Single wall carbon nanotubes enter cells by endocytosis and not membrane penetration
© Yaron et al; licensee BioMed Central Ltd. 2011
Received: 13 May 2011
Accepted: 30 September 2011
Published: 30 September 2011
Carbon nanotubes are increasingly being tested for use in cellular applications. Determining the mode of entry is essential to control and regulate specific interactions with cells, to understand toxicological effects of nanotubes, and to develop nanotube-based cellular technologies. We investigated cellular uptake of Pluronic copolymer-stabilized, purified ~145 nm long single wall carbon nanotubes (SWCNTs) through a series of complementary cellular, cell-mimetic, and in vitro model membrane experiments.
SWCNTs localized within fluorescently labeled endosomes, and confocal Raman spectroscopy showed a dramatic reduction in SWCNT uptake into cells at 4°C compared with 37°C. These data suggest energy-dependent endocytosis, as shown previously. We also examined the possibility for non-specific physical penetration of SWCNTs through the plasma membrane. Electrochemical impedance spectroscopy and Langmuir monolayer film balance measurements showed that Pluronic-stabilized SWCNTs associated with membranes but did not possess sufficient insertion energy to penetrate through the membrane. SWCNTs associated with vesicles made from plasma membranes but did not rupture the vesicles.
These measurements, combined, demonstrate that Pluronic-stabilized SWCNTs only enter cells via energy-dependent endocytosis, and association of SWCNTs to membrane likely increases uptake.
Carbon nanotubes (CNTs) have recently been explored for potential uses in biology and medicine. Their small size, high surface area, inert chemical composition, and unique physical properties have made them extensively investigated for transport of DNA, nucleic acids, drugs, and a variety of other potential therapeutics. Single wall CNTs (SWCNTs) with a 1-2 nm outer diameter have variable length and unique optical and electrical properties desirable for biological applications. Cytotoxicity of SWCNTs depends on SWCNT length, impurities, and dispersion quality (isolated vs. bundles). SWCNTs dispersed in a biocompatible Pluronic triblock copolymer reorganize sub-cellular structures without inducing cell death[8, 9]. To better understand the toxicological effects posed by SWCNTs and to develop SWCNT-related cellular biotechnologies, unambiguous determination of the mechanism of uptake into the cell is essential.
Competing hypotheses exist regarding the mechanism by which SWCNTs enter cells: non-specific physical penetration of the cell membrane, endocytosis or both. Numerous studies have imaged CNTs inside cells and have shown that CNTs are endocytosed[10–14]. Theoretical and simulation studies on CNT uptake into cells provided contradictory results: some theoretical reports have suggested that CNTs may not be able to trigger endocytosis due to their small diameter and the kinetics of endosome formation[15, 16]. Simulation studies have shown that CNTs have affinity for membranes, but suggest that CNTs have insufficient energy to pierce through both leaflets of a membrane. While endocytosis is the commonly suggested mechanism of cellular uptake, physical penetration has not been rigorously considered and may account for significant uptake. In particular, alteration or disruption of sub-cellular membranous structures or CNT affinity to membranes may also be responsible for altering cellular uptake and architecture.
Here, we employed complementary methods including in vitro model membranes and cellular imaging to investigate mechanisms of cellular uptake of short (145 ± 17 nm), Pluronic F-127 (PF-127) triblock copolymer-dispersed SWCNTs in Millipore-filtered deionized water to discern whether cellular uptake occurs only via an active endocytosis process or passive physical penetration through the membrane. To minimize confounding effects due to SWCNT sample preparation, such as contamination from metal catalysts, undesirable carbon polymorphs, distribution of SWCNT lengths, and defects resulting from functionalization methods, we utilized highly purified, length-selected, dispersed, pristine SWCNTs that have been previously developed in our group[9, 18, 19]. We have determined uptake and localization of Pluronic copolymer-stabilized SWCNTs into cells through temperature dependent cell studies with confocal Raman spectroscopy and fluorescence lifetime imaging (FLIM). We also used electrochemical impedance spectroscopy (EIS) of sparsely-tethered bilayer lipid membranes (stBLMs) and Langmuir monolayers (LMs) of synthetic phospholipids to model the plasma membrane. We further verified our results by examining interactions between SWCNTs and giant plasma membrane vesicles (GPMVs). GPMVs are produced from cell membranes, are more complex than synthetic lipid systems, but circumvent biological complications of cells. stBLMs, LMs and GPMVs have been utilized previously as model systems with great success to determine spatial localization, binding affinities, dissociation constants, and insertion pressures necessary for membrane association and cellular incorporation of materials[20–22].
