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One-Dimensional Protein-Based Nanoparticles Induce Lipid Bilayer Disruption: Carbon Nanotube Conjugates and Amyloid Fibrils Atsushi Hirano,† Ken Uda,† Yutaka Maeda,‡,§ Takeshi Akasaka, and Kentaro Shiraki*,† Institute of Applied Physics, University of Tsukuba, Tsukuba, Ibaraki 305-8573, Japan, ‡Department of Chemistry, Tokyo Gakugei University, Koganei, Tokyo 184-5801, Japan, §PRESTO, Japan Science and Technology Agency, Chiyoda-ku, Tokyo 102-0075, Japan, and Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan )
†
Received April 1, 2010. Revised Manuscript Received September 23, 2010 Along with recent progress of nanotechnology, concern has risen about biological impacts of nanoparticles deriving from their interaction with cell membranes. Nanoparticles tend to adsorb proteins in vivo. Therefore, the physical properties of the conjugates to cell membranes must be investigated to elucidate and assess their properties. We examined whether one-dimensional protein-based nanoparticles induce liposome leakage in physiological saline. Carbon nanotube conjugates with adsorbed lysozyme interacted with the liposome through electrostatic interaction, leading to liposome leakage. Surprisingly, amyloid fibrils of lysozyme resembled the conjugate in terms of their effects on liposome leakage. Results described herein provide new insight into the interaction between nanoparticles and cell membranes in terms of their shape, mechanical properties, and noncovalent interactions.
Introduction
*To whom correspondence should be addressed. Mailing address: Institute of Applied Physics, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8573, Japan. Telephone: þ81-29-853-5306. E-mail:
[email protected]. ac.jp.
attention. Uptake of the nanoparticles into cells through the environment can cause cytotoxicity.3,12-16 Because carbon nanotubes, as a potential type of nanoparticle, have unique physicochemical properties, they have been used in several applications including biomedical materials such as peptide, protein, and nucleic acid delivery vehicles.17-20 Before carbon nanotubes are put to practical use as novel delivery vehicles, their biological impacts must be assessed properly, as has been done with other nanoparticles. Furthermore, the impacts of the release of carbon nanotubes from research and industrial outlets into the environment should be considered carefully. Pristine industrially synthesized carbon nanotubes have higherthan-usual cytotoxicity.21 Therefore, the diffusion of carbon nanotubes into the environment can severely harm living bodies through their absorption. Their subsequent uptake into cells is attributable to endocytosis19,22-25 or spontaneous insertion and diffusion across the cell membranes.17,18,20 Although various simulation studies addressing interaction of carbon nanotubes and lipids have been performed using model systems with simple lipid bilayers,26 little experimental knowledge related to physical mechanisms in the system has been reported.
(1) Leroueil, P. R.; Hong, S.; Mecke, A.; Baker, J. R.; Orr, B. G.; Banaszak Holl, M. M. Acc. Chem. Res. 2007, 40, 335–342. (2) Oberdorster, G. Int. Arch. Occup. Environ. Health 2001, 74, 1–8. (3) Oberdorster, G.; Oberdorster, E.; Oberdorster, J. Environ. Health Perspect. 2005, 113, 823–839. (4) Hong, S.; Leroueil, P. R.; Janus, E. K.; Peters, J. L.; Kober, M. M.; Islam, M. T.; Orr, B. G.; Baker, J. R., Jr.; Banaszak Holl, M. M. Bioconjugate Chem. 2006, 17, 728–734. (5) Ginzburg, V. V.; Balijepalli, S. Nano Lett. 2007, 7, 3716–3722. (6) Qiao, R.; Roberts, A. P.; Mount, A. S.; Klaine, S. J.; Ke, P. C. Nano Lett. 2007, 7, 614–619. (7) Lee, H.; Larson, R. G. J. Phys. Chem B 2008, 112, 12279–12285. (8) Roiter, Y.; Ornatska, M.; Rammohan, A. R.; Balakrishnan, J.; Heine, D. R.; Minko, S. Nano Lett. 2008, 8, 941–944. (9) Leroueil, P. R.; Berry, S. A.; Duthie, K.; Han, G.; Rotello, V. M.; McNerny, D. Q.; Baker, J. R., Jr.; Orr, B. G.; Banaszak Holl, M. M. Nano Lett. 2008, 8, 420– 424. (10) Roiter, Y.; Ornatska, M.; Rammohan, A. R.; Balakrishnan, J.; Heine, D. R.; Minko, S. Langmuir 2009, 25, 6287–6299. (11) Verma, A.; Stellacci, F. Small 2010, 6, 12–21. (12) Colvin, V. L. Nat. Biotechnol. 2003, 21, 1166–1170. (13) Donaldson, K.; Aitken, R.; Tran, L.; Stone, V.; Duffin, R.; Forrest, G.; Alexander, A. Toxicol. Sci. 2006, 92, 5–22.
