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Clustering of Stimuli on Single-Walled Carbon Nanotube Bundles Enhances Cellular Activation Tarek R. Fadel,† Michael Look,‡ Peter A. Staffier,§ Gary L. Haller,† Lisa D. Pfefferle,† and Tarek M. Fahmy*,†,‡ †

Departments of Chemical Engineering and ‡Biomedical Engineering and §Mechanical Engineering, Yale University, P.O. Box 208284, New Haven, Connecticut 06520 Received June 9, 2009. Revised Manuscript Received July 28, 2009

Functionalized single-walled carbon nanotube bundles (f-bSWNT) adsorbed with T-cell-stimulating antibodies are shown to enhance both the kinetics and magnitude of T cell stimulation compared to the same concentration of free antibodies in solution. This enhancement is unique to f-bSWNT compared to other artificial substrates with high surface area and similar chemistry. We explored the origins of this enhanced activity with FRET microscopy and found the preferential formation of large antibody stimuli clusters (5 to 6 μm) on the surface of functionalized versus untreated nanotubes. This highlights the important aspect that antigen clusters can be formed on f-bSWNT, impacting the potency of the T cell stimulus. Clustering of T cell antigens on artificial substrates impacts the avidity of interaction with cells facilitating rapid stimulation dynamics and an overall greater magnitude of response. These findings support the use of chemically treated nanotube bundles as an efficient substrate for the presentation of antigens and point to their potential in clinical applications involving artificial antigen-presentation for ex vivo T cell expansion in adoptive immunotherapy.

Introduction Since their discovery by Ijima,1 single-walled carbon nanotubes (SWNTs) have been of great interest in a variety of scientific disciplines. The unique properties of SWNTs endows them with a number of applications ranging from electronics2,3 to imaging,4-6 biosensing,7,8 and drug delivery.9,10 The use of SWNTs in biomedical applications is especially attractive given their tunable material properties, facility for different biochemical functionalization, and capacity for protein adsorption.11-14 Our group previously reported that T cell antigens (anti-CD3ε antibodies, which are a nonspecific stimulus for T cell activation15) induce effective T cell stimulation in vitro when adsorbed onto singlewalled carbon nanotube bundles treated with 3 M HNO3/ LiBH4.16 This treatment produced functionalized bundled SWNT (f-bSWNT) with hydroxyl end groups and an enhanced capacity to adsorb proteins. This treatment was also shown to result in nanotube bundles that were nontoxic in T cell cultures at *Corresponding author. E-mail: [email protected]. (1) Iijima, S.; Ichihashi, T. Nature 1993, 363, 603–605. (2) Kang, S. J.; et al Nat. Nanotechnol. 2007, 2, 230–236. (3) Odom, T. W.; Huang, J. L.; Lieber, C. M. Ann. N.Y. Acad. Sci. 2002, 960, 203–215. (4) Al Faraj, A.; Cieslar, K.; Lacroix, G.; Gaillard, S.; Canet-Soulas, E.; Cremillieux, Y. Nano Lett. 2009, 9, 1023–1027. (5) Sitharaman, B. et al. Chemical Communications; Royal Society of Chemistry: Cambridge, England, 2005; pp 3915-3917. (6) Hartman, K. B.; et al Nano Lett. 2008, 8, 415–419. (7) Chen, Z.; et al Nat. Biotechnol. 2008, 26, 1285–1292. (8) Barone, P. W.; Baik, S.; Heller, D. A.; Strano, M. S. Nat. Mater. 2005, 4, 86–92. (9) Kam, N. W.; O’Connell, M.; Wisdom, J. A.; Dai, H. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 11600–5. (10) Cai, D.; et al Nat. Methods 2005, 2, 449–454. (11) Shi Kam, N. W.; Jessop, T. C.; Wender, P. A.; Dai, H. J. Am. Chem. Soc. 2004, 126, 6850–6851. (12) Chen, R. J.; Zhang, Y.; Wang, D.; Dai, H. J. Am. Chem. Soc. 2001, 123, 3838–3839. (13) Prato, M.; Kostarelos, K.; Bianco, A. Acc. Chem. Res. 2008, 41, 60–68. (14) Pantarotto, D.; et al Chem. Biol. 2003, 10, 961–966. (15) Kruisbeek, M. A.; Shevach, E.; Thornton, M. A. Current Protocols in Immunology; John Wiley & Sons: New York, 2004. (16) Fadel, T. R.; et al Nano Lett. 2008, 8, 2070–2076.