SWCNTs added to the extracellular media localize within cells
Lipid bilayer association of SWCNTs
To determine if SWCNTs perforate bilayers, and by a similar mechanism might penetrate the plasma membrane of the cell, we examined SWCNT interactions with synthetic lipid bilayers. We monitored the electric responses of sparsely-tethered bilayer lipid membranes (stBLMs) to SWCNT addition using electrochemical impedance spectroscopy (EIS; Additional Methods and Additional Figure 1A). It was recently shown that stBLMs prepared by rapid solvent exchange form complete bilayers on solid substrates that are contiguous and virtually defect-free. These stBLMs are decoupled from the substrates by a highly hydrated, nanometer-thin stratum to retain their in-plane fluidity and, therefore, constitute realistic models of lipid membranes. Specifically, they form fluid, disordered membranes with a resistance in the MΩcm2 regime that permits the sensitive detection of changes in their electrical properties by membrane-targeting enzymes or channel-forming toxins[27, 28].
The results (Figure 2B-D and Additional Information) showed that the stBLMs possess near-ideal capacitive behavior. The exponents of the constant phase element (CPE; see Methods and Additional Methods) were always near unity with αstBLM > 0.95, (Figure 2B) allowing the stBLM CPE in the ECM to be approximated as a capacitance CPEstBLM ≈ CstBLM. Tracking CstBLM versus incubation time (Figure 2C) or concentration (Additional Information) showed a slight increase, indicating a small change in local dielectric constant in the hydrophobic core or in bilayer thickness. The second CPE in the ECM (see Additional Figure 1B), CPEdefect, depends on electrical conductivity of the sub-membrane space. Increased electric resistance of the aqueous reservoir leads to a higher electric field penetration into this space and, consequently, a smaller CPEdefect. Assuming that ion mobility remains the same in the sub-membrane space as in the bulk of the electrolyte, this defect density in the stBLM may be estimated from Rdefect. Changes in Rdefect versus incubation time (Figure 2D) and concentration (Additional Information) were negligible, suggesting no change in bilayer defect density due to changes induced by incubation with SWCNTs. For comparison, Rdefect may change by more than two orders of magnitude when stBLMs are perforated by protein membrane pores or reduced in their thickness, leading to a lower hydrophobic barrier to ion transfer across the bilayer[27, 28]. While the SWCNTs did not affect the capacitance or resistance of the membrane significantly, there were long-term changes in the membrane properties in the presence of PF-127 and SWCNTs suggesting some reordering of the membranes.
Interaction of SWCNTs with Langmuir monolayers
MIP calculated from linear fits of DPPC monolayers exposed to SWCNTs dispersed in PF-127 and to PF-127 alone.
At room temperature, DPPC forms phase-separated LMs in a coexistence regime, which complicates the determination of the SWCNTs' MIP. To confirm MIP results in a purely fluid lipid phase, similar experiments were performed using DOPC LMs. However, due to its higher fluidity than DPPC, DOPC LMs are intrinsically less stable, making reproducibility an issue. We never observed any pressure increase ΔΠ at initial pressures exceeding 22 mN/m, but we were unable to produce a MIP plot of similar accuracy to that obtained using DPPC.
PF-127 has a higher MIP than the PF-127 stabilized SWCNTs (Table 1), suggesting that the free polymer has a higher affinity for the membrane. To determine if the PF-127 dispersant was separating from the SWCNTs and formed aggregates in the experiments, we took samples from the subphase of the Langmuir film balance after experiments and checked for SWCNT aggregates using Vis-NIR absorbance spectroscopy. There was no significant change observed compared with a freshly-dispersed nanotube solution.
