Protein Binding by Functionalized Multiwalled Carbon Nanotubes Is

Feb 12, 2008 - St. Jude Children's Research Hospital, Memphis, Tennessee 38105, and School of Pharmaceutical Sciences,. Shandong UniVersity, Jinan ...
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J. Phys. Chem. C 2008, 112, 3300-3307

Protein Binding by Functionalized Multiwalled Carbon Nanotubes Is Governed by the Surface Chemistry of Both Parties and the Nanotube Diameter Qingxin Mu,†,‡ Wei Liu,‡ Yuehan Xing,‡ Hongyu Zhou,†,‡ Zhenwei Li,‡ Ying Zhang,‡ Leihua Ji,‡ Fang Wang,‡ Zhikun Si,‡ Bin Zhang,‡ and Bing Yan*,†,‡ St. Jude Children’s Research Hospital, Memphis, Tennessee 38105, and School of Pharmaceutical Sciences, Shandong UniVersity, Jinan, Shandong, China ReceiVed: NoVember 1, 2007; In Final Form: December 11, 2007

The protein binding propensity of nanoparticles determines their in vivo toxicity and their fate to be opsonized and cleared by human defense systems. In this work, protein-binding mechanisms of pristine and functionalized multiwalled carbon nanotubes (f-MWNTs) were investigated by varying f-MWNTs’ diameters, nanotube surface chemistry, and proteins using steady-state and time-resolved fluorescence, and circular dichroism (CD) spectroscopies. The f-MWNTs with a larger diameter (∼40 nm) generally exhibited stronger protein binding compared to those with a smaller diameter (∼10 nm), demonstrating that the curvature of nanoparticles plays a key role in determining the protein binding affinity. Negative charges or steric properties on f-MWNTs enhanced binding for some proteins but not others, indicating that the electrostatic and stereochemical nature of both nanotubes and proteins govern nanotube/protein binding. Protein fluorescence lifetime was not altered by the binding while the intensity was quenched indicating a static quenching through complex formation. The binding-induced conformational changes were further confirmed by CD studies.

1. Introduction The potential use of nanotherapeutics and nanodevices in the human body signal the need for in-depth studies of the impact of nanoparticles on living systems.1-10 At the same time, the rapid expansion of the number of nanomaterial-based consumer products in average households (website: http://www.nanotechproject.org/44) further magnifies the urgency for nanotoxicity research. Among various nanomaterials, carbon nanotubes are intensively investigated for various biological and medical applications in addition to the vast interest they generate in other technological areas.1,7,11 However, the possible in vivo toxicity, opsonization, and quick clearance of nanotubes remain a major concern.12-14 When injected into animals, single- and multiwalled carbon nanotubes (SWNTs, MWNTs) quickly partition intovariousorgansandcellularcompartmentsbeforeclearance.15-22 As the building block of life, the cell contains complex biological machinery. Numerous cellular enzymes, receptors, and functional modulators form a highly interweaving system for cell function and integrity. By perturbing a single protein, nanoparticles may alter enzyme function or generate unexpected cryptic epitopes in signaling proteins that may trigger some undesirable physiological consequences.10,23,49,50 Therefore, a clear understanding of how nanotubes interact with proteins is a prerequisite for nanomedicine and nanotoxicity research. Structure, size, surface chemistry, charge, and shape are key parameters in determining the bioactivity of nanomaterials. Although the interaction between SWNTs and proteins has been widely reported, studies on MWNTs are still lacking. When SWNTs interact with peptides and proteins, certain binding selectivity has been observed.24-28 Hydrophobic interactions, * Corresponding author. E-mail: [email protected]. Phone: +9014952797. Fax: +9014955715. † St. Jude Children’s Research Hospital. ‡ Shandong University.

especially π-π stacking interactions,25,29-31 and electrostatic interactions,32,33 are reported to play key roles in SWNT/protein binding. Protein conformational changes and protein partial denaturation34,35 all promote SWNT/protein binding. This suggests that the more hydrophobic protein interior has a greater propensity to bind to SWNTs. In this work, we studied MWNTs of different diameters with altered surface hydrophobic, electrostatic, and steric properties through chemical modifications (Figure 1). Proteins with different biological functions and surface properties were selected to probe f-MWNT/protein interactions using various biophysical techniques. We demonstrated that selective protein binding by f-MWNTs was both size (diameter) and surface chemistry dependent for both proteins and f-MWNTs. We also found that high protein affinity in carboxylated MWNT was significantly reduced by its derivatization. These findings opened opportunities for modulating biocompatibility and toxicity of MWNTs through size variation and chemical modification of the nanotube surface. 2. Materials and Methods 2.1. Materials. Bovine serum albumin (BSA) was purchased from Bio Basic Inc. (Markham Ontario, Canada). Carbonic anhydrase (CA), bovine hemoglobin (Hb), hexokinase (HK), and ovalbumin were purchased from Worthington Biochemical Corporation (Lakewood, NJ). Multiwalled carbon nanotubes were synthesized by chemical vapor deposition. The purity was higher than 95%, and the catalyst residue was less than 0.2%. They were purchased from Nanotech Port Co., Ltd. (Shenzhen, China). All multiwalled carbon nanotubes were purified by acids in the oxidation reaction and the subsequent repetitive washing and centrifugation separations to remove metals or other catalyst residues. Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