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concentrations up to 150 μg/mL.16 We observed an enhanced cellular response unique to f-bSWNT as compared to other highsurface-area materials such as activated carbon, polystyrene nanoparticles, and antibodies alone.16 We investigated the role of surface area and chemical environment offered by these materials and their effects on protein adsorption and T cell activation and found that f-bSWNT was a superior substrate for cellular stimulation. We hypothesized that the unique enhancement in T cell response facilitated by f-bSWNT adsorbed with anti-CD3 (RCD3) may involve the clustering of T cell antigens in defect regions created by chemical treatment. Here, we investigate this phenomenon and show that it enhances not only the magnitude of the T cell response but also the kinetics by which it occurs. A ubiquitous biological feature in cell-cell interactions is the clustering17a of cell surface receptor proteins. This phenomenon enables enhanced recognition through high avidity interactions and may also amplify the responses of otherwise weak interactions.17b-21 For example, in immunity, antigen-presenting cells are known to cluster antigenic ligands upon interfacing with T cells to enhance the response of T cells and increase their sensitivity for detection.18,22-25 This biophysical phenomenon has been studied extensively with T cells and because its biological implications have been termed a supramolecular activation (17) (a) Kentner, S.; et al. Science Signaling 2009, 2, ra15. (b) Perelson, A.; DeLisi, C.; Wiegel, F. Cell Surface Dynamics: Concepts and Models; Marcel Dekker: New York, 1984; pp 223-276. (18) Fahmy, T. M.; Bieler, J. G.; Edidin, M.; Schneck, J. P. Immunity 2001, 14, 135–143. (19) Fooksman, D. R.; Gronvall, G. K.; Tang, Q.; Edidin, M. J. Immunol. 2006, 176, 6673–6680. (20) Boniface, J. J.; et al Immunity 1998, 9, 459–466. (21) Minguet, S.; et al Immunity 2007, 26, 43–54. (22) Vyas, J. M.; Van der Veen, A. G.; Ploegh, H. L. Nat.e Rev. Immunol. 2008, 8, 607–618. (23) Giannoni, F.; et al J. Immunol. 2005, 174, 3204–3211. (24) Gonzalez, P. A.; Carreno, L. J.; Figueroa, C. A.; Kalergis, A. M. Cytokine Growth Factor Rev. 2007, 18, 19–31. (25) Vogt, A. B.; Spindeldreher, S.; Kropshofer, H. Immunol. Rev. 2002, 189, 136–151.

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cluster (SMAC) or immunological synapse.26,27 Spatial reorganization of membrane proteins on these cells facilitates a higher sensitivity to antigens because clustered receptors require less ligand for recognition17b,18 and indeed lead to dynamic regulation of the immune response.25,26,28,29 Inducing clusters of stimuli on a variety of biomaterial substrates was successfully used to create artificial antigenic surfaces for the enhancement of T cell activation.23,30,31 A feature of these substrates is their capability to present a high density of antigens in a modular fashion for efficient stimulation.16,31 Thus, different ligands can be added to the surface of the substrates and their concentration on the surface can be tuned, allowing for consistent, reproducible results and potentially eliminating the variability with cell-based antigenpresenting systems. Such artificial antigen-presenting platforms represent an off-the-shelf technology that can be easily standardized and potentially sterilized and is stable for immunotherapeutic applications requiring the expansion of T cells against a variety of antigens.32,33

