Letter pubs.acs.org/Langmuir
Functionalization of Multiwalled Carbon Nanotubes and Their pHResponsive Hydrogels with Amyloid Fibrils Chaoxu Li and Raffaele Mezzenga* Food & Soft Materials, Institute of Food, Nutrition & Health, ETH Zurich, Switzerland S Supporting Information *
ABSTRACT: New biocompatible, pH-responsive, and fully fibrous hydrogels have been prepared based on amyloid fibrils hybridized and gelled by functionalized multiwalled carbon nanotubes (MWNTs) far below the gelling concentration of amyloid fibrils. Sulfonic functional groups were introduced on the surfaces of MWNTs either by a covalent diazonium reaction or by physical π−π interactions. The presence of the isoelectric point of amyloid fibrils allows a reversible gelling behavior through ionic interactions with functionalized MWNTs.
1. INTRODUCTION Due to an extraordinary combination of thermal, mechanical, optical and electrical properties, carbon nanotubes (NTs) have drawn significant attention in both industrial and academic fields.1 In particular, the discovery of antibacterial activity, the ability of penetrating cells, and the near-infrared-sensitivity of NTs has triggered great interest in the biological electronic/ optical devices and biomedical fields, such as biosensor, drug release, tissue engineering, and biomolecule assembly.2 Developing composites which combine the exceptional properties of NTs has become one of the most important and active subjects under investigation. For instance, NTs-hybrid hydrogels are used to deliver therapeutic drugs in vivo, or even to selectively target and destroy cancer cells as near-infrared responsive agents.3 NTs were incorporated into hydrogels to reinforce both their mechanical and electrical features desirable in tissue engineering.4 However, notwithstanding these encouraging findings, the poor biological compatibility and aqueous stability of NTs still severely hinder their practical applications in biomaterials and biotechnology. Despite the recent improvement in stability of aqueous dispersions of NTs,5 few reports have been published concerning biological hydrogels based on NTs. Typically, functionalized NTs are incorporated into aqueous solution before the gelling process. Li et al. reported a gelatin hybrid hydrogel in which multiple-walled NTs (MWNTs) were mixed physically to enhance the mechanical properties of the gel.6 © 2012 American Chemical Society
Bhattacharyya et al. reinforced hyaluronic acid hydrogels by incorporating carboxylated single-walled NTs (SWNTs).7 Properly functionalized NTs were also proposed as gelators and to induce hydrogel formation. Ogoshi et al. dispersed SWNTs through π−π interactions with pyrene-modified βcyclodextrins, and host−guest interactions of β-cyclodextrins with other guest molecules resulted in supramolecular SWNT hydrogels.8 Wang et al. dispersed SWNTs by a Pluronic copolymer and incorporated them into α-cyclodextrin solutions, leading to a hybrid hydrogel mediated by interactions between the poly(ethylene oxide) blocks of the Pluronic copolymers and α-cyclodextrin.9 Bayazit et al. synthesized pyridine-functionalized SWNTs via diazonium chemistry, which can act then as cross-linkers for poly(acrylic acid) hydrogel.10 SWNTs wrapped by DNA or bile salts are also reported to be able to act as cross-linkers for hydrogels.11 Amyloid fibrils based hydrogels, as an important type of supramolecular fibrous hydrogel, have also attracted increasing attention in the literature.12 They are advantageous over other synthetic hydrogels not only owing to their high biocompatibility, but also because of their structural stability, wide accessibility, inexpensiveness, and diverse peptide and protein sources. They can be engineered to gel at relatively low Received: April 16, 2012 Revised: June 15, 2012 Published: June 19, 2012 10142
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Letter
Figure 1. Schematic illustrations of functionalization of carbon nanotubes used in this work for enabling their interactions with amyloid fibrils.