10.1021/jp710541j CCC: $40.75 © 2008 American Chemical Society Published on Web 02/12/2008

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Figure 2. TEM photographs of p-MWNT (A,C) and f-MWNT 2 (B,D) of two different diameters: 10 nm (panels A,B) and 40 nm (panels C,D).

Figure 1. Chemical structures of MWNT 1-4 (10 or 40 nm).

2.2. Functionalization of MWNTs. The pristine MWNTs in HNO3/H2SO4 (v/v ) 1:3) were placed in an ultrasonic bath at 60 °C for 3 h. The mixture was poured into deionized water, and a well-dispersed black suspension was formed. The mixture was then filtered through a 0.22 µm Millipore polycarbonate membrane and thoroughly washed with deionized water until the pH of the filtrate was about 7.0. The solid was dried under vacuum for 24 h to give f-MWNT 2. The further chemical functionalization of f-MWNT 2 was based on amidation or esterification of carboxylic groups. The f-MWNT 2 was first activated with EDC and NHS in tetrahydrofuran (THF) for 30 min. Then a 10-fold excess of amine or Fmoc-Tyr were added to the mixture and the mixture was stirred for 24 h. The mixture was filtrated through 0.2 µm poly(tetrafluoroethylene) membrane and thoroughly washed with THF and ethanol. Then the products were dried under vacuum overnight and characterized by Fourier transform infrared (FTIR), magic-angle spinning (MAS) 1H NMR, and element analysis. 2.3. Steady-State Fluorescence Spectroscopy. Steady-state fluorescence spectra were measured using a Hitachi F-4500 spectrofluorometer (Hitachi Co. Ltd., Tokyo, Japan). Intrinsic fluorescence of proteins was measured by gradual addition of f-MWNT stock solutions. All nanotubes formed uniform suspension in ethanol (Supporting Information, Figure 4). The nanotube concentration was calculated by nanotube weight and the subsequent dilutions. Final concentrations of carbon nanotubes in protein solutions were 0, 1.1, 2.3, 4.6, 9.3, and 18.7 µg/mL, respectively. Concentration of all proteins was 50 µg/ mL in deionized water (pH ∼ 7). Proteins were excited at 280 nm, and emission wavelength was set from 300 to 400 nm. Scanning speed was 1200 nm/min. Excitation and emission slit was set to 5.0 and 5.0 nm, respectively. PMT voltage was set to 700 volt. Fluorescence intensities at 340 nm were used for calculation in Stern-Volmer equation. All measurements were performed at room temperature (23 °C). 2.4. Time-Resolved Fluorescence Spectroscopy. Fluorescence lifetimes were measured by phase-modulation approach using an ISS K2 multifrequency phase fluorometer (ISS, Inc., Champaign, IL). The final MWNTs’ concentrations were 9.3

µg/mL, and the protein concentration was 500 µg/mL except for BSA, which was 800 µg/mL. The excitation wavelength was set at 280 nm. The reference was glycogen from oyster (1 mg/mL). The fluorescence lifetimes were analyzed with a two exponential decay rate equation using a VINCI-Analysis software. Measurements were performed at room temperature (23 °C). 2.5. CD Spectroscopy. CD measurements were performed on a Jasco J-810 circular dichroism spectrometer (Jasco Co. Ltd., Tokyo, Japan). Concentrations of f-MWNTs and proteins were the same as in steady-state fluorescence assay. The spectra were measured between 190 and 250 nm with a bandwidth of 2.0 nm and a scan speed of 500 nm/min. Cell length was 10 mm. Three scans were averaged. All CD measurements were performed at 23 °C. 3. Results and Discussion 3.1. Diverse Functionalization of MWNT. Molecular recognition and binding specificity can be achieved by combining many unique molecular properties such as hydrophobicity, electrostatic interactions, hydrogen bonding, and steric properties. Using pristine MWNT 1 (p-MWNT 1, nonfunctionalized MWNT, 10 and 40 nm in diameter) as a starting material, we diversified surface chemistry by synthesizing functionalized MWNT 2, 3, and 4 (f-MWNT 2, 3 and 4; Figure 1). The f-MWNT 2 contained carboxyl groups at termini and along the side walls40 and carried negative charges at our experimental (neutral) pH. On the other hand, f-MWNT 4 had a neutral molecule on the surface. The f-MWNT 3 also carried negative charges, but it also exhibited distinct hydrophobic and steric properties compared to f-MWNTs 1, 2, and 4. Pristine MWNT 1 and f-MWNT 2 of different diameters were first characterized by transmission electron microscopy (TEM) to confirm their integrity after the harsh oxidation reaction (Figure 2). The chemical transformation on the surface was further characterized by FTIR, MAS 1H NMR (Figure 3) and elemental analysis (Table 1). The f-MWNT 2 formation was supported by the appearance of an IR band at 1713 cm-1. In f-MWNT 3, the IR band at 1713 cm-1 remained. Elemental analysis indicated an increase in nitrogen content from 0.08% in f-MWNT 2 to 0.63% in f-MWNT 3, providing the evidence of f-MWNT 3 as a distinct nitrogen-containing product. The disappearance of the IR peak at 1713 cm-1 and the increased peak intensity at 2957 and 2850 cm-1 for the methyl and