Materials and Methods Materials. RCD3 and RCD28 (with and without biotin), mouse IL-2, and IFN-γ ELISA reagents were all obtained from BD Biosciences-Pharmingen (San Jose, CA). Goat anti-mouse IgG-2a tagged with Alexa Fluor 555 and Alexa Fluor 647 were obtained from Molecular Probes, Inc. (Eugene, OR). Activated carbon (AC), fluoresceinyl-aminomethyldithiolano-phalloidin (phalloidin-FITC), and 40 ,6-diamidino-2-phenylindole (DAPI) was obtained from Sigma-Aldrich. Paraformaledyde and Vectashield were obtained, respectively, from USB Corporation (Cleveland, OH) and from Vector Laboratories, Inc. (Burlingame, CA). Polybead hydroxylate microspheres (0.20 μm) were obtained from Polysciences, Inc. Cell titer-blue assay reagents were obtained from Promega, Inc. (Madison, WI). Trypan blue was purchased from Fisher Science, Inc. (Waltham, MA). Streptavidin gold nanoparticles (10 nm, 0.05% Au) were purchased from NANOCS, Inc. (New York, NY). Nitric acid was obtained from J. T. Baker (Phillipsburg, NJ). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Preparation of 3 M HNO3/LiBH4 SWNT Bundles (f-bSWNT). Bundled SWNTs (bSWNT) were initially synthesized from cobalt-incorporated MCM-41 (Co-MCM-41) in order to obtain bulk low-defect-density nanotubes.34a The carbon nanotubes were purified using a mild, four-step treatment procedure consisting of NaOH reflux, HCl wash, and oxidation by 4 mol % molecular oxygen.34b,35 Samples were washed twice in sodium hydroxide for 1 h, followed by subsequent filtration using a PTFE filter. A second cleaning step was carried out using hydrochloric acid (HCl) at 60 °C overnight. To remove amorphous carbon particulates, samples were heated in 4 mol % oxygen stream in a quartz reactor at 300 °C followed by repeated HCl washing, filtration, and drying steps. Functionalized bSWNT (f-bSWNT) was produced by stirring samples in 3 M HNO3 at 70 °C for 1 h, followed by filtration using a 5 μm pore size PTFE membrane and drying at 45 °C overnight. (26) Grakoui, A.; et al. Science 1999, 285, 221–227. (27) Monks, C. R.; et al. Nature 1998, 395, 82–86. (28) Dustin, M. L. Semin. Immunol. 2005, 17, 400–410. (29) Lee, K. H.; et al. Science 2003, 302, 1218–1222. (30) Zappasodi, R.; Di Nicola, M.; Carlo-Stella, C. Haematologica 2008, 93, 1523–1534. (31) Steenblock, E. R.; Fahmy, T. M. Mol. Ther. 2008, 16, 765–772. (32) Oelke, M.; Krueger, C.; Giuntoli, R. L.; Schneck, J. P. Trends Mol. Med. 2005, 11, 412–420. (33) Kim, J. V.; Latouche, J. B.; Riviere, I.; Sadelain, M. Nat. Biotechnol. 2004, 22, 403–410. (34) (a) Nan, L.; Xiaoming, W.; Fang, R.; Haller, G.; Pfefferle, L. J. Phys. Chem. C 2009, 113, 10070–10078. (b) Chen, Y.; Wei, L.; Wang, B.; Lim, S.; Ciuparu, D.; Zheng, M.; Chen, J.; Zoican, C.; Yang, Y.; Haller, G. L.; Pfefferle, L. D. ACS Nano 2007, 1, 327–336. (35) Song, J.; Lim, S. J. Phys. Chem. C 2008, 112, 12442–12454.

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Samples were then reduced by the addition of LiBH4 solution in THF (200 mg SWNT þ 125 mL THF þ 400 mg LiBH4) and sonication for 1.5 h.