Figure 2. (a) Photograph and TEM images of aqueous dispersions of (b) covalently and (c) noncovalently functionalized MWNTs. In part (a), the letters A, B, and C identify pristine MWNTs, covalently functionalized MWNTs, and noncovalently functionalized MWNTs, respectively.
concentrations and have been recently tailored to gel reversibly due to temperature changes.13 Nonetheless, pH-responsiveness is typically inefficient in pure amyloid gels, as aggregation often results in irreversible aggregates.14 A great number of natural proteins are reported to be able to self-assemble into amyloid fibrils, such as lysozyme and β-lactoglobulin.15 The fibrils consist of several filaments intertwined and twisted in a hierarchical way and exploring several length scales.16 Over a critical concentration, they associate together into hydrogels via interfibril or intrafibril interactions.14 Despite intensive study concerning NTs functionalized and dispersed by proteins in water,17 to the best of our knowledge, no study has been reported on pH-responsive hydrogels made of hybrids of amyloid fibrils and NTs, and we disclose this here for the first time.
Previous attempts in NT−proteins hybrids have focused on binding and solubilizing NTs.18 Eventually, further studies also investigated the growth of amyloid fibrils from several types of nanoparticles, including NTs, but without evidence of gelation.19 In order to gel amyloid fibrils under their critical gelling concentration, multiple chemical groups capable of bridging different fibrils are required.20 In this study, we use ionic interactions by synthesizing negatively charged MWNTs to gel amyloid fibrils of β-lactoglobulin. β-Lactoglobulin is the major whey protein present in cow’s milk. By thermal denaturation at pH 2, it can yield pure, long, thin, and highly positively charged amyloid fibrils. Although the procedure described is equally applicable to SWNTs, MWNTs were preferred here due to their distinguishable cross section from that of amyloid fibrils. 10143
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2. RESULTS AND DISCUSSION The preparation protocol for the materials is summarized in Figure 1. As shown in Figure 1, MWNTs were sulfonated covalently through a mild diazonium reaction in aqueous media at 80 °C for 20 h. This environmentally friendly reaction functionalized NTs to a similar degree as reported using other environmentally dangerous methods, which rely on expensive organic solvents or strong acids, and therefore, it offers an ideal alternative process for biological applications.21 The final dispersion was washed several times by filtering with deionized water and ethanol until the filtrate was clear. For the sake of comparison, noncovalently functionalized MWNTs were also prepared. Pyrene sulfonic acid was left interacting with MWNTs via π−π interactions. Free acid molecules, which did not bond on NTs, were removed by a redispersion of the MWNTs and filtration process. After functionalization in either the covalent or the physical method, MWNTs gain sufficient solubility in water due to the negative charges on the surfaces, which can then promote complex formation in the presence of highly positively charged amyloid fibrils. The existence of functional groups was confirmed by FTIR in Figure S2 of Supporting Information. Figure 2a shows the dispersion efficiency of functionalized MWNTs in water. Pristine MWNTs were also tested as comparison. After centrifugation, while pristine MWNTs gave a transparent colorless supernatant, with a dark phase segregated on the bottom, both the covalently and noncovalently functionalized MWNTs gave a homogeneous, dark supernatant even without ultrasonication, which remained stable up to several months. This indicates that both functionalizing methods provide a good amount of hydrophilic groups on the surface of MWNTs, allowing a fine dispersion in water. To investigate the structural details of the dispersed MWNTs, the TEM images of covalently and noncovalently functionalized MWNT dispersions are shown in Figure 2b,c, respectively. It can be seen that both methods give well-dispersed MWNTs. Small bundles consisting of 2−3 tubes account only for a very small proportion of the observed MWNTs, while the great majority consist of individual, separated nanotubes, which confirms the successful functionalization in both methods. Furthermore, Figure 2c shows no indication of existence of aggregated free pyrene sulfonic acid molecules, which infers a complete adsorption onto MWNT walls. This confirms our expectation that the inherent van der Waals interactions between MWNTs could be suppressed by the negatively charged sulfonated groups on the surfaces, yielding sufficiently hydrophilic surfaces, which do not phase segregate. The electrophoretic mobility, measured in the range of −2 to −3 μm·cm/(V s) for the MWNTs, also completed the evidence of existing electrostatic repulsive interactions. The degrees of functionalization were probed by thermogravimetric analysis (TGA). As show in Figure 3, pristine MWNTs give pI. Since many proteins can form amyloid fibrils with various properties, this study provides the potential to prepare responsive hydrogels on demand, which could serve in biological applications, drug release, sensors, and tissue engineering.