(14) Hardman, R. Environ. Health Perspect. 2006, 114, 165–172. (15) Nel, A.; Xia, T.; Madler, L.; Li, N. Science 2006, 311, 622–627. (16) Lewinski, N.; Colvin, V.; Drezek, R. Small 2008, 4, 26–49. (17) Lu, Q.; Moore, J. M.; Huang, G.; Mount, A. S.; Rao, A. M.; Larcom, L. L.; Ke, P. C. Nano Lett. 2004, 4, 2473–2477. (18) Pantarotto, D.; Briand, J. P.; P. Chem. Commun. 2004, 1, 16–17. (19) Shi Kam, N. W.; Jessop, T. C.; Wender, P. A.; Dai, H. J. Am. Chem. Soc. 2004, 126, 6850–6851. (20) Bianco, A.; Hoebeke, J.; Godefroy, S.; Chaloin, O.; Pantarotto, D.; Briand, J. P.; Muller, S.; Prato, M.; Partidos, C. D. J. Am. Chem. Soc. 2005, 127, 58–59. (21) Sayes, C. M.; Liang, F.; Hudson, J. L.; Mendez, J.; Guo, W.; Beach, J. M.; Moore, V. C.; Doyle, C. D.; West, J. L.; Billups, W. E.; Ausman, K. D.; Colvin, V. L. Toxicol. Lett. 2006, 161, 135–142. (22) Cherukuri, P.; Bachilo, S. M.; Litovsky, S. H.; Weisman, R. B. J. Am. Chem. Soc. 2004, 126, 15638–15639. (23) Kam, N. W.; Dai, H. J. Am. Chem. Soc. 2005, 127, 6021–6026. (24) Kam, N. W.; Liu, Z.; Dai, H. Angew. Chem., Int. Ed. 2006, 45, 577–581. (25) Liu, Q.; Chen, B.; Wang, Q.; Shi, X.; Xiao, Z.; Lin, J.; Fang, X. Nano Lett. 2009, 9, 1007–1010. (26) Monticelli, L.; Salonen, E.; Ke, P. C.; Vattulainen, I. Soft Matter 2009, 5, 4433–4445.
Nanoparticles are currently anticipated for use in biological applications such as drug and gene delivery materials, medical imaging, cancer targeting and therapeutics, catalysis, and other devices. Because of the recent expansion of nanoparticle applications in biological systems, their potential for biological impact has become an important concern. Controlling the balance between effectively crossing the cell membranes and inducing cytotoxicity is a key challenge in this field.1 One determinant of nanoparticle bioavailability and cytotoxicity is the interaction between cell membranes and nanoparticles, which depends on the electrostatic interaction, shape, size, surface area, flexibility, and amphipathic character of the nanoparticles.2-11 Among them, electrostatic interaction accounts for interplay between cationic nanoparticles and lipid bilayers.4,7 Environment, health, and safety issues related to nanoparticles have also received increasing
17256 DOI: 10.1021/la103615b
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Considering the absorption of the carbon nanotubes into biological systems through their release into environment or medical treatment, one can assume them to be conjugated with proteins and other biopolymers in the systems because of nonspecific adsorption onto the sidewalls of carbon nanotubes. The possibility of adsorption is supported not only by dispersibility of carbon nanotubes by the biopolymers27-34 but also by the effect of serum protein on the intracellular uptake and cytotoxicity of carbon nanotubes.35 The conjugate comprises, so to speak, carbon nanotubes that are naturally modified by proteins. Such adsorbed proteins associated with nanoparticles make up the “protein corona”.36,37 The positively charged proteins are spontaneously adsorbed onto the nanoparticles in biological systems, and consequently, they seem to disrupt lipid bilayers.7 In this work, single-walled carbon nanotubes (SWNTs) with adsorbed positively charged protein, that is, lysozyme from chicken egg white, were used to assess their effect on leakage of liposome contents. The liposomes were composed of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC). As control samples, monomer, amorphous aggregate, and amyloid fibril formed by lysozyme were used separately. The solution used for the assay contained sodium chloride in proportions closely matching those of physiological saline. The results provide meaningful insight into nanoparticle effects on biological systems in terms of their shape, mechanical properties, and noncovalent interactions.