High-Resolution Scanning Electron Microscopy (SEM). SEM images were obtained on a Hitachi SU-70 high-resolution microscope with an accelerating voltage of 10 kV. Twenty-five micrograms per milliliter of bSWNT solution dissolved in phosphate buffered saline (PBS) was washed and resuspended in deionized water. The washing step was repeated three times using a microcentrifuge at 13 200 rpm for 8 min. Twenty microliters of the washed solution was applied to 0.25 cm2 of carbon tape mounted on an aluminum stub. The stub was placed at -80 °C for 2 h and then lyophilized overnight using a Labonco Free Zone1 lyophilizer (0.057 mbar vacuum, -44 °C collector). Raman Spectroscopy. Raman spectroscopy measurements were performed on a Jasco Raman spectrometer. The instrument is equipped with an Olympus confocal microscope. An excitation wavelength of 785 nm was used. For data analysis, each peak from the defect region (D, typically at 1300 cm-1) or the Raman breathing mode (RBM, typically at [100-350] cm-1) was reduced to a common baseline (red dashed line) and then normalized to the graphitic band (G, typically at 1580 cm-1). Tubes of specific diameters can be sorted as given by dx = 248/ωRBM(x) where x stands for peak 1, 2, or 3 and ωRBM(x) is the Raman shift value at the selected peak.36 Adsorption of Antibodies on Substrates. RCD3 and RCD28 were added at an equal ratio and serially diluted in RPMI media in a 96-well plate to a volume of 100 μL. One hundred microliters of previously sterilized f-bSWNT was then added at a final concentration of 25 μg/mL to each well. Other materials were added as a separate control group at a volume of 100 μL per well and at a calculated concentration approximately matching the f-bSWNT surface area (as estimated previously using nitrogen physisorption;16 see Supporting Information Figure S0: A[ f-bSWNT] = 1560 m2/g, A[bSWNT] = 845 m2/g, and A[AC] = 1762 m2/g). The particle count for PS-OH is 5.68  1012 particles/mL at a density of 25 mg/mL and a particle diameter of 200 nm. The surface was estimated to be A[PS-OH] = 29 m2/g.16 No washing of unbound proteins was involved for the substrate groups. For soluble control, only media were added at a volume of 100 μL/well. The 96-well plates were then placed on a shaker for 1 h at room temperature. Adsorption of a Nanogold-Labeled Antibody. Streptavidin nanogold particles were added in molar excess (1:6 molar ratio) to biotinylated antibodies. Once mixed, the nanogold-labeled antibody was added to a solution of carbon nanotubes in PBS (final nanotube concentration 25 μg/mL) to yield a final concentration of 5 μg/mL. After mixing at room temperature for 1 h, the substrate was washed twice, resuspended in deionized water, and mounted for SEM analysis. Activation of Primary T Cells. Primary splenocytes were isolated from the spleen of a C57BL/6 mouse. The spleen was mashed using a sterile syringe plunger on a cell strainer to isolate cells from fatty tissue. Cells were washed and resuspended in ACK buffer to lyse red blood cells. Splenocytes were washed a second time in sterile PBS and resuspended in RPMI T cell media supplemented with 10% fetal bovine serum (FBS). One hundred microliters of splenocytes was then added to a final concentration of 2  105 cells/mL to each well within the plates containing antibody-adsorbed substrates or soluble antibody. Culture conditions were 5% CO2 at 37 °C. Cell Proliferation. The cell titer-blue (CTB) cell metabolic activity assay is a fluorescent method for monitoring cellular activity on the basis of the ability of living cells to convert a redox dye (resazurin) into a fluorescent end product (resorufin). CTB was used to measure cell viability37 after cell exposure to the biomaterials used in this study. Groups were cultured in triplicate (36) Jorio, A.; et al. Phys. Rev. Lett. 2001, 86, 1118–1121. (37) Nakagawa, T.; et al. Nature 2005, 434, 652–658.

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Fadel et al. at 5% CO2 and 37 °C. Stimulatory (RCD3) and costimulatory (RCD28) components were used in a molar ratio of 1:1. All cultures in this study were performed in 96-well flat-bottom plates in a final volume of 200 μL RPMI media supplemented with 10% FBS. For the f-bSWNT group, previously sterilized 3 M HNO3/ LiBH4 SWNT bundles were added to a final concentration of 25 μg/mL. Splenocytes isolated from a wild type C57BL/6 mouse were RBC-lysed and added to all groups at a concentration of 1.3  105 cells/mL. Aliquots at the indicated time points were removed for cytokine analysis. In some experiments, another aliquot was removed for the trypan blue exclusion test, imaging of proliferation, and viability testing by the CTB assay. Assays with CTB and f-bSWNT took into account the relative adsorption of CTB by f-bSWNT, which was corrected for by using cellfree f-bSWNT in CTB. CTB measurements were made 2 h after incubation of the substrate with the respective groups. Fluorescence was recorded at an excitation of 560 nm and an emission of 590 nm using a Spectra Max M5 spectrometer from Molecular Devices (Sunnyvale, CA). mIL-2/mIFN-γ ELISAs. A sandwich ELISA protocol was used to measure mIL-2 and mIFN-γ cytokine production. One hundred microliters of capture antibody was added per well in a flat bottom 96-well ELISA plate (high protein binding). The ELISA plate was then incubated overnight at 4 °C. Assay diluent (AD), 10% FBS in PBS, was added at 200 μL per well and incubated for 2 h at room temperature as a blocking step. Plates were next washed three times in 0.05% Tween-20 buffer. Standard (either mIL-2 or mIFN-γ) was added and serially diluted in AD to reach its linear range. Samples from the T cell activation plates were centrifuged at 3000 rpm for 8 min to pellet cells and substrates and diluted 5-fold in AD, and then aliquots were added to the ELISA plate. Plates were incubated at room temperature for 2 h and then washed three times in 0.05% Tween-20 buffer followed by the addition of 100 μL of secondary antibody reagent and incubation for 1 h at room temperature. After a final wash, substrate solution was added to each well and incubated while being protected from light. When the colorimetric reaction was visible, ELISA was quenched by the addition of a stop solution (2 N H2SO4), and absorbance was then measured in a plate reader at 450 nm. Confocal Microscopy. Cells were imaged using a Leica SP5 microscope and processed using Leica LAS AF software. Threedimensional rendering was performed using Volocity software from Improvision, Inc. Before analysis, cells were incubated for 72 h with an equal ratio of RCD3 and RCD28, which were previously adsorbed on carbon-nanotube-bound coverslips. Cell culture coverslips were then rinsed with PBS, immersed in 1% AD for 5 min, and fixed in 4% paraformaledyde. For actin staining, phalloidin-FITC was added to the culture slides in a ratio of 1:200 in PBS. For nuclear staining, DAPI was added to a final concentration of 545 nM. One drop of Vectashield was then added to a microscope slide before mounting the coverslip. FRET Sample Preparation. Imaging was performed on several samples per group. Donor-only, acceptor-only, and donor-acceptor samples were prepared by incubating the substrates with goat anti-mouse IgG-2a tagged with Alexa Fluor 555 (donor), goat anti-mouse IgG-2a tagged with Alexa Fluor 647 (acceptor), or a mixture of both (donor-acceptor). All samples were allowed to tumble at room temperature in a rotator protected from light. The samples were then microcentrifuged at 13 200 rpm for 10 min. The supernatant was removed, and the samples were resuspended in an equal volume of PBS. A fourth group of f-bSWNT without antibody was included as a control. FRET-Acceptor Photobleaching (FRET-AP). All images were collected on a Leica SP5 microscope and processed using the Leica LAS AF software. HeNe-I (543 nm) and HeNe-II (633 nm) lasers were used for Alexa Fluor 555 and Alexa Fluor 647, respectively. The exposure times were kept the same within each Langmuir 2010, 26(8), 5645–5654