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Figure 4. Three-dimensional (3D) models of proteins. Threedimensional proteins structures were obtained from Protein Data Bank and 3D models were plotted using PyMOL software. PDB codes: Hexokinase-1IG8; Hemoglobin-1FSX; Carbon anhydrase-1V9E; Ovalbumin-1OVA; HSA (using BSA as a model)-1BM0.

Figure 3. FTIR spectra for MWNT 1-4 and MAS 1H NMR spectra for f-MWNT 2 and 3.

TABLE 1: Elemental Analysis of f-MWNTs 1-4 f-MWNT

C%

H%

1 2 3 4

95.77 84.94 90.7 ( 0.200 93.1 ( 0.17

0.18 0.63 1.00 ( 0.014 0.79 ( 0.008

N%

Loading (mmol/g)

0.09 0.08 0.63 ( 0.008 0.45 ( 0.07 0.63 ( 0.005 0.45 ( 0.04

methylene groups indicated the conversion from f-MWNT 2 to f-MWNT 4. Elemental analysis provided quantitative data for the loading of functional groups: around 0.45 mmol/g for 3 and 4, both have one nitrogen atom (Table 1). This result was further substantiated through the Fmoc release analysis in f-MWNT 3 by UV absorption at 300 nm. By first reacting f-MWNT 2 with a 10-fold excess of Fmoc-Tyr to completely convert f-MWNT 2 to f-MWNT 3 and then quantifying the amount of Fmoc released in a 30% pyridine/dimethylformamide solution, the loading of carboxyl groups on f-MWNT 2 was determined to be 0.42 ( 0.09 mmol/g. Reacting MWNT 2 with 10-fold excess of Fmoc-Tyr cannot guarantee a complete reaction. Therefore, the loading should be a lower limit of the carboxyl group loading. The consistent results with the elemental analysis suggest that the coupling reaction was near completion. On the basis of the functional loading, carbon nanotube diameter, and carbon nanotube surface area, we estimated that the average density of modification is approximately ∼5000 modifications per 1000 nm length for 10 and 40 nm carbon nanotubes. NMR is the gold standard for identifying structures of organic molecules. Although NMR of molecules attached to MWNTs often showed no detectable signals due to the line broadening induced by the heterogeneity of the samples and their magnetic susceptibility differences, the magnetic susceptibility-induced

line broadening in heterogeneous samples can be eliminated by spinning the sample at the magic angle (54.7° from Z).41-42 We used MAS 1H NMR with Nano probe to characterize nanomaterial-bound molecules in this investigation. We first acquired the 1H NMR spectrum of MWNT 2. This is the first 1H NMR spectrum of carboxylated MWNT to our knowledge. Defect sites in nanotubes always exist, and those sites make the oxidation possible. Therefore, this NMR data provides a realistic view of the chemical status of sites in nanotubes. Unfortunately, the attempt to suspend pristine MWNT and get its 1H NMR spectrum was not successful, and we could not observe the defect sites in pristine MWNT. The MAS 1H NMR spectrum of MWNT-COOH 2 (top spectrum in Figure 3B) showed signals of NMR solvent d6-dimethyl sulfoxide at 2.5 ppm, water at 3.33 ppm, and a number of small NMR signals, possibly from hydroxyl groups and some defect sites. Some of these signals were also seen in the spectrum of MWNT 3 (bottom spectrum in Figure 3B). MAS 1H NMR spectrum (Figure 3B) analysis for MWNT 3 showed the formation of aromatic proton signals corresponding to tyrosine and Fmoc groups, confirming the success of the reaction in f-MWNT 3 synthesis and its purity. The p-MWNT and f-MWNTs were dispersed well in ethanol after sonication for 5 min, and suspensions were stable for at least 3 h. 3.2. Protein Binding by f-MWNTs Is Dependent on Nanotube Diameter, Surface Chemistry and Protein Type. Fluorescence spectroscopy is sensitive to protein dynamics because the excited fluorescent state persists for nanoseconds, which is exactly the time scale of many important biological processes such as the rotational motion of proteins, molecular binding, and protein conformational changes.43 We first studied a panel of proteins44-48 (Figure 4) involved in various biological functions for their binding with f-MWNTs using steady-state fluorescence spectroscopy. Protein intrinsic fluorescence was quenched to a different degree (Figure 5 and Supporting

