K. Lackey c, Christine H. Moran a, Valerie C. Moore c, Jodie L. Conyers c, Richard E. Smalley a,d,z, Howard K. Schmidt d, R. Bruce Weisman a, Paul S. Engel a,* a Department of Chemistry, Rice University, P.O. Box 1892, Houston, TX 77251, United States Department of Chemical and Biomolecular Engineering, Rice University, P.O. Box 1892, Houston, TX 77251, United States c Department of Internal Medicine—Cardiology, University of Texas Health Science Center at Houston, 7000 Fannin Street, Suite 1690, Houston, TX 77030, United States Carbon Nanotechnology Laboratory, R.E. Smalley Institute for Nanoscale Science and Technology, Rice University, P.O. Box 1892, Houston, TX 77251, United States b

d

Received 8 May 2007; accepted 9 July 2007 Available online 21 July 2007

Abstract Pristine, individualized single-walled carbon nanotubes (SWCNTs) have been noncovalently captured within PEG-terminated block copolymer amphiphiles. Two cross-linkable amphiphiles were evaluated: polyethylene glycol-polyacrylic acid-polystyrene (PEG-PAAPS) and polyethylene glycol-polybutadiene (PEG-PB). The resulting self-assembled PEG-PAA-PS structures, called PEG-eggs, are freely soluble in water and stable in physiological media. SWCNTs in PEG-eggs retain their intrinsic near-infrared fluorescence, resist exchange with serum proteins, and are non-cytotoxic to mouse macrophage and human renal cells based on in vitro viability assays.  2007 Elsevier Ltd. All rights reserved.

1. Introduction Single-walled carbon nanotubes (SWCNTs) are hollow graphitic cylinders (ca. 1 nm in diameter and ca. 1 lm in length) that have recently been explored as unique nanoscale particles with potential for intravenous pharmaceutical applications such as gene transfection and targeted thermoablation [1–5]. However, the significant hydrophobicity of SWCNTs requires chemical functionalization with water-solubilizing moieties in order to produce stable aqueous SWCNT suspensions. While covalent functionalization of SWCNTs with ionic moieties confers aqueous solubility, it offers limited stability in vivo due to charge screening, which results in pro*

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Corresponding author. Fax: +1 713 348 5155. E-mail address: [email protected] (P.S. Engel). Deceased October 28, 2005.

0008-6223/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.carbon.2007.07.006

nounced flocculation [6,7]. Furthermore, covalent sidewall functionalization perturbs the electronic structure of the nanotube, quenching the intrinsic near-infrared (NIR) fluorescence emission from semiconducting SWCNTs. Therefore, in order to preserve the nanotubes’ electronic properties while providing water compatibility, self-assembly of uncharged amphiphilic biocompatible polymers has been used to noncovalently encapsulate the nanotube [8–11]. Noncovalent steric stabilization using copolymers such as poloxamers provides an attractive route for delivering SWCNTs in vivo. Poloxamers (e.g. pluronic) are nonionic di-block copolymers of polypropylene oxide and polyethylene glycol (PEG) with high conformational flexibility. PEG is a well-established, benign bio-passivating material [12–14] and SWCNTs encapsulated in PEGylated micelles should be stably dispersible. In vivo, the PEG sheath prevents activation of the reticuloendothelial system, providing for longer circulation times, and

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maximizing the concentration of SWCNTs in target tissues for diagnostic or therapeutic uses [12,13]. However, we have recently shown that intravenous injection of poloxamer-encapsulated SWCNTs results in rapid displacement of the micelle coating by endogenous proteins found in blood sera [15]. This displacement and exchange is attributed to intravenous dilution of the poloxamer as well as the relatively weak noncovalent attraction between the di-block copolymers and the nanotube sidewall. Therefore, in order to obtain a stable and biocompatible SWCNT micelle, we have prepared noncovalently encapsulated SWCNTs within a shell-crosslinked, PEGylated micelle. This structure, which we term the ‘‘PEG-egg’’ and illustrate schematically in Fig. 1, is intended to preserve the electronic character of the SWCNT, be resistant to protein displacement, and retain the biostabilizing properties of PEG [14,16,17]. 2. Experimental methods 2.1. Preparation of PEG-PAA-PS Based on an elegant approach developed by Wooley [18] and by Kang and Taton [19], we have synthesized the tri-block copolymer poly(ethylene glycol)-b-poly(acrylic acid)-b-(polystyrene) (PEG-PAA-PS). This material is similar to the polymers prepared by Niu et al. [20] and by Tang [21] via atom transfer radical polymerization (ATRP) [22,23]. As shown in Fig. 2, poly(ethylene glycol) monomethyl ether 1 was treated with 2-bromoisobutyryl bromide 2 to give a PEG macroinitiator 3 [24], which was reacted with tert-butyl acrylate under copper (I) bromide/pentamethyldiethylenetriamine (PMDETA) catalysis to give a di-block copolymer initiator (PEG-b-PtBA-Br) 4. A similar ATRP approach was used to prepare the