Article series of images and chosen such that all pixel intensities were within the linear range. A 63 oil immersion lens was used. The background was eliminated by subtracting the f-bSWNT control from both channels. Each sample was imaged with HeNeI, HeNe-II, and brightfield to ensure the absence of cross-talk. The acceptor photobleaching module within Leica LAS AF was used to measure the FRET efficiency. The 633 nm laser line was used to bleach Alexa Fluor 647 selectively. A minimum of 10 regions of interest4 was selected at random on the f-bSWNT FRET sample for acceptor photobleaching. The bleaching procedure was set at 60% laser intensity and at 30 frames. These settings were kept uniform across all samples. FRET efficiency (FRET Eff) was calculated as follows: FRET Eff = Dpost - Dpre/ Dpost for all Dpost > Dpre, where Dpre and Dpost are the donor fluorescence intensities before and after bleaching, respectively. FRET Efficiency Analysis. FRET efficiency images obtained from the FRET-AP studies were replotted in MATLAB using a custom-coded script. The eight-bit grayscale intensity values of the FRET efficiency images were rescaled so that FRET efficiency values were mapped to a single color scale. FRET efficiency maps were then assessed on the average cluster size per sample and by estimating an average distance separating the two FRET fluorescent probes involved. For cluster size determination, we assigned any FRET efficiency >50% to a cluster structure. On the basis of images collected from FRET efficiency analysis, we calculated an average cluster size for each platform (if FRET was present). Next, we estimated an average FRET efficiency score for each cluster and deduced the FRET radius separating the donor and acceptor using the F€ orster equation: R = (R06/FRET Eff - R06)1/6, where R0 is the F€ orster radius for the selected FRET pair (Alexa Fluor 555 and Alexa Fluor 647) estimated to be 52 A˚ (5.2 nm). The F€ orster radius is defined as the distance at which energy transfer is 50% efficient (i.e., 50% of excited donors are deactivated by FRET). Fitting of Data. Model fits were performed using Prism version 4.0b from Graphpad Software (2004). For mIl-2 release from T cells, a sigmoidal response was used: Y = B þ (A - B)/ (1 þ 10((log(EC50) - X)H)), where X is the logarithm of concentration, Y is the response, and A and B are the initial and final responses, respectively, H is the hill slope, and EC50 is the halfmaximal stimulation value. For protein adsorption, a one-phase exponential association fit was used: y = y0 þ (ymax - y0) 3 (1-e-k 3 x), where y0 was set to zero. Student t Test. Statistical analysis was performed using Prism version 4.0b from Graphpad Software (2004). All Student t tests were unpaired with a two-tailed p value and a 95% confidence interval.