FLIM of CellMask orange-labeled GPMVs with SWCNTs
To determine if SWCNTs have a preferential association with bilayer membranes and to provide a link between synthetic lipid and cell experiments, we visualized SWCNT interactions with vesicles derived from cell plasma membranes. GPMVs, produced from NIH-3T3 cells (see Methods), were labeled with CellMask orange and exposed to SWCNTs at similar time points and concentration as the other experiments. GPMVs were imaged via widefield microscopy for > 2 hours, and no vesicle rupture was observed.
Fluorescence lifetime imaging microscopy (FLIM) values obtained from CellMask Orange-treated GPMVs exposed to SWCNTs.
FLIM of GPMVs with CellMask Orange (+) SWCNTs
721 ± 90
0.829 ± 0.186
2055 ± 464
0.171 ± 0.066
890 ± 32
1.357 ± 0.089
505 ± 62
0.859 ± 0.099
1861 ± 462
0.141 ± 0.032
674 ± 20
1.462 ± 0.073
Temperature-dependent endocytosis assay
To quantify SWCNT distribution in the cells at 37°C and 4°C, we collected confocal Raman spectra in the x-y plane and then scanned along z-direction (height of the cell) from -20 to 20 μm with a 1 μm z-step size. We arbitrarily designated z = 0 to be ~2 μm from the basal plane. We independently determined the cell height to be ~7 μm (z = -2 μm to z = 5 μm) using the laser scanning confocal microscope. SWCNTs for the 37°C sample were located in a z region approximately corresponding to the height of the cell (Figure 5C). Conversely, the 4°C sample did not show SWCNT intensity for height regions corresponding to the cell height (Figure 5C). Minimal SWCNT intensity was observed below z = -2 μm, corresponding to the SWCNTs on the substrate, and above z = 5 μm, corresponding to SWCNTs on the cell membrane.
Altered endocytosis rates in SWCNT treated cells
FLIM of endosomes
Fluorescence lifetime imaging microscopy (FLIM) values obtained from cells transfected with GFP-endo and GFP and exposed to SWCNTs.
GFP-endo (+) "Short" (145 ± 17 nm) SWCNTs
0.574 ± 0.081
0.426 ± 0.074
1.200 ± 0.037
1588 ± 117
0.601 ± 0.105
2757 ± 116
0.399 ± 0.068
1.291 ± 0.088
1918 ± 124
0.553 ± 0.109
2560 ± 101
0.447 ± 0.104
1.346 ± 0.072
GFP (+) "Short" (145 ± 17 nm) SWCNTs
1638 ± 83
0.558 ± 0.133
2803 ± 107
0.442 ± 0.125
1.261 ± 0.082
1306 ± 146
0.624 ± 0.173
3054 ± 136
0.376 ± 0.132
1.364 ± 0.206
1574 ± 186
0.568 ± 0.163
2527 ± 118
0.432 ± 0.138
1.342 ± 0.099
GFP-endo (+) "Long" (1.25 ± 0.75 μm) SWCNTs
1945 ± 131
0.550 ± 0.111
2606 ± 107
0.450 ± 0.109
1.231 ± 0.063
2026 ± 167
0.534 ± 0.110
2515 ± 121
0.466 ± 0.101
1.456 ± 0.101
1827 ± 151
0.583 ± 0.097
2639 ± 111
0.417 ± 0.088
1.337 ± 0.064
Quantification of τm from the different experimental conditions (Figure 7A-C) showed time-dependent changes (Figure 7D). Treatment with short SWCNTs generated a statistically significant (p < 0.01) reduction in τm after 5 minutes for both GFP-endo and GFP compared to their respective controls. At 25 minutes after treatment, the GFP τm remained significantly reduced. However, the GFP-endo τm increased at 25 minutes. This suggests that HeLa cells had begun increasing the number of endosomes after 25 minutes (such as in Figure 6) which returned some of the signal to a more-baseline level. Also SWCNTs may have escaped from the endosomes, so they would no longer quench GFP-endo, but rather soluble GFP in the cytoplasm. The long SWCNTs showed no change in τm at 5 minutes of treatment and only a slightly decrease in τm at 25 minutes suggesting incubation with long SWCNTs does not alter the GFP fluorescence lifetime. Raw FLIM data is available in Table 3: A1 and A2 are the average relative contributions of τ1 and τ2 to τm (Eq. 1 in Methods); average τm cannot be calculated directly from averaging Ai and τi.