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Figure 5. Fluorescence spectra of BSA and CA before and after f-MWNTs 1-4 titration. Final concentrations of f-MWNT in protein solutions from top to bottom is 0, 1.1, 2.3, 4.6, 9.3, and 18.7 µg/mL, respectively.

Figure 6. Pseudo Stern-Volmer plots of fluorescence quenching for proteins with f-MWNTs 1-4. (A-E) MWNTs with a diameter of 10 nm; (F-J) MWNTs with a diameter of 40 nm; (K,M) MWNTs with a diameter of 10 nm in water; (L,N) for MWNTs with a diameter of 10 nm in 120 mM NaCl. f-MWNTs were dissolved in ethanol, and therefore ethanol was used as a titration control and plotted in the Stern-Volmer plots.

Information, Figure 1) indicating molecular interactions between f-MWNTs and proteins. The potential effect of nanotube scattering on protein fluorescence was studied by measuring fluorescence spectrum of MWNT 2. The scattering/fluorescence of MWNT 2 at the concentration used in this study was about 1/200 of the protein fluorescence intensity under our experimental conditions, and furthermore it was supposed to increase, which is in the opposite direction of the fluorescence quenching. The classical Stern-Volmer equation51 relates the quenching of fluorescence to the concentration of quencher in the relationship Fo/F ) τo/τ ) 1 + Ksv[Q], where Fo, F, and τo, τ represent initial or modified fluorescence intensity or lifetimes, respec-

tively. Ksv is the Stern-Volmer constant and [Q] is the quencher concentration. Because the molecular weight and molar concentration of f-MWNTs cannot be accurately determined, we used µg/mL instead of molar concentration for f-MWNTs. Figure 6A-J shows the pseudo Stern-Volmer plots for the fluorescence quenching data between five proteins and four MWNTs at two different diameters (10 and 40 nm). Figure 6K-N shows the pseudo Stern-Volmer plots between two proteins and MWNT 1 and 2 at normal buffer and an elevated ionic strength. From these plots, we noted the following: (1) Comparing Figure 6F-J with Figure 6A-E, the f-MWNTs with a 40 nm diameter exhibited stronger protein binding than those

3304 J. Phys. Chem. C, Vol. 112, No. 9, 2008 with a smaller diameter (10 nm). This may be due to the difference in curvature on the nanotube surface and is in line with previous findings that larger silica nanoparticles bound protein stronger than smaller ones.23,49,50 Careful examination of TEM of nanotubes (Figure 2) indicated that there were more kinks on the tube and less bundle formation. These might also contribute to more protein binding although the curvature effect might be the dominant factor. (2) The f-MWNT 2 quenched protein fluorescence significantly more than other f-MWNTs, and f-MWNT 3 is the second strongest quencher in most cases, suggesting a dominant role of electrostatic interaction in protein binding. (3) The quenching of protein fluorescence by f-MWNT 2 was affected (inhibited BSA and enhanced in HK and hemoglobin (HG)) at higher ionic strengths (Figure 6K-N), indicating that f-MWNT 2 was bound to protein mainly through electrostatic interactions. However, the effect also depends on the protein surface properties, because the binding can be either strengthened or weakened. All proteins (pI < 7) in this study had net negative charges under our experimental conditions (pH ) 7.0). In the case of MWNT 2, the electrostatic interactions might take place between the negatively charged carbon nanotubes and the local neutral or positively charged surface residues on the proteins. The detailed interactions between carbon nanotubes and residues/domains on protein surface await further investigation. (4) Larger proteins seemed to bind stronger with nanotubes (Figure 6) suggesting that more functional groups on a larger surface might favor a stronger binding. (5) The upward curving of the Stern-Volmer plots that was more pronounced in f-MWNT 2 suggests the involvement of accelerated quenching or static quenching, which is often related to a conformational change51 or complex formation.52 Small molecules quench protein fluorescence through collisional or dynamic quenching mechanism, and this results in a decrease in both fluorescence intensity and lifetime. Static quenching, unlike collisional or dynamic quenching, would not change the lifetime of the fluorophores. To investigate the molecular origin of the fluorescence quenching by f-MWNTs, we further carried out fluorescence lifetime measurements. 3.3. Experimental Proof for f-MWNT/Protein Complex Formation. Multifrequency phase and modulation data for BSA and CA are shown in Figure 7, and data for other protein/fMWNTs are included in Supporting Information, Figure 3. The fluorescence lifetimes for all the binding pairs were analyzed with a double exponential decay rate equation. Lifetimes and the relevant pre-exponential factors are summarized in Table 2. Upon binding, most proteins showed negligible change in both long and short lifetimes except for ovalbumin, which exhibited a small (∼18%) reduction in lifetime indicating a minor dynamic quenching component. Small molecules quench protein fluorescence mainly through collisional or dynamic quenching mechanisms, characterized by a decrease of the protein fluorescence lifetime. In this experiment, no significant change in fluorescence lifetime was detected. Therefore, fMWNTs quench protein fluorescence mainly through the static quenching mechanism involving the formation of binary complexes. 3.4. The f-MWNTs Selectively Induced Protein Secondary Structure Changes. Steady-state and time-resolved fluorescence experiments indicated that the f-MWNTs bound to proteins by forming a complex. The stronger fluorescence quenching and the upward curving in the Stern-Volmer plot in the case of f-MWNT 2 suggested the possibility of a conformational change induced by the nanotube binding. We carried out circular dichroism (CD) measurements and protein secondary-structure