Experimental Section Preparation of Protein Conjugated SWNTs. The SWNTs conjugated with lysozyme (SWNT-LSZ) were prepared as follows. Solutions containing 1 mg/mL lysozyme, 50 mM citratephosphate buffer (pH 3.4), and 3 M guanidine hydrochloride were mixed with SWNT powder. Although dispersion using lysozyme can be accomplished at neutral pH and in the absence of 3 M guanidine chydrochloride,33 this method was used for enhancing the yield of SWNT-LSZ.38 The SWNTs were dispersed by ultrasonication for 30 min at 20 °C (including stirring after 0 and 15 min) using an ultrasonic processor (UT-250S; Sharp Corp., Osaka, Japan). Subsequently, the dispersed SWNT-LSZ solutions were dialyzed against large volumes of distilled water for 34 h at room temperature to reduce the nonadsorbed lysozyme completely using cellulose ester dialysis membranes (MWCO 100 000). Nondispersed SWNTs were removed by filtration of the solution using Whatman filter paper (no. 41). Amounts of the dispersed SWNTs in the solutions were assessed by absorbance at 600 nm corresponding to the S22 transition using a spectrophotometer (V550 UV/vis; Jasco Corp., Tokyo, Japan). (27) Dieckmann, G. R.; Dalton, A. B.; Johnson, P. A.; Razal, J.; Chen, J.; Giordano, G. M.; Munoz, E.; Musselman, I. H.; Baughman, R. H.; Draper, R. K. J. Am. Chem. Soc. 2003, 125, 1770–1777. (28) Zheng, M.; Jagota, A.; Semke, E. D.; Diner, B. A.; McLean, R. S.; Lustig, S. R.; Richardson, R. E.; Tassi, N. G. Nat. Mater. 2003, 2, 338–342. (29) Zorbas, V.; Ortiz-Acevedo, A.; Dalton, A. B.; Yoshida, M. M.; Dieckmann, G. R.; Draper, R. K.; Baughman, R. H.; Jose-Yacaman, M.; Musselman, I. H. J. Am. Chem. Soc. 2004, 126, 7222–7227. (30) Karajanagi, S. S.; Yang, H.; Asuri, P.; Sellitto, E.; Dordick, J. S.; Kane, R. S. Langmuir 2006, 22, 1392–1395. (31) Matsuura, K.; Saito, T.; Okazaki, T.; Ohshima, S.; Yumura, M.; Iijima, S. Chem. Phys. Lett. 2006, 429, 497–502. (32) Nepal, D.; Geckeler, K. E. Small 2006, 2, 406–412. (33) Nepal, D.; Geckeler, K. E. Small 2007, 3, 1259–1265. (34) Wang, D.; Chen, L. Nano Lett. 2007, 7, 1480–1484. (35) Zhu, Y.; Li, W. X.; Li, Q. N.; Li, Y. G.; Li, Y. F.; Zhang, X. Y.; Huang, Q. Carbon 2009, 47, 1351–1358. (36) Cedervall, T.; Lynch, I.; Lindman, S.; Berggard, T.; Thulin, E.; Nilsson, H.; Dawson, K. A.; Linse, S. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 2050–2055. (37) Lynch, I.; Cedervall, T.; Lundqvist, M.; Cabaleiro-Lago, C.; Linse, S.; Dawson, K. A. Adv. Colloid Interface Sci. 2007, 134-135, 167–174. (38) Hirano, A.; Maeda, Y.; Yuan, X.; Ueki, R.; Miyazawa, Y.; Fujita, J.; Akasaka, T.; Shiraki, K. Chem.;Eur. J. 2010, 16, 12221-12228.