Previous studies involving several different cell types have demonstrated uptake of various types of functionalized CNTs[38–43]. Most studies show that CNTs enter cells via endocytosis[10–14]. However, given that millions of CNTs can enter cells, a small fraction of CNTs piercing the membrane could be significant. Some theoretical analyses suggested that SWCNTs may be insufficient to trigger endocytosis due to their small diameter and the kinetics of endosome formation[15, 16]. Experiments using nanoparticles of varying size and composition determined that uptake of nanoparticles depends strongly on their size[11, 15, 45]. However, given the unique anisotropy of SWCNTs, it is unclear which orientation governs interactions with cells[16, 46]. Using complementary membrane and cellular techniques we have shown unambiguously that polymer-dispersed SWCNTs were unable to penetrate complete bilayers. However, the affinity of the SWCNTs for membranes did appear to increase rates of endocytosis in the cell.
Endocytosis of SWCNTs
Confocal Raman spectroscopy and imaging of HeLa cells revealed that SWCNTs were preferentially localized within the cell. SWCNTs were observed throughout the cell with SWCNT intensity located in the perinuclear region, consistent with previous observations[10, 24], possibly suggesting that SWCNTs entered cells via endocytosis and were deposited in or near the endoplasmic reticulum. This is also supported by a reduction in cellular uptake of SWCNTs at 4°C when energy-dependent processes, including endocytosis, are reduced. We suggest that SWCNTs or PF-127 by means of their membrane activity were able to disrupt some of the endosomes as they shrank into lysosomes, thus depositing SWCNTs throughout the cell and enabling them to interact with other cellular components.
SWCNTs enter cells via an energy-dependent endocytosic process. However, endocytosis may proceed by a number of different mechanisms including clathrin-mediated endocytosis, calveoli-mediated endocytosis and pinocytosis (see review of nanomaterial uptake by endocytosis ). In this study we have not examined the specific type of endocytosis relevant for SWCNT uptake which could be further clarified by targeting integrins and other cell-specific receptors on the plasma membrane.
SWCNTs associate with but do not penetrate membranes
Synthetic membrane experiments indicated that SWCNTs cannot completely penetrate membrane bilayers, but changes in monolayer pressure suggested that SWCNTs might penetrate the outer leaflet of lipids. We hypothesize that the association of SWCNT with lipids may alter membrane tension locally and stimulate endocytosis[47, 48]. Exposure of cells to SWCNTs significantly increased the number of endosomes when compared to control (Figure 4). Therefore, we suggest that SWCNTs affect the tension of the cell membrane, which may lead to a significant increase in endocytotic activity.
We have shown that short (145 nm) SWCNTs altered the fluorescence lifetime of GFP-endo, which label endosomes. While it is possible that these SWCNTs only indirectly alter the fluorophore's nanoenvironment, we have also shown that long (1,250 nm) SWCNTs had no effect on lifetime compared to control, and these long SWCNTs are too long for endocytosis[11, 15, 45]. Further, confocal Raman imaging confirmed that short SWCNTs entered HeLa cells, probably via endocytosis as evidenced by the perinuclear localization. We propose that short SWCNTs in endosomes directly altered the τm of the GFP-endo fluorophore; long SWCNTs, unable to enter via endocytosis, did not alter the τm of GFP-endo. Soluble GFP was quenched 5 and 25 min after treatment, suggesting that SWCNTs were liberated from endosomes and quenched non-localized cellular GFP, further confirming the SWCNT sub-cellular localization, independently demonstrated via confocal Raman imaging. Unlike the GFP-endo, the GFP remained quenched after 25 min, suggesting that some endosomes may have ruptured and released SWCNTs into the intracellular space and that the rate of entry of SWCNTs into the cell is decreased at long time.
Potential mechanisms of SWCNT uptake
In combination, the wide range of techniques used in this work shows conclusively that short Pluronic-coated SWCNTs enter the cell via endocytosis and not via membrane penetration. From our results we suggest that the mechanism of SWCNT uptake into cells includes the following steps: (A) SWCNTs adsorb onto the cell surface and penetrate into the outer leaflet of the bilayer. (B) This association increases membrane tension and induces an imbalance between the outer leaflet and the inner leaflet. (C) Localized disturbances in membrane tension can stimulate endocytosis as the membrane attempts to regulate tension. (D) Once endocytosis is stimulated, the SWCNTs localize in the endosomes, which shrink during processing. (E) By disruption of the endosomes or lack of lysosomal processing, SWCNTs enter the cytoplasm.