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tri-block copolymer (PEG-b-PtBA-b-PS-Br) 5 by reacting 4 with styrene and then hydrolyzing the product (5) with trifluoroacetic acid (TFA) to afford the PEG-b-PAA-b-PS 6.

2.2. Preparation of SWCNT PEG-eggs Water was slowly added to a DMF solution of PEG-PAA-PS containing raw HiPco SWCNTs under tip sonication. 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide (EDC) was then added followed by 2,2 0 -(ethylenedioxy)diethylamine 7 to crosslink the polymer (Fig. 3). Interestingly, PEG-egg formation in the reaction vial is visible to the eye (see Fig. 1S). After adding the carbodiimide activator, the micelle-trapped SWCNTs precipitated out. However, adding the diamine immediately turned the heterogeneous two-layer mixture to a homogeneous, clear black suspension, indicating that crosslinking takes place rapidly and that the resulting PEG-egg is water-soluble. This was confirmed by brief and gentle shaking of the lyophilized, purified material in water, which led to a dark suspension. (Fig. 4a and b). 1H NMR analysis (3 sec pulse delay) of the empty PEG-egg revealed that the polymer was PEG(17)-PAA(35)-PS(14).

2.3. Near-infrared fluorescence spectra and imaging NIR fluorescence spectra were measured by using 685-nm diode laser excitation with a model NS-1 NanoSpectralyzer (Applied NanoFluorescence, Houston, TX). NIR fluorescence microscopy was performed using a customized fluorescence microscope described previously [25].

2.4. Biocompatibility testing After demonstrating that a solution of our SWCNT PEG-eggs in phosphate buffered saline (PBS) was stable for months (showing no visible flocculation), biocompatibility was assessed on mouse macrophage and human kidney epithelial cells. These two cell lines were incubated overnight at 37 C in growth media to which SWCNT PEG-eggs were added

Fig. 1. Schematic illustration of the encapsulation of a SWCNT inside a PEG-egg. Blue (outermost wiggles) represents the PEG segment, pink (center wiggles) is the PAA, and gray (innermost wiggles) is the PS.

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R. Wang et al. / Carbon 45 (2007) 2388–2393 O Br

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Fig. 3. PEG-egg formation – crosslinking of the tri-block copolymer 6 using a water-soluble diamine 7 and EDC coupling reagent.

to give a SWCNT concentration of 33 mg/L. PBS was used as a positive control, while a 0.1% Triton X aqueous solution (lysis reagent) served as the negative control for biocompatibility. Finally, a LIVE/DEAD viability/cytotoxicity assay (Invitrogen L-3224) was performed on all cells and fluorescence images were obtained.

3. Results and discussion The method we employed to form PEG-eggs amounts to ‘‘covalent capture,’’ wherein a supramolecular structure is

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Fig. 4. Micelle stability tests on PEG-PAA-PS PEG-eggs: (a) lyophilized, purified PEG-PAA-PS encapsulated SWCNTs; (b) dried PEG-eggs after re-suspension in water by mild shaking; (c) PEG-eggs in water diluted 25fold with THF and (d) uncrosslinked PEG-PAA-PS SWCNT suspension diluted 25-fold with THF.