Mu et al.

Figure 7. Phase-modulation and the double exponential fit data for BSA and CA with and without f-MWNTs 2-4 (10 nm diameter). The final MWNTs’ concentrations were 9.3 µg/mL, and protein was 500 µg/mL proteins in deionized water except for BSA, which was 800 µg/mL. (A) BSA with f-MWNT 2; (B) BSA with f-MWNT 3; (C) BSA with f-MWNT 4; (D) BSA; (E) CA with f-MWNT 2; (F) CA with f-MWNT 3; (G) CA with f-MWNT 4; and (H) CA.

calculations based on CD spectra. CD spectra of BSA and CA with or without the addition of f-MWNTs (same concentration as in the fluorescence measurements) and the photomultiplier tube (PMT) voltage during the wavelength scans are shown in Figure 8. The wavelength-dependent changes in the PMT voltage, which was often ignored in previous CD studies of nanoparticle/protein interactions, rose rapidly below 200 nm and caused noises in the CD spectra, compared to longer wavelengths. Therefore, some CD spectral changes below 200 nm did not correspond to binding-induced conformational changes and was not included in our secondary-structure calculations. Protein-binding induced CD changes were more evident between BSA and f-MWNT 2 for nanotubes of both diameters and f-MWNT 3 for the nanotube with a larger diameter. Specifically, interactions between f-MWNT 2 (larger diameter) and BSA (Figure 8) caused a change from -80 to -60 mdegree at 222 nm. On the basis of secondary-structure calculations using the Yang method,53 these changes correlated to a 10% decrease in the R-helix structure, a 5% decrease in turns and random coil, and a concomitant 20% increase in the β-sheet structure. Other f-MWNTs induced CD changes, and the related secondarystructure calculations are all included in Supporting Information, Table 2. The order of protein/f-MWNT binding-induced CD changes corroborated with the fluorescence quenching results. 4. Conclusions In summary, we investigated the effects of surface chemistry, surface charge, and diameter of MWNTs on their interaction with proteins in this investigation. Spherical nanoparticles were found to selectively interact with proteins54 depending on particle size23,49,50 and the nature of their surface chemistry55,56 Electrostatic57-60 or neutral61 molecular interactions, as well as binding-induced protein conformational change,62 have been reported. Proteins tend to bind to an elongated unmodified SWNT through hydrophobic patches on their surface (aromatic residues).29-31 Binding-induced conformational changes,34 or

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TABLE 2: Fluorescence Lifetime of Proteins Bound to f-MWNTsa

a

title

τ1 (ns)

f1

τ2 (ns)

f2 (fixed)

χ2

BSA (800 ug/mL) + f-MWNT 2 f-MWNT 3 f-MWNT 4 carbonic anhydrase (500 ug/mL) + f-MWNT 2 f-MWNT 3 f-MWNT 4 hexokinase (500 ug/mL) + f-MWNT 2 f-MWNT 3 f-MWNT 4 ovalbumin (500 ug/mL) + f-MWNT 2 f-MWNT 3 f-MWNT 4

0.28 ( 0.009 0.07 ( 0.003 0.08 ( 0.004 0.11 ( 0.007 0.08 ( 0.008 0.02 ( 0.005 0.18 ( 0.007 0.12 ( 0.006 0.44 ( 0.032 1.79E-008 ( 0.008 0.05 ( 0.016 1.73E-008 ( 0.005 1.10 ( 0.018 0.09 ( 0.006 0.10 ( 0.005 0.17 ( 0.009