The fate of the Pluronic polymer outside or inside the cell remains unknown. Polymer-coated SWCNTs have been shown to adsorb protein when injected into the circulation of animals. We did not observe the Pluronic being displaced by protein in solution over 6 days with agitation (Additional Figure 2). However, the complex mixture of different proteins, surfactants and cellular secretions in the circulation and in cell culture media may prove to displace Pluronic from the SWCNTs. As such we cannot assess at what point PF-127 is lost from the tubes.
The pluronic coating likely increases the association of the SWCNTs with membranes, enhancing endocytosis. Since surfactant molecules are generally used to disperse SWCNTs in solutions, we believe that our results are generic features of SWCNT uptake into cells. The specific targeting of SWCNTs for use in therapeutics should also focus on endocytosis as the preferential method for the cellular uptake.
We have shown that short, polymer-dispersed SWCNTs enter the cells by endocytosis, and SWCNTs do not penetrate through bilayers non-specifically. There is a preferential interaction of SWCNTs with membranes, which likely increases rates of endocytosis after ~30 minutes. Thus the membrane affinity of SWCNTs control cellular uptake, but incorporation does not occur by passive membrane penetration.
Synthesis of SWCNTs
Cell Culture and Imaging
HeLa cells were grown in Dulbecco's Modified Eagle Medium (DMEM; Hyclone catalog number SH30021) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (both Invitrogen), collectively referred to as cell culture media (CCM). Cells were passaged onto #1.5 coverslips in 35 mm dishes, incubated for 12 - 24 hours and exposed to SWCNTs in 2 mL of CCM. Solutions were equally distributed over the cells, and CCM was pre-heated for most experiments. For 4°C cell culture experiments, the CCM was removed and the cells washed with phosphate buffer saline (PBS) at room temperature before 2 mL of 4°C CCM with SWCNTs was added, and the plate was immediately transferred to a 4°C cold-room for 15 min.
Cells were transferred to coverslips in 35 mm dishes or 6-well plates and were treated with 1 mL of SWCNTs diluted in CCM; SWCNT concentrations and times are reported in the results section for particular experiments. For imaging, cells on coverslips were washed twice with PBS at room temperature, fixed for 10 min with 3.7% formaldehyde (Sigma-Aldrich) and mounted onto slides. For some Raman imaging experiments, hematoxylin (Sigma-Aldrich) was added before mounting to enhance contrast of cellular features, but most cells were not labeled.
Brightfield and widefield fluorescence imaging of the cells was performed with 40 × (oil, 1.25 N.A.) or 63 × (oil, 1.4 N.A.) objectives on a Leica DMI6000 B inverted microscope. Confocal Raman spectroscopy was performed on an inverted Raman confocal microscope (inVia, Renishaw) with a 785 nm laser (100 mW) using either a 40 × (oil, 1.25 N.A.) or 100 × (oil, 1.4 N.A.) objective (Leica Microsystems). The in-plane (x-y) resolution of the Raman microscope with the 100 × objective is ~250 nm and the z resolution is ~300 nm. Confocal Raman spectra between 1327 and 1819 cm-1 were collected with a 0.82 cm-1 resolution. We then quantified the Raman intensity at 1590 ± 17 cm-1 to obtain intensity maps of SWCNTs; the tangential mode of graphene, called the G-band, is located at 1590 cm-1 and is widely used to identify SWCNTs[13, 23]. Image analysis was performed using WiRE (Renishaw), and z-section data were analyzed using MATLAB (MathWorks) code developed in the lab.
Cell Transfection and Imaging of Endosomes
For transient transfection, cells were grown to ~ 60% confluence and transfected with either pAc-GFP1-Endo or pEGFP-C1 (Clontech) using PolyFect (Qiagen) according to manufacturer's recommendations, and cells were incubated overnight. To measure changes in endocytosis, cells were incubated with 100 μg of SWCNTs in CCM for 0 (added and immediately washed), 5, 10, 15, 20 and 25 min at 37°C in the tissue culture incubator. At the selected time points, the CCM with SWCNTs was removed, and cells were washed, fixed and mounted as described above. All procedures were performed in low light to prevent GFP photobleaching during incubation, fixation, mounting, and transportation.