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data). Fluorescence observed from currently produced samples of SWCNT PEG-PAA-PS PEG-eggs is weaker than from Pluronic SWCNT suspensions, probably because the nanotubes were less well debundled prior to encapsulation. Nevertheless, we have successfully observed images of SWCNT PEG-eggs on glass slides using a NIR fluorescence microscope (cf. Supplementary data Fig. 4S). This suggests that, if they are taken up by cells, SWCNT PEG-eggs should be suitable fluorophores for microscopic cellular imaging. Finally, we observed that SWCNT emission features shifted to longer wavelengths by approximately 7 nm when the PEG-PAA-PS coating was crosslinked to form a PEG-egg. This shift probably reflects a change in the nanotubes’ local dielectric environment [9,10]. 3.1. Stability of SWCNT PEG-eggs

stabilized by the formation of covalent bonds [26,27]. Since our tri-block copolymer system contains a PEG terminus, it possesses the advantages of steric stablization of the micelle, avoidance of inter-micelle crosslinking [27], biocompatibility, and the potential for enhanced circulation time in vivo [13]. After noncovalently suspending SWCNTs in water using an amphiphilic polymeric surfactant (PEGPAA-PS or PEG-PB), we added a crosslinker or radical initiator to permanently entrap the SWCNTs in the hydrophobic core of the micelle without chemical functionalization of the sidewalls (see Fig. 1). The retention of NIR fluorescence, shown in Fig. 5, confirms that the encapsulated SWCNTs retain a relatively pristine electronic structure, in contrast to non-fluorescent covalently functionalized SWCNTs [28,29]. As we found that SWCNTs encapsulated in PEG-PAA-PS PEG-eggs fluoresced much more intensely than those in PB-PEGeggs, this paper will be mainly concerned with the former (details about PEG-PB are presented in the Supplementary

Fig. 5. Normalized NIR emission spectra of SWCNT PEG-eggs excited at 658 nm. The solid line shows spectral data measured before addition of BSA to the sample; open circles show data measured from the same sample after BSA addition. The two spectra are nearly identical.

The PEG-egg surrounding a SWCNT should act as a micelle whose stability does not depend on the presence of amphiphiles in the surrounding solution. Additionally, the PEG-egg should resist protein displacement in biological environments, and may provide some shielding of the encapsulated SWCNT from external chemical agents. We observed that aqueous dilution (1:2) of SWCNT suspensions in PEG-PAA-PS caused flocculation after several days in an uncrosslinked sample while a crosslinked (PEGegg) sample remained fully suspended. As shown in frames a and b of Fig. 4, a SWCNT PEG-egg suspension could be lyophilized to dryness and then readily re-suspended by mild shaking. Frames c and d of Fig. 4 show that addition of THF to an aqueous suspension caused immediate flocculation of an uncrosslinked sample, but not of a crosslinked SWCNT PEG-egg sample. These results indicate that SWCNT PEG-egg suspensions are highly stabilized against changes in the surrounding solvent. To test the resistance of SWCNT PEG-eggs to coating displacement by proteins, we measured the NIR emission spectrum of a sample before and after adding bovine serum albumin (BSA), a common blood protein. As shown in Fig. 5, the two spectra are virtually identical. Even after more than one week of exposure, the spectrum showed no peak broadening or red shifts of several nanometers that are typically observed when proteins displace or perturb SWCNT surfactant coatings [10,15]. We conclude that the crosslinked PEG-eggs have good stability in proteinrich environments. The final benefit of the PEG-egg should be to make the enclosed SWCNT less accessible to chemical reactants. To assess the redox reactivity of SWCNT PEG-eggs [30], we rapidly injected 20 lL of 1 lM Fe(III) solution into 1 mL of SWCNT suspension and measured NIR emission spectra at one-second intervals (oxidation of SWCNTs is known to quench the NIR emission [30]). Fig. 6 compares kinetic data (at the (7, 6) emission peak) for SWCNT samples suspended in uncrosslinked PEG-PAA-PS and (crosslinked) PEG-eggs. The apparent rate constant for

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3.2. Microscopic imaging The samples were also characterized by atomic force microscopy. AFM images showed elongated structures in samples containing SWCNTs suspended in PEG-PAAPS, but not in control samples containing only PEGPAA-PS. As can be seen from Fig. 7, the coating has a globular structure. Analysis of the AFM images showed that the average length of nanotubes in the SWCNT PEG-egg sample was 120 nm. AFM also indicated that approximately 50% of the nanotube objects were bundles, with an average height of 12 nm, whereas the features identified as individual SWCNTs averaged 4 nm high. 3.3. Biocompatibility Fig. 6. Quenching kinetics of NIR fluorescence from (7, 6) SWCNTs after addition of Fe3+ ions. Solid circles show data for SWCNTs suspended in non-crosslinked PEG-PAA-PS; open triangles show data for SWCNTs in crosslinked PEG-PAA-PS (PEG-eggs). Solid curves are single-exponential fits to the data.

oxidation of the PEG-egg sample is lower than that of a non-crosslinked sample by a factor of 1.8. We attribute this slower oxidation to reduced permeability of the crosslinked micelle core. We also note that the emission intensities do not approach zero on the time scale of our measurements. Instead, the normalized asymptotic levels are 0.18 in the crosslinked sample and 0.09 without crosslinking. These components represent slowly oxidized portions of the samples and likely reflect inhomogeneity of the SWCNT surface coating. The higher asymptote of the PEG-egg sample may indicate a larger fraction of fully encapsulated SWCNTs with that coating.