0.06 ( 0.001 0.21 ( 0.001 0.13 ( 0.001 0.08 ( 0.001 0.17 ( 0.001 0.25 ( 0.001 0.19 ( 0.002 0.24 ( 0.002 0.06 ( 0.003 0.14 ( 0.001 0.12 ( 0.002 0.12 ( 0.001 0.23 ( 0.005 0.25 ( 0.002 0.27 ( 0.002 0.16 ( 0.002

5.85 ( 0.021 5.99 ( 0.025 5.88 ( 0.022 5.81 ( 0.021 5.14 ( 0.022 5.09 ( 0.025 5.18 ( 0.024 4.99 ( 0.025 4.14 ( 0.021 3.89 ( 0.014 4.06 ( 0.019 4.25 ( 0.014 5.51 ( 0.042 4.75 ( 0.024 4.27 ( 0.025 4.68 ( 0.022

0.94 0.79 0.87 0.93 0.83 0.75 0.82 0.76 0.94 0.86 0.88 0.88 0.77 0.75 0.73 0.84

157 84.6 83.8 116 401 273 403 271 27.7 27.1 24.1 180 99.3 275 42.2 115

τ1, τ2 are two fluorescence lifetimes of proteins. f1 and f2 are fractions of each lifetime component.

Figure 8. CD spectra of BSA and CA titrated with f-MWNTs (10 and 40 nm diameter) using concentrations indicated in the top left panel and the wavelength-dependent high voltage on the photomultiplier tube.

proteins-unfolding caused by light sonication63 or their instability,34 all intensified protein binding to SWNTs. Peptides reoriented their structures to optimize their interactions with SWNT through their aromatic residues.25,26 Although the evidence for π-π stacking interactions as a main driving force is compelling, the involvement of charge in protein/SWNT binding has also been reported.32,33 The goal of the current study was to investigate molecular mechanisms involved in protein/ f-MWNTs interactions. Regarding the influence of nanotube diameter on their affinity to proteins, f-MWNTs with larger diameter consistently showed

stronger protein bindings than those with a smaller diameter. Our data proved experimentally that the smoother curvature can induce larger protein conformational changes while the protein adapts to the “unfamiliar” surface curvature (Figure 9) in onedimensional nanoparticles. This is in line with a previous findings on spherical silica nanoparticles,23,49,50 With negatively charged and neutral surfaces on f-MWNTs (via chemistry modifications), although p-MWNT binds all proteins through mostly π-π stacking interactions, negatively charged f-MWNT 2 and 3 both showed increased protein binding. Neutralizing the surface charges with an amide

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Figure 9. Cartoon of protein binding to f-MWNT with different diameters.

formation in f-MWNT 4 reduced the protein binding compared to f-MWNT 2 and 3. Drastically different from the fluorescence quenching in protein/small-ligand interactions (in which collisional quenching is the major cause), static quenching mechanisms dominate protein/f-MWNT binding, as shown in fluorescence lifetime measurements. The nanotube-binding-induced protein conformational changes shown in the CD studies support the formation of binary complexes. The affinity of f-MWNTs to proteins in a large part also depends on the surface electrostatic and stereochemical properties of the proteins. An f-MWNT often exhibited different binding-induced fluorescence quenching and CD changes with different protein counterparts. Therefore, by modulating the size, shape, surface charges, or surface chemistry of MWNTs, the protein-binding capability and their in vivo toxicity can be regulated and biocompatibility improved, optimizing their potential application in nanomedicine. Acknowledgment. We thank Dr. Shumei Zhai and Ms. Xifeng Ma for their technical assistance, Ms. Yuyan Chen and Dr. Gang Liu for assistance in acquiring MAS NMR spectra, and Dr. Gopal Murti for acquiring TEM images. This work was supported by the American Lebanese and Syrian Associated Charities (ALSAC), St. Jude Children’s Research Hospital, and Shandong University. Supporting Information Available: Details regarding protein binding results using steady-state and time-resolved fluorescence spectroscopy and circular dichroism. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Mattson, M. P.; Haddon, R. C.; Rao, A. M. J. Mol. Neurosci. 2000, 14, 175-182. (2) Lin, Y.; Taylor, S.; Li, H.; Fernando, K. A. S.; Qu, L.; Wang, W.; Gu, L.; Zhou, B.; Sun, Y.-P. J. Mater. Chem. 2004, 14, 527-541. (3) Bianco, A.; Kostarelos, K.; Prato, M. Curr. Opin. Chem. Biol. 2005, 9, 674-679. (4) Kam, N. W. S.; Liu, Z.; Dai, H. J. Am. Chem. Soc. 2005, 127, 12492-12493. (5) Gao, L.; Nie, L.; Wang, T.; Qin, Y.; Guo, Z.; Yang, D.; Yan, X. ChemBioChem. 2006, 7, 239-242. (6) Klumpp, C.; Kostarelos, K.; Prato, M.; Bianco, A. Biochim. Biophys. Acta 2006, 1758, 404-412. (7) Liu, Z.; Cai, W.; He, L.; Nakayama, N.; Chen, K.; Sun, X.; Chen, X.; Dai, H. Nat. Nanotechnol. 2007, 2, 47-52. (8) Nel, A.; Xia, T.; Ma¨dler, L.; Li, N. Science 2006, 311, 622627. (9) Colvin, V. L. Nature Biotech. 2003, 21, 1166-1170. (10) Lynch, I.; Dawson, K. A.; Linse, S. Sci. STKE, 2006, 327, pe14. (11) Baughman, R. H.; Zakhidov, A. A.; de Heer, W. A. Science 2002, 297, 787-792. (12) Shvedova, A.; Castranova, V.; Kisin, E. R.; Schwegler-Berry, D.; Murray, A. R.; Gandelsman, V. Z.; Maynard, A.; Baron, P. J. Toxicol. EnViron. Health, A 2003, 66, 1909-1926. (13) Fiorito, S.; Serafino, A.; Andreola, F.; Togna, A.; Togna, G. J. Nanosci. Nanotechnol. 2006, 6, 591-599.