Confocal imaging caused photobleaching of small spots and could not be used to image endosomes in the entire cell, so we used widefield fluorescence imaging. To minimize artifacts of out-of-plane light, cells were imaged in the central plane, as identified by the z-position at which the nucleus was in best focus. Previous analysis has shown that there was no change in overall height in SWCNT-treated cells. Images were processed with ImageJ to identify GFP-labeled endosomes by their fluorescence intensity and minimum and maximum sizes.
Giant Plasma Membrane Vesicles (GPMVs)
Vesicles were produced from NIH-3T3 cells grown in DMEM (Hyclone SH30022) supplemented with 10% calf serum and 1% penicillin/streptomycin in 60 mm dishes at 80-90% confluence using a method described elsewhere[50, 51]. Briefly, each dish was washed twice with 10 mL of a freshly prepared and thoroughly mixed buffer containing 10 mM HEPES, 0.15 M NaCl, 2 mM CaCl2, and 0.05% v/v gelatin. Cells were then incubated in 5 mL of buffer plus 1 mM dithiotheritol (DTT) and 25 mM formaldehyde for 1 hour at 37°C on an orbital shaker at 75 rpm. The solution was gently decanted and allowed to sit for 15 min at 4°C in a conical tube. Samples were taken from the middle portion of the solution to minimize extracellular material and necrotic cells. GPMVs were imaged directly using widefield microscopy or were treated with the membrane-active CellMask orange fluorophore (Invitrogen) with gentle agitation for 5 min. Then, 0.5 mL of the GPMV solution was transferred to a 35 mm glass-bottom dish (MatTek) pre-incubated with poly-L-lysine solution (Sigma-Aldrich).
Fluorescence Lifetime Imaging (FLIM)
FLIM was performed using a Leica TCS SP5 inverted laser scanning confocal microscope with a 100 × (oil, 1.4 N.A.) objective with a pixel resolution of 256 × 256 and a scan rate of 400 Hz. A tunable (720-950 nm), mode-locked Ti:sapphire pulsed infrared laser (Chameleon, Coherent) served as the multiphoton (MP) excitation source (1 W, average). Pulse-widths of < 140 fs were delivered at 90 MHz. For GFP, the MP laser was tuned to 942 nm, and the FLIM-dedicated photomultiplier tube (PMT) was tuned to 481-615 nm to detect the full range of GFP emission. For CellMask orange, the MP laser was tuned to 900 nm with the PMT tuned to 550-700 nm.
Time-correlated single photon counting (TCSPC) was implemented using a Becker & Hickl SPC-830 acquisition package with 10 ps resolution that includes three software programs: DCC (controls DCC 100 hardware), SPCM (controls image acquisition), and SPCImage (controls lifetime data analysis). Lifetime images were acquired for 180 s to minimize the coefficient of variation to ~2.5%[52, 53]. 220 time channels and a measurement window of 10.8 ns were used to minimize the variance of lifetime over a wide range of ratios of the measurement window to lifetime. The rates of the detected, converted, and stored photons were maintained between 1 × 104 and 1 × 106 (< 1% of laser repetition rate) to prevent errors in lifetime determination due to the pileup effect, to achieve the required signal-to-noise ratio after 3 min of detection, and to minimize photobleaching.
FLIM Data Processing
Fluorescence lifetime analysis was performed using SPCImage (Becker & Hickl). Instrument response functions were estimated and manually verified through the SPCImage software for each data set. Lifetime images were binned to achieve a peak photon count of ≥ 1000 (≥ 50,000 photons over all time channels), ensuring that two exponential decays could be accurately resolved[53, 54].
Each pixel was separately modeled as a single and a double exponential decay, and the corresponding average lifetimes (τ1, τ2 and τm), goodness of fits (χ2), and standard deviations were calculated in MATLAB. Reported lifetime values (Tables 2 and 3) were averaged and errors were calculated using the derivative method of error propagation. Thus, the spatially average values presented in Table 3 will comply with Eq. 2 for the individual values. Fluorescence lifetime images were then generated using SPCImage.