Figure 5. Photographs and frequency dependence of the storage modulus (G′, filled symbols) and loss modulus (G″, open symbols) for hybrid hydrogels containing 0.8 wt % amyloid fibrils and 0.35 wt % covalently functionalized MWNTs at pH 2 (●○) and 7 (■□).
measured before gelling or in other types of hydrogels made of amyloid fibrils.20 At pH 7, both the storage and loss modulus dropped by orders of magnitude, with the loss modulus dominating the storage modulus, as expected for a liquid fluid. It is interesting to note that, due to a lower density of sulfonated groups on the surfaces and their dynamic nature, noncovalently functionalized MWNTs mixed with amyloid fibrils at the same concentrations, gave weaker hydrogels (see Figure S4 in Supporting Information). A rough comparison of the connectivity of the hybrid hydrogels discussed here with that of other types of hydrogels made by pure amyloid fibrils can be made using the theory of fractal rigid gels.22 At 1 Hz or 2π rad/s, our hydrogels have storage modulus of ∼200 Pa, that is, around ∼6 times higher than that reported at the same frequency and similar concentration for β-lactoglobulin fibrils gelled by Ca2+.20 The fractal gel theory applied to rigid gels considers the bending energy contribution of each gel strand to the overall modulus G. Accordingly, each strand contributes with a term inversely proportional to its contour length, the latter proportional to the mass of the strand MS. Thus, the total energy associated within the volume of a mesh ξ3 is no longer the purely entropic contribution KBT of an entropic springs network, but rather a term proportional to the inverse of the mass of the loadcarrying strand, e.g., MS−1. As the strand covering the entire mesh space has its own fractal dimensionality d, one gets MS ∼ ξd and finally obtains
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ASSOCIATED CONTENT
S Supporting Information *
Experimental Section, conductivity measurements, FTIR spectra, and rheology of hydrogels.This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: raff
[email protected]. Notes
The authors declare no competing financial interest. 10145
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Tool. Small 2006, 2, 406−412. Nepal, D.; Geckeler, K. E. Proteins and Carbon Nanotubes: Close Encounter in Water. Small 2007, 3, 1259− 1265. Nepal, D.; Geckeler, K. E. In Advanced Nanomaterials, Advanced Nanomaterials, Geckeler, K. E., Nishide, H., Eds.; Wiley-VCH Verlag GmbH & Co.: KGaA, 2010; pp 715. (18) Li, C.; Adamcik, J.; Zhang, A.; Mezzenga, R. Twofold pH and temperature stimuli-responsive behaviour in block copolypeptidedecorated single wall carbon nanotubes. Chem. Commun. 2011, 47, 262−264. Balavoine, F.; Schultz, P.; Richard, C.; Mallouh, V.; Ebbesen, T. W.; Mioskowski, C. Helical Crystallization of Proteins on Carbon Nanotubes: A First Step towards the Development of New Biosensors. Angew. Chem., Int. Ed. 1999, 38, 1912−1915. (19) Linse, S.; Cabaleiro-Lago, C.; Xue, W. -F.; Lynch, I.; Lindman, S.; Thulin, E.; Radford, S. E.; Dawson, K. A. Nucleation of protein fibrillation by nanoparticles. Proc. Natl Acad. Sci. U.S.A. 2007, 104, 8691−8696. (20) Bolder, S.; Hendrickx, H.; Sagis, L.; van der Linden, E. Ca2+induced cold-set gelation of whey protein isolate fibrils. Appl. Rheol. 2006, 16, 258−264. (21) Price, B. K.; Tour, J. M. Functionalization of Single-Walled Carbon Nanotubes On Water. J. Am. Chem. Soc. 2006, 128, 12899− 12904. (22) Kantor, Y.; Webman, I. Elastic Properties of Random Percolating Systems. Phys. Rev. Lett. 1984, 52, 1891−1894. (23) Pouzot, M.; Nicolai, T.; Durand, D.; Benyahia, L. Structure Factor and Elasticity of a heat-set Globular Protein Gel. Macromolecules 2004, 37, 614−620.
ACKNOWLEDGMENTS The Electron Microscopy Centre of the ETH Zurich (EMEZ) and Jingyi Rao are kindly acknowledged for the facility support and FTIR experiments, respectively.
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