The live/dead assay described above showed that 95% (±5%) of cultured macrophage and kidney cells survived in the presence of SWCNT PEG-eggs. As expected, nearly 99% of both cell types survived the PBS control and none survived the lysis negative control. From these results, we conclude that the PEG-eggs show no acute cytotoxicity at 33 lg/mL for the cell types studied. Further experiments are underway to assess toxicity from chronic exposure. 4. Conclusions In summary, we have noncovalently encapsulated single-walled carbon nanotubes inside crosslink-stabilized PEG shells. These ‘‘SWCNT PEG-eggs’’ show the high aqueous dispersability and resistance to coating displacement typically attained by covalent sidewall derivatization, yet they retain their intrinsic NIR fluorescence because the

Fig. 7. AFM images of SWCNT PEG-eggs after purification by dialysis. Left frame: height mode; Right frame: amplitude mode. Each frame is 2l · 2l.

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nanotube p-electron system remains intact. In vitro assays show minimal cytotoxicity. We believe that SWCNT PEG-eggs will prove important in biomedicine as a platform for developing novel diagnostic and therapeutic nanomaterials. Acknowledgments The authors thank the following funding agencies for support: the National Aeronautics and Space Administration (NASA) Alliance for NanoHealth (Grant NNJ05HE75A), the United States Army, Telemedicine and Advanced Technology Research Center (TATRC) (Grant W81XWH-04-2-0035 to JLC), the National Science Foundation (Grant CHE-0314270 to RBW) and the Robert A. Welch Foundation (Grant C-0499 to PSE and Grant C-0807 to RBW). We also thank Professor Matteo Pasquali for suggesting the PEG-PB system. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.carbon.2007.07.006. This information includes synthesis of PEG-PAA-PS, the preparation of PEG-eggs from PEGPAA-PS and PEG-PB, and details of the biocompatibility testing. References [1] Kam NWS, Jessop TC, Wender PA, Dai H. Nanotube molecular transporters: internalization of carbon nanotube-protein conjugates into mammalian cells. J Am Chem Soc 2004;126:6850–1. [2] Kam NWS, O’Connell MO, Wisdom JA, Dai H. Carbon nanotubes as multifunctional biological transporters and near-infrared agents for selective cancer cell destruction. Proc Natl Acad Sci USA 2005;102(33):11600–5. [3] Pantarotto D, Briand J-P, Prato M, Bianco A. Translocation of bioactive peptides across cell membranes by carbon nanotubes. Chem Commun 2004:16–7. [4] Pantarotto D, Singh R, McCarthy D, Erhardt M, Briand J-P, Prato M, et al. Functionalized carbon nanotubes for plasmid DNA gene delivery. Angew Chem Int Ed Engl 2004:5242–6. [5] Singh R, Pantarotto D, Lacerda L, Pastorin G, Klumpp C, Prato M, et al. Tissue biodistribution and blood clearance rates of intravenously administered carbon nanotube radiotracers. Proc Natl Acad Sci USA 2006;103(9):3357–62. [6] Chattopadhyay J, Sadana AK, Liang F, Beach JM, Xiao Y, Hauge RH, et al. Carbon nanotube salts. Arylation of single-wall carbon nanotubes. Org Lett 2005;7:4067–9. [7] Hudson JL, Casavant MJ, Tour JM. Water-soluble, exfoliated, nonroping single-wall carbon nanotubes. J Am Chem Soc 2004;126:11158–9. [8] Hirsch A, Vostrowsky O. Functionalization of carbon nanotubes. Top Curr Chem 2005;245:193–237. [9] Weisman RB, Bachilo SM, Tsyboulski D. Fluorescence spectroscopy of single-walled carbon nanotubes in aqueous suspension. Appl Phys A 2004;78:1111–6.