Mu et al. (14) Lam, C.-W.; James, J. T.; McCluskey, R.; Arepalli, S.; Hunter, R. L. Critical ReV. Toxicol. 2006, 36, 189-217. (15) Wang, H.; Wang, J.; Deng, X.; Sun, H.; Shi, Z.; Gu, Z.; Liu, Y.; Zhao, Y. J. Nanosci. Nanotechnol. 2004, 4 (8), 1019-1024. (16) Singh, R.; Pantarotto, D.; Lacerda, L.; Pastorin, G.; Klumpp, C.; Prato, M.; Bianco, A.; Kostarelos, K. Proc. Nat. Acad. Sci. U.S.A. 2006, 103, 3357-3362. (17) Cherukuri, P.; Bachilo, S. M.; Litovsky, S. H.; Weisman, R. B. J. Am. Chem. Soc. 2004, 126, 15638. (18) Kam, N. W. S.; Jessop, T. C.; Wender, P. A.; Dai, H. J. Am. Chem. Soc. 2004, 126, 6850-6851. (19) Pantarotto, D.; Briand, J-P.; Prato, M.; Bianco, A. Chem. Commun. 2004, 16-17. (20) Kam, N. W. S.; O’Connell, M.; Wisdom, J. A.; Dai, H. Proc. Nat. Acad. Sci. U.S.A. 2005, 102, 11600-11605. (21) Pantarotto, D.; Singh, R.; McCarthy, D.; Erhardt, M.; Briand, J. P.; Prato, M.; Kostarelos, K.; Bianco, A. Angew. Chem., Int. Ed. 2004, 43, 5242. (22) Kam, N. W. S.; Liu, Z.; Dai, H. Angew. Chem., Int. Ed. 2006, 45, 577-581. (23) Vertegel, A.; Siegel, R. W.; Dordick, J. S. Langmuir 2004, 20, 6800-6807. (24) Erlanger, B. F.; Chen, B. X.; Zhu, M.; Brus, L. Nano Lett. 2001, 1, 465-467. (25) Wang, S.; Humphreys, E. S.; Chung, S. Y.; Delduco, D. F.; Lustig, A. R.; Wang, H.; Parker, K. N.; Rizzo, N. W.; Subramoney, S.; Chiang, Y.-M.; Jagota, A. A. Nat. Mater. 2003, 2, 196-200. (26) Zorbas, V.; Ortiz-Acevedo, A.; Dalton, A. B.; Yosida, 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. (27) Lin, Y.; Allard, L. F.; Sun, Y. P. J. Phys. Chem. B 2004, 108, 3760-3764. (28) Su, Z.; Leung, T.; Honek, J. F. J. Phys. Chem. B 2006, 110, 2362323627. (29) Chen, R.; Zhang, Y.; Wang, D.; Dai, H. J. Am. Chem. Soc. 2001, 123, 3838-3839. (30) Zoebas, V.; Smith, A. L.; Xie, H.; Ortiz-Acevedo, A.; Dalton, A. B.; Dieckmann, G. R.; Draper, R. K.; Baughman, R. H.; JoseYacaman, M.; Musselman, I. H. J. Am. Chem. Soc. 2005, 127, 1232312328. (31) Li, X.; Chen, W.; Zhan, Q.; Dai, L.; Sowards, L.; Pender, M.; Naik, R. R. J. Phys. Chem. B 2006, 110, 12621-12625. (32) Bradley, K.; Star, M. B.; Gru¨ner, G. Nano Lett. 2004, 4, 253256. (33) Nepal, D.; Geckeler, K. E. Small 2006, 2, 406-412. (34) Karajanagi, S. S.; Vertegel, A. A.; Kane, R. S.; Dordick, J. S. Langmuir 2004, 20, 11594-11599. (35) Matsuura, K.; Saito, T.; Okazaki, T.; Ohshima, S.; Yumura, M.; Iijima, S. Chem. Phys. Lett. 2006, 429, 497-502. (36) Davis, J. J.; Green, M. L. H.; Hill, H. A. O.; Leung, Y. C.; Sadler, P. J.; Sloan, J.; Xavier, A. V.; Tsang, S. C. Inorg. Chim. Acta 1998, 272, 261-266. (37) Balavoine, F.; Schultz, P.; Richard, C.; Mallouh, V.; Ebbesen, T. W.; Mioskowski, C. Angew. Chem., Int. Ed. 1999, 38, 1912-1915. (38) Tsang, S. C.; Guo, Z.; Chen, Y. K.; Green, M. L. H.; Hill, A. A. O.; Hambley, T. W.; Sadler, P. J. Angew. Chem., Int. Ed. Engl. 1997, 36, 2198-2200. (39) Guo, Z.; Sadler, P. J.; Tsang, S. C. AdV. Mater. 1998, 10, 701703. (40) Azamian, B. R.; Coleman, K. S.; Davis, J. J.; Hanson, N.; Green, M. L. Chem. Comm. 2002, 366-367. (41) Drain, L. E. Proc. Phys. Soc. 1962, 80, 1380-1382. (42) Doskocilovfi, b. and Schneider, B. Chem. Phys. Lett. 1970, 6, 381384. (43) Gratton, E.; Jameson, D. M.; Hall, R. D. Ann. ReV. Biophys. Bioeng. 1984, 13, 105-124. (44) Kim, C. Y.; Chang, J. S.; Doyon, J. B.; Baird, T. T., Jr.; Fierke, C. A.; Jain, A.; Christianson, D. W. J. Am. Chem. Soc. 2000, 122, 1212512134. (45) Anderson, C. M.; Stenkamp, R. E.; Steitz, T. A. J. Mol. Biol. 1978, 123, 15-33. (46) Ghuman, J.; Zunszain, P. A.; Petitpas, I.; Bhattacharya, A. A.; Otagiri, M.; Curry, S. J. Mol. Biol. 2005, 353, 38. (47) Paoli, M.; Liddington, R.; Tame, J.; Wilkinson, A.; Dodson, G. J. Mol. Biol. 1996, 256, 775-792. (48) Stein, P. E.; Leslie, A. G.; Finch, J. T.; Carrell, R. W. J. Mol. Biol. 1991, 221, 941-959. (49) Shang, W.; Nuffer, J. H; Dordick, J. S.; Siegel, R. W. Nano Lett. 2007, 7, 1991-1995. (50) Lundqvist, M.; Sethson, I.; Jonsson, B. H. Langmuir 2004, 20, 10639-10647.