Results and Discussion Functionalized Bundled Single-Walled Carbon Nanotubes (f-bSWNTs) Are Effective Antigen-Presenting Substrates. We previously demonstrated16 the enhanced activation of T cells using antibody-adsorbed f-bSWNT. Chemical treatment of bundled SWNT (bSWNT) had two effects: first, it increased the overall surface area of the bundle via the introduction of defects38,39 and increased the capacity for protein adsorption;16 second, this treatment introduced a more electronegative40,41 microenvironment favorable for T cell interactions.42 Chemical treatment involved a reflux of SWNT in nitric acid to introduce defect sites and carbonyl or carboxylic groups38 and then a reduction in lithium borohydride that reduced carboxylic groups to hydroxyls16 (Supporting Information Figure S1). However, increased surface area was not a determinant of T cell response because materials with equivalent (38) Hu, H.; Zhao, B.; Itkis, M. E.; Haddon, R. C. J. Phys. Chem. B 2003, 107, 13838–13842. (39) Cinke, M.; et al. Chem. Phys. Lett. 2002, 365, 69–74. (40) Esumi, K.; et al. Carbon 1996, 34, 279–281. (41) Hu, H.; et al. J. Phys. Chem. B 2005, 109, 11520–11524. (42) Andersen, P. S.; et al. J. Biol. Chem. 2001, 276, 49125–49132.

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Figure 1. Schematic of T cell activation studies with antibody adsorbed onto treated SWNT bundles (f-bSWNT). The first step in these studies is equimolar adsorption of stimulatory (RCD3) and costimulatory (RCD28) antibodies43 onto f-bSWNT. In a second step, splenocytes isolated from a C57BL/6 mouse are incubated with f-bSWNT þ Ab. Subsequently, T cell activation is assessed at multiple time points, from 24 to 96 h, following the time of incubation. Two time points (t1 and t2) are schematically presented in the presence of f-bSWNT þ Ab (blue) depicting the proliferation of T cells (white).

areas were not as efficient at activating T cells as f-bSWNT.16 Similarly, surface chemistry was not a determining factor because hydroxylated or carboxylated substrates (polystyrene beads) added to the T cell culture did not enhance activation to the same extent as did f-bSWNT.16 Importantly, these properties of presentation and the subsequent magnitude of T cell responses were unique to functionalized SWNT bundles when compared to other materials.16 To determine the kinetics of the T cell responses on the surface of the nanotubes, we incubated mouse splenocytes with T cell antigens and co-stimulatory ligands (RCD3 and RCD28) adsorbed on these substrates. In the scheme shown in Figure 1, SWNT bundles were incubated with an equal ratio of RCD3 and RCD28 monoclonal antibodies.43 The concentration of nanotube bundles used was 25 μg/mL. At this concentration, there were no cytotoxic effects on T cells in culture with or without nanotube functionalization (Supporting Information Figure S2A,B) and in agreement with previous findings.16,44,45 Stimulation of primary T cells isolated from the spleen of a wild-type mouse by these antibody-adsorbed substrates was quantified by cytokine secretion of the classical activation determinants such as interleukin-2 (mIL-2) and, interferon gamma (mIFN-γ)15,46, and by endpoint proliferation at each 24 h time point (up to 96 h). Enhanced Stimulation Kinetics with Antibodies Adsorbed to f-bSWNT. We compared the kinetics of T cell activation by soluble Ab versus Ab adsorbed to functionalized SWNT bundles over the course of 4 days by measuring the secretion of mIL-2 from cultured T cells. The results are shown in Figure 2A. The effects of f-bSWNT can be seen as early as 24 h after incubation. Here, mIL-2 secretion was observed for doses of antibody (Ab) as low as 0.05 μg/mL for f-bSWNT as compared to soluble antibody, which showed little or no response at that time point. An early plateau for mIL-2 secretion in f-bSWNT þ Ab was also observed at intermediate concentration ranges (∼0.3 μg/mL). In the absence of antibody, secreted mIL-2 was not detected from T cells incubated with f-bSWNT or alone at 24 h as well as other (43) Lee, K. H.; et al. Science 2003, 302, 1218–1222. (44) Bottini, M.; et al. Toxicol. Lett. 2006, 160, 121–126. (45) Zhang, J.; Ji, X.; Liu, C.; Shen, S.; Wang, S.; Sun, J. Frontiers of Materials Science in China. 2008, 2, 228–232. (46) Murphy, K. M.; Travers, P.; Walport, M.; Janeway, C. Janeway’s Immunobiology, 7th ed.; Garland Science: New York, 2008.