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[10] Cherukuri P, Bachilo SM, Litovsky SH, Weisman RB. Near-infrared fluorescence microscopy of single-walled carbon nanotubes in phagocytic cells. J Am Chem Soc 2004;126:15638–9. [11] Lee JU, Huh J, Kim KH, Park C, Jo WH. Aqueous suspension of carbon nanotubes via non-covalent functionalization with oligothiophene-terminated poly(ethylene glycol). Carbon 2007;45:1051–7. [12] Moghimi SM, Szebeni J. Stealth liposomes and long circulating nanoparticles: critical issues in pharmacokinetics, opsonization and protien-binding properties. Prog Lipid Res 2003;42:463–78. [13] Molineux G. PEGylation: Engineering improved pharmaceuticals for enhanced therapy. Cancer Treat Rev 2002;28(Suppl. A):13–6. [14] Roberts MJ, Bentley MD, Harris JM. Chemistry for peptide and protein PEGylation. Adv Drug Deliver Rev 2002;54:459–76. [15] Cherukuri P, Gannon CJ, Leeuw TK, Schmidt HK, Smalley RE, Curley SA, et al. Mammalian pharmacokinetics of carbon nanotubes using intrinsic near-infrared fluorescence. Proc Natl Acad Sci USA 2006;103:18882–6. [16] Parrish B, Breitenkamp RB, Emrick T. PEG- and peptide-grafted aliphatic polyesters by click chemistry. J Am Chem Soc 2005;127:7404–10. [17] Dumortier H, Lacotte S, Pastorin G, Marega R, Wu W, Bonifazi D, et al. Functionalized carbon nanotubes are non-cytotoxic and preserve the functionality of primary immune cells. Nano Letters 2006;6(7):1522–8. [18] Huang H, Kowalewski T, Remsen EE, Gertzmann R, Wooley KL. Hydrogel-coated glassy nanospheres: a novel method for the synthesis of shell cross-linked knedels. J Am Chem Soc 1997;119:11653–9. [19] Kang Y, Taton TA. Micelle-encapsulated carbon nanotubes: a route to nanotube composites. J Am Chem Soc 2003;125:5650–1. [20] Niu H, Zhang L, Gao M, Chen Y. Amphiphilic ABC triblock copolymer-assisted synthesis of core/shell structured CdTe nanowires. Langmuir 2005;21:4205–10. [21] Tang Y, Liu SY, Armes SP, Billingham NC. Solubilization and controlled release of hydrophobic drug using novel micelle-forming ABS triblock copolymers. Biomacromolecules 2003;4:1636–45. [22] Matyjaszewski K, Spanswick J, Sumerlin BS. Preparation, characterization, and applications of polymers synthesized by atom transfer radical polymerization. In: Jagur-Grodzinski J, editor. Living and controlled polymerization. New York: Nova Science Publishers; 2006. p. 1–38. [23] Matyjaszewski K, Xia J. Atom transfer radical polymerization. Chem Rev 2001;101:2921–90. [24] Du J, Chen Y. Atom-transfer radical polymerization of a reactive monomer: 3-(trimethoxysilyl)propyl methacrylate. Macromolecules 2004;37:6322–8. [25] Tsyboulski DA, Bachilo SM, Weisman RB. Versatile visualization of individual single-walled carbon nanotubes with near-infrared fluorescence microscopy. Nano Letters 2005;5:975–9. [26] Hartgerink JD. Covalent capture: a natural complement to selfassembly. Curr Opin Chem Biol 2004;8:604–9. [27] O’Reilly RK, Hawker CJ, Wooley KL. Cross-linked block copolymer micelles: functional nanostructures of great potential and versatility. Chem Soc Rev 2006;35(11):1068–83. [28] Dukovic G, White BE, Zhou Z, Wang F, Jockusch S, Steigerwald ML, et al. Reversible surface oxidation and efficient luminescence quenching in semiconductor single-wall carbon nanotubes. J Am Chem Soc 2004;126:15269–76. [29] Weisman RB. Fluorescence spectroscopy of single-walled carbon nanotubes. In: Rotkin SV, Subramoney S, editors. Applied physics of nanotubes: fundamentals of theory, optics, and transport devices. Berlin: Springer; 2005. p. 183–202. [30] O’Connell MJ, Eibergen EE, Doorn SK. Chiral selectivity in the charge-transfer bleaching of single-walled carbon-nanotube spectra. Nature Mater 2005;4:412–8.