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Dispersibility of the SWNT-LSZ solution was retained for this experiment. Our previous study performed in similar conditions demonstrated that the mass ratio of lysozyme to SWNTs was close to unity. The secondary structure of lysozyme on the SWNTs differed from that of the nonadsorbed one measured by circular dichroism spectra. The changed structure of protein was retained for more than 1 week.38 Preparation of Amyloid Fibrils. Amyloid fibrils of lysozyme were formed as follows. A stock solution containing 2.0 mg/mL lysozyme, 136.7 mM sodium chloride, and 2.7 mM potassium chloride was prepared and adjusted to pH 2.0 by addition of HCl. The samples were incubated for 5 h at 37 °C with continuous agitation using a stirrer. After fibril formation, sample solutions were ultrasonicated for 30 min at room temperature using the ultrasonic processor. They were subsequently readjusted to pH 7.4 or 11 using NaOH. Preparation of Liposome. Unilamellar liposomes composed of various DOPG/DOPC components were prepared using the extrusion method with 200 nm pore size polycarbonate membranes. The appropriate amounts of lipids in chloroform were mixed; subsequently, the solvent was completely removed in a vacuum desiccator connected to a rotary vacuum pump for 12 h. An appropriate amount of 70 mM calcein (pH 7) was added to this dry lipid film, and then the suspension was vortexed for several seconds at room temperature. Then the solution was extruded sufficiently through 200 nm pore size polycarbonate membranes (Avanti Mini-Extruder; Avanti Polar Lipids, Inc., Alabaster, AL). Subsequently, to remove the untrapped calcein, the liposome suspension was eluted through a Sephadex G-75 column with 140 mM sodium chloride. Concentrations of the phospholipids in the samples were determined based on the Fiske-Subbarow method and the Bartlett method.39,40 Liposome Leakage Assay. Calcein leakage from the liposomes induced by various concentrations of SWNT-LSZ or the amyloid fibril was detected using a spectrofluorometer (FP6500; Jasco Corp., Tokyo, Japan). The 400 μM liposome suspensions (10 μL) prepared above were mixed with 10 mM phosphate buffer (pH 7.4) containing 140 mM sodium chloride (1460 μL); subsequently, 30 μL of SWNT-LSZ or the amyloid fibril was added to the solutions. The fluorescence intensity of the solution in the absence and presence of 6 mM sodium dodecyl sulfate (SDS) was designated, respectively, as 0% and 100% leakage.
Results and Discussion The SWNT-LSZ was mixed with 1:1 DOPG/DOPC liposome in the physiological saline at pH 7.4, where the net charges of DOPG and DOPC were, respectively, a negative value of -1 and zero. The conjugate induced leakage of liposome content calcein (Figure 1). The leakage rate of calcein depended on the SWNT-LSZ concentration. The concentrations depicted in the figure are of SWNTs composing SWNT-LSZ quantified spectroscopically. Increment of the leakage rate with concentration was saturated around 80 ng/mL. Irrespective of the concentration, the leakage reached respective maximum by 4 h. The leakage was slightly caused by SWNTs only (data not shown). This smaller effect on the leakage can be attributed to the following two properties of SWNTs. One is the reduction of the effective surface areas of SWNTs because SWNTs are readily aggregated in aqueous solution unlike SWNT-LSZ. The other is the smaller effect of SWNT itself on the leakage. This result shows that SWNT-LSZ dispersed through the adsorption of lysozyme on SWNTs enhances the interaction between SWNTs and the membranes in the physiological saline. Here, SWNT-LSZ is charged positively in the solution because of the isoelectric point (39) Fiske, C. H.; Subbarow, Y. J. Biol. Chem. 1925, 66, 375–400. (40) Bartlett, G. R. J. Biol. Chem. 1959, 234, 466–468.
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Figure 1. Representative data for leakage of 1:1 DOPG/DOPC liposome contents in the presence of various concentrations of SWNT-LSZ.
Figure 2. Final amount of liposome leakage with various concentrations of SWNT-LSZ obtained by changing the membrane components.
(pI ∼ 11), whereas the membranes are charged negatively. Therefore, the electrostatic interaction between SWNT-LSZ and the membranes might be a predominant factor in the leakage. To support this hypothesis, the mixing ratio of DOPG and DOPC of the membranes was changed. As expected, the leakage decreased concomitantly with the decrease of the membrane charge, independent of the concentration of SWNT-LSZ (Figure 2). The pH dependence of the leakage supports the consideration explained above: the leakage at pH 11, where lysozyme is not charged, was not induced by the addition of SWNT-LSZ (Supporting Information Figure S1A). Although these results imply that SWNTs have a disruptive effect on cell membranes in the biological systems through the adsorption of proteins, the effect is expected to depend on the charge of the protein, as described above. Reportedly, serum proteins adsorbed onto carbon nanoparticles attenuated their cytotoxicity on the cells.35 The existing proteins in the media can account for the attenuation: that is, most serum proteins are negatively charged in the solution. Here, it was noted that leakage by 80 ng/mL SWNT-LSZ was less than that by 40 ng/mL SWNT-LSZ (Figure 2), which is attributable to their aggregation because of their excess. This result might be related with the fact that individually dispersed SWNTs have greater cytotoxicity than aggregated SWNTs.41 It is also noteworthy that the monomer of native lysozyme at the same concentration did not induce leakage at pH 7.4 (data not shown). The disruption effect of the conjugate is expected to be derived through the alignment of lysozyme with the linear SWNTs. (41) Liu, S.; Wei, L.; Hao, L.; Fang, N.; Chang, M. W.; Xu, R.; Yang, Y.; Chen, Y. ACS Nano 2009, 3, 3891–3902.