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time points. Soluble antibodies first induced a response in T cells at 48 h with a maximal secretion of 80 pg/mL, but this response was lower than for f-bSWNT þ Ab (maximal secretion of 240 pg/ mL). Similar observations were noted for longer incubation times. For all time points, we observed a greater enhancement in T cell stimulation via f-bSWNT þ Ab compared to soluble forms at the same concentration. Thus, treated SWNT bundles enhanced the dynamics of the T cell activation process. To verify that this enhancement in kinetic stimulation was not restricted to mIL-2 secretion, we measured the levels of mIFN-γ production. IFN-γ is a type II interferon secreted by T cells after activation within the first 24 h. Time-dependent enhancement in the secretion of mIFN-γ followed a trend similar to that for mIL-2 (Figure 2B, top panel) with increased activation at the 72 h time point and a maximal value reaching ∼2000 pg/mL at 72 h with the f-bSWNT group compared to ∼1000 pg/mL at 96 h for the soluble antibody group. Cells without stimulation did not produce a measurable amount of mIFN-γ. The proliferation of T cells (Figure 2B, center panel) paralleled cytokine secretion with a peak population of T cells at 72 h in both f-bSWNT þ Ab and soluble Ab and a significant ∼30% increase in the T cell population observed with the f-bSWNT þ Ab group as compared to that for soluble Ab. Imaging the interaction of the T cells with f-bSWNT þ Ab compared to soluble Ab showed increased clustering around f-bSWNT and the formation of large lymphoblast aggregates, which is observed primarily at the 72 h time point (Figure 2B, bottom panel; dead cells appear as dark blue using the trypan blue exclusion test). Consistent with this activity, confocal images of cells (Figure 2C) indicate the presence of a lymphoblast cluster forming around f-bSWNT þ Ab at the 72 h incubation time point (Supporting Information Figure S5B,C). In culture and in the absence of stimulation, T cells are known to commit to a program of apoptosis, and this is observed as early as 24 h (Supporting Information Figure S5A).15 Comparing T Cell Stimulation via Antibody Adsorbed to f-bSWNT versus Other Equivalent Substrates. We compared the dynamics of T cell stimulation by antibodies adsorbed on four selected substrates: f-bSWNT; bSWNT; activated carbon (AC), a porous carbon material with high surface area; and hydroxylated polystyrene bead substrates with functional groups similar to Langmuir 2010, 26(8), 5645–5654

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Figure 2. Enhancement in the kinetics of T cell activation with antibody adsorbed on f-bSWNT. (A) Progression of mIL-2 secretion from T cells stimulated with treated bSWNT with and without Ab, soluble Ab, and cells alone over the course of 4 days. The final concentration of treated bSWNT in the cell culture is 25 μg/mL. RCD3 and RCD28 are presented in an equal ratio (x axis). (B) Measurements of mIFN-γ (top panel) and T cell proliferation (center panel). The total antibody concentration in soluble and treated SWNT groups is 1.25 μg/mL. Bottom panel: brightfield images of cellular proliferation at the 72 h time point. The scale bar represents 100 μm. Dead cells appear as dark-blue post-trypan blue staining. (**) in the mIFN-γ graph represents a p value of 0.0017, (**) and (*) in the T cell proliferation graph represents p values of 0.0024 and 0.0324, respectively. The analysis was performed using an unpaired Student t test. (C) Confocal imaging of cellular proliferation after 72 h on f-bSWNT þ Ab, Sol Ab, and f-bSWNT groups. DAPI nuclear stain is blue, and phalloidin FITC stain is green. The white scale bar represents 50 μm. Langmuir 2010, 26(8), 5645–5654

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Figure 3. Enhancement of the kinetics of T cell activation is a unique feature of functionalized-bundled SWNTs (f-bSWNT). (A) Comparative study of mIL-2 secretion over the course of 4 days from T cells after incubation with f-bSWNT, bSWNT, PS-OH, and AC substrates. RCD3 and RCD28 are presented in equal amounts (x axis). The final concentration of f-bSWNT in the cell culture is 25 μg/mL. Data are fit to a sigmoidal dose-response (variable slope) equation (Materials and Methods). (B) Maximal mIL-2 secreted as a function of time for the various substrates. (C) High-resolution SEM images of substrates with similar overall surface area and chemical functionality: f-bSWNT (top panel), bSWNT (second panel from the top), AC (third panel from the top), and PS-OH (hydroxylated polystyrene beads, bottom panel). The inset within each panel represents a higher magnification of the selected region of interest.