Sparsely-Tethered Bilayer Lipid Membranes (stBLMs)
The synthetic, zwitterionic phospholipids used in the model membrane experiments, i.e., dipalmitoyl-sn-glycro-3-phosphocholine and dioleoyl-sn-glycero-3-phosphocholine (DPPC and DOPC, respectively), were from Avanti Polar Lipids. stBLMs were prepared on gold-coated Si wafers by precipitation of DOPC onto a preformed self-assembled monolayer (SAM) as described. For further details, see Additional Methods.
Electrochemical Impedance Spectroscopy (EIS)
EI spectra of stBLMs on Si/gold in Teflon (PTFE) sample cells of local design were obtained between 1 Hz and 65 kHz with ten logarithmically distributed measurements per decade using a Solartron (Farnborough) potentiostat and frequency analyzer. The gold-coated Si wafer at the bottom of a buffer-filled compartment served as the electrode in connection with a saturated [Ag|AgCl|NaCl] reference and a Pt auxiliary electrode immersed into the buffer. Details are given in Additional Methods.
Fitting of the EI spectra was performed using the electrical equivalent circuit model (ECM) represented in Additional Figure 1B. CPE, or constant-phase element, refers to an electrical element with an impedance, ZCPE = 1/T(iω)α, where T is a coefficient measured in Farad per unit area × sα-1, and the exponent α varies between 0 and 1. The ECM in Additional Figure 1B shows two CPEs, one associated with the capacitive nature of the bilayer (CPEstBLM) and one associated with the capacitive properties of defects in the bilayer (CPEdefect). Such defects also give rise to a residual conductance, Y = 1/Rdefect. In addition, Rsolvent and Cstray describe the resistivity of the buffer and stray capacitance of the sample configuration. Two ECM parameters, CPEstBLM and Rdefect, obtained from the model were used to quantify the quality of as-prepared bilayers and changes in bilayer properties upon incubation with PF-127-dispersed SWCNTs or, as a control, equivalent concentrations of PF-127 alone. If the CPE exponent α equals 1, the CPE simply reduces to the capacitance, C. Since this is the case here (see Table 3), the two ECM parameters represent the bilayer capacitance and the inverse of the residual conductance due to defects.
Langmuir Monolayers (LMs)
A custom-built Teflon trough (Nima Technology, U.K.) with a surface area of 250 cm2 and a depth of 0.5 cm on an active anti-vibration table was filled with filtered H2O (Milli-Q, Millipore). The surface pressure, Π, due to a deposited LM was monitored using a Wilhelmy plate, cut from ashfree filter paper and connected to an electronic microbalance. LMs were formed by spreading either DPPC or DOPC dissolved in chloroform using a microsyringe (Hamilton) and allowing the solvent to evaporate for at least 15 min. The resulting LMs were compressed by two symmetrically moving barriers to a desired surface pressure, Π0. Subsequently, dispersed SWCNT in aqueous solution were injected into the subphase underneath a LM and the ensuing surface pressure change ΔΠ(t) was recorded at constant surface area over time, t, until it reached at stable plateau. Such experiments, performed at a series of increasing values of Π0, were used to determine the maximum insertion pressure (MIP), i.e., the minimum value of Π0 that just suppresses any insertion of the adsorbent into the LM.
Dulbecco's Modified Eagle Medium
equivalent circuit model
electrical impedance spectroscopy
fetal bovine serum
fluorescence lifetime imaging microscopy
giant plasma membrane vesicle
phosphate buffer saline
sparsely tethered bilayer lipid membrane
single wall carbon nanotube.
We thank Matteo Broccio, Rima Buvytyte (Vilnius University) and Haw-Zan Goh for help with the EIS studies and David Vanderah at the NIST-CSTL/IBBR for a sample of the tether lipid HC18. This work was supported by the NSF (CBET-0708418 & DMR-0619424 to KND and MFI), the Sloan Foundation (MFI), the NIH (1P01 AG032131 to ML), the Bertucci Graduate Fellowship (BDH), and the National Defense Science and Engineering Graduate Fellowship (BDH).
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