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Figure 3. Representative data for leakage of 1:1 DOPG/DOPC liposome contents obtained using various concentrations of the amyloid fibril.
Interaction of spherical nanoparticles with a lipid bilayer is affected not only by their charge density but also by their size.7,42 According to these data, it can be inferred that the onedimensional shape of SWNT-LSZ accounts for the liposome disruption. The liposomes used here have a diameter of 200 nm; the length of SWNT-LSZ is longer than the diameter of the liposome, although the diameter of SWNT-LSZ is shorter than that of the liposome. Therefore, SWNT-LSZ might create anisotropic stress on the membranes, that is, a difference in the disruptive effects along the longitudinal and tangential directions of SWNTs. In terms of their one-dimensional shape, investigating the interaction between nanoparticles and the lipid membranes is meaningful because there might be some characteristic that is common to the one-dimensional nanoparticle in biological systems. Furthermore, nanoparticle flexibility affects interactions with lipid bilayers.7 The SWNTs have a much higher Young’s modulus,43 thereby confining the configuration of lysozyme on SWNTs. Such a stronger mechanical stiffness can induce a unique disruptive property of the conjugate and not the monomer protein. Lysozyme particles of two additional types were prepared for the liposome leakage assay: amyloid fibril and heat-induced aggregate. Most amyloid fibrils have a similar shape to that of carbon nanotubes, which have generally nanometer-order diameter and micrometer-order length.44,45 It is particularly interesting that the leakage assay by the amyloid fibril of lysozyme appears to resemble that by SWNT-LSZ in terms of their profile (Figure 3). The leakage rate increased concomitantly with increasing concentration of the amyloid fibril. Irrespective of their concentrations, the leakage reached their respective maxima by 4 h. Changing the component of the membranes, the percentage of the leakage was measured (Figure 4). Dependence of the leakage on the charge of the membranes was also similar to that of SWNT-LSZ: the less negatively charged the membranes, the less the leakage. It is noteworthy that the liposome composed of 1:4 DOPG/DOPC was unaffected by the amyloid fibril in contrast to SWNT-LSZ (Figure 2). The stronger effect of SWNTLSZ might be explained by the stronger electrostatic interaction attributed to the nanostructure. Amorphous aggregates and monomer protein induced marginal leakage (Figure 4), where the former was prepared by heating 2 mg/mL lysozyme at 98 °C and (42) Mecke, A.; Majoros, I. J.; Patri, A. K.; Baker, J. R., Jr.; Banaszak Holl, M. M.; Orr, B. G. Langmuir 2005, 21, 10348–10354. (43) Salvetat, J. P.; Bonard, J. M.; Thomson, N. H.; Kulik, A. J.; Forro, L.; Benoit, W.; Zuppiroli, L. Appl. Phys. A: Mater. Sci. Process. 1999, 69, 255–260. (44) Hoppener, J. W.; Ahren, B.; Lips, C. J. N. Engl. J. Med. 2000, 343, 411–419. (45) Sipe, J. D.; Cohen, A. S. J. Struct. Biol. 2000, 130, 88–98.
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Figure 4. Final amounts of the liposome leakage in 28 μg/mL amyloid fibril, amorphous aggregate, and monomer.
Figure 5. Concentration-dependence of amyloid fibril on the final amount of leakage obtained by changing the membrane components.