Protein/f-MWNT Binding Mechanism (51) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: Berlin, Germany, 2006. (52) Eftink, M. R.; Ghiron, C. A. Anal. Biochem. 1981, 114, 199227. (53) Yang, J. T.; Wu, C. S. C.; Martinez, H. M. Methods Enzymol. 1986, 130, 208-269. (54) Karlsson, M.; Carlsson, U. Biophys. J. 2005, 88, 3536-3544. (55) Fischer, N. O.; McIntosh, C. M.; Simard, J. M.; Rotello, V. M. Proc. Nat. Acad. Sci. U.S.A. 2002, 99, 5018-5023. (56) You, C. C.; De, M.; Han, G.; Rotello, V. M. J. Am. Chem. Soc. 2005, 127, 12873-12881. (57) Verma, A.; Simard, J. M.; Rotello, V. M. Langmuir 2004, 20, 4178-4181.

J. Phys. Chem. C, Vol. 112, No. 9, 2008 3307 (58) Hong, R.; Fischer, N. O.; Verma, A.; Goodman, C. M.; Emrick, T.; Rotello, V. M. J. Am. Chem. Soc. 2004, 126, 739-743. (59) Aubin-Tam, M. E.; Hamad-Schifferli, K. Langmuir 2005, 21, 12080-12084. (60) Rezwan, K.; Meier, L. P.; Gauckler, L. J. Biomaterials 2005, 26, 4351-4357. (61) Peng, Z. G.; Hidajat, K.; Uddin, M. S. J. Colloid Interface Sci. 2004, 271, 277-283. (62) Karlsson, M.; Mårtensson, L. G.; Jonsson, B. H.; Carlsson, U. Langmuir 2000, 16, 8470-8479. (63) Karajanagi, S. S.; Yang, H.; Asuri, P.; Sellitto, E.; Dordick, J. S.; Kane, R. S. Langmuir 2006, 22, 1392-1395.