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described that SWNTs are more toxic toward bacteria with less stiff surfaces, might be partially explained by this mechanism.41 Consequently, stiffer nanoparticles with adsorbed positively charged proteins possibly induce cytotocixity through their interaction with the cell membranes. The absorption of nanoparticles into biological systems and subsequent adsorption of proteins onto them might occur via the environment once they are exposed to it. Another perspective of this study is an understanding of the biophysical properties of amyloid fibrils. Amyloid fibril deposits are associated with numerous diseases.49 Recent reports about the diseases have specifically addressed prefibrillar oligomers as primary cytotoxic species. In this context, mature amyloid fibrils such as those used for this study tend to be dismissed as inert species. More recently, however, it was reported that amyloid fibrils have cytotoxic potential and that they disrupt liposome membranes.50 Shorter amyloid fibrils have greater cyotoxicity, which is attributable to the enhancement of fibril-membrane surface interaction and reduction of fibril-fibril association. It is particularly interesting that individually dispersed SWNTs also have more cytotoxicity than aggregated SWNTs.41 Therefore, both phenomena are apparently based on the common physical mechanism suggested by results of this study. Finally, other biomembrane components, such as cholesterol and sphingomyelin, can produce complex phase behavior in lipid bilayers.51 Although these components should play important roles in the biological impact,52 their effects on the lipid bilayer disruption are still unclear at present. This issue will be investigated in future studies.
Conclusion Proteins adsorbed readily onto SWNTs; the conjugates are apparently similar to amyloid fibrils in their interaction with lipid bilayers. Results of this study suggest that these protein-based nanoparticles can be treated as identical physical objects in biological systems in terms of their shape, mechanical properties, and noncovalent interactions with lipid bilayers. Actually, the protein did not disrupt the lipid bilayers in physiological saline unless the protein formed a one-dimensional shape. Their interaction with the lipid bilayers also depends on their electrostatic interaction. Although the one-dimensional nanoparticles (carbon nanotubes and amyloid fibrils) have been discussed only rarely in the same terms, this study offers a common insight into the biological impacts of these nanoparticles. Physical mechanisms must be understood to support future development and risk assessment of nanotechnology.
subsequent ultrasonication at 25 °C. These results support the hypothesis presented above: the one-dimensional shape plays a crucial role in the liposome leakage. Irrespective of the membrane components, the leakage reached respective maxima around 10 μg/mL (Figure 5). A possible reason for the saturation of the leakage is the restructuring of the liposome on the amyloid fibril, similar to that on SWNTs,46 leading to protection of the leakage. Similarly to SWNT-LSZ, the liposome leakage by the amyloid fibril was reduced at pH 11 (Supporting Information Figure S1B). Therefore, leakage by the amyloid fibril is also attributable to the electrostatic interaction. In this study, DOPG and DOPC, as unsaturated lipids, were used as membrane components. The membranes were in a liquid crystalline phase at room temperature. It was therefore assumed that the lipid bilayers have less mechanical stiffness than SWNT-LSZ and the amyloid fibril. Actually, the Young’s modulus of the lipid bilayer is less than that of SWNTs or amyloid fibrils, although that of amyloid fibrils is less than that of SWNTs.43,47,48 The rigidity of cationic nanoparticles can affect the interaction with the membranes.7 The mechanism suggested here is that stiffer and rigid nanoparticles, which are positively charged, disrupt the soft membranes. Previous reports, which
Supporting Information Available: Materials and pH dependence of the liposome leakage obtained by the addition of SWNT-LSZ and the amyloid fibril. This material is available free of charge via the Internet at http://pubs.acs.org.
(46) Wang, H. W.; Michielssens, S.; Moors, S. L. C.; Ceulemans, A. Nano Res. 2009, 2, 945–954. (47) Rutkowski, C. A.; Williams, L. M.; Haines, T. H.; Cummins, H. Z. Biochemistry 1991, 30, 5688–5696. (48) Smith, J. F.; Knowles, T. P.; Dobson, C. M.; Macphee, C. E.; Welland, M. E. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 15806–15811.
(49) Chiti, F.; Dobson, C. M. Annu. Rev. Biochem. 2006, 75, 333–366. (50) Xue, W. F.; Hellewell, A. L.; Gosal, W. S.; Homans, S. W.; Hewitt, E. W.; Radford, S. E. J. Biol. Chem. 2009, 284, 34272–34282. (51) Veatch, S. L.; Keller, S. L. Biochim. Biophys. Acta 2005, 1746, 172–185. (52) Gabriel, G. J.; Som, A.; Madkour, A. E.; Eren, T.; Tew, G. N. Mater. Sci. Eng., R 2007, 57, 28–64.
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Acknowledgment. This work was supported by a Grant-in-Aid for JSPS Fellows (20 3 3087).
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