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those of the functionalized nanotubes (PS-OH, 200 nm). We investigated the effect of these four substrates on T cell stimulation after antibody adsorption onto their surfaces. Results are shown in Figure 3A. Here, the quantity of substrates used was selected to match the surface area on all materials as previously calculated16 (Materials and Methods), thus normalizing for the density of antibody presentation. At 24 h, the T cell response induced by antibody-adsorbed bSWNT is analogous to the response induced by f-bSWNT, whereas activated carbon and polystyrene beads resulted in a lower T cell response as indicated by the amount of mIL-2 released. This comparable effect is clearly observed after 48 h where mIL-2 release from T cells in f-bSWNT þ Ab reached 240 pg/mL versus 145 pg/mL for bSWNT þ Ab, 160 pg/mL for AC þ Ab, and 118 pg/mL for PS-OH þ Ab. In the absence of Ab, secreted mIL-2 was not detected in all groups. At the two subsequent time points, it appeared that, whereas the T cell response measured with f-bSWNT þ Ab continued to increase to a peak at 72 h (as previously described in Figure 2A) the T cell responses mediated by other platforms peaked earlier at 48 h with higher Ab concentrations. These differences in maximal stimulation are summarized in Figure 3B, which shows that the enhanced release of mIL-2 in f-bSWNT þ Ab for the first 72 h is a unique feature of functionalized SWNT bundles and is not dependent on surface area. High-resolution SEM images of all four groups are shown in Figure 3C. High magnification of these images (shown in the Figure 3C insets) points toward differences in the surface morphology of all three carbon substrates: activated carbon shows a porous nanostructure whereas images of the SWNT groups corroborate their bundled tubular nanostructure. In addition, the effect of SWNT chemical treatment can also be seen qualitatively in these images. f-bSWNT shows a rougher structure after treatment, with slightly different curvature and topography (as illustrated in Supporting Information Figure S6). Enhanced response kinetics with f-bSWNT could be due to better avidity of the interaction with T cells. Increased protein avidity is promoted via surfaces that are convoluted or “rough” on the molecular length scales. Previous studies point to the importance of rough surfaces in contributing to steric stabilization of proteins in solution47 by lowering the average interaction energy of proteins with the adsorbing surface. Rough structures can also modulate the 2D effective affinity or avidity of bound proteins.42,48,49 In addition to surface roughness, changes in the chemical microenvironment of the ligand-binding surface through oxidation (hence rendering it more electronegative40,41) could enhance the 2D functional affinity of bound proteins.42 To determine the actual chemical differences between the carbon nanotube substrates, we studied the Raman spectra of bSWNT and f-bSWNT. The Raman spectra at an excitation wavelength of 785 nm indicate important differences in the bSWNT structure before and after chemical treatment (Figure 4A). First, the chemical treatment produced defects on the surface of bSWNT as indicated by an increase in the defect to graphitic (D/G) ratio from 0.18 to 0.27. The D/G ratio is used to compare the relative change in defect density triggered by chemical treatment50 on bSWNT. Second, additional changes in tube diameters can be observed as the lower-wavelength Raman breathing mode peaks appear to shift to higher tube diameters (tube diameters at (47) Roth, C. M.; Neal, B. L.; Lenhoff, A. M. Biophys. J. 1996, 70, 977–987. (48) Bell, G. I.; Dembo, M.; Bongrand, P. Biophys. J. 1984, 45, 1051–1064. (49) Williams, T. E.; Nagarajan, S.; Selvaraj, P.; Zhu, C. J. Biol. Chem. 2001, 276, 13283–13288. (50) Cinke, M.; Li, J.; Chen, B.; Cassell, A.; Delzeit, L. Chem. Phys. Lett. 2002, 365, 69–74.

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peak 1 = 1.7 nm, at peak 2 = 1 nm, and at peak 3 = 0.9 nm) as a result of SWNT treatment. The induction of defects during oxidation at the tube ends and the selective destruction of smaller-diameter tubes at the oxidation and reduction steps both support the observed increase in tube diameter.50,51 We investigated the effect of chemical treatment of bSWNT on antibody adsorption. Figure 4B shows an enhanced adsorption of a typical antibody isotype (IgG) due to chemical treatment.16 Using a one-phase exponential association model fit, we estimated the maximum antibody adsorption for f-bSWNT at 265 ( 32 μg/mg SWNT and for bSWNT at 99 ( 6 μg/mg SWNT (Supporting Information Table S2). Chemical treatment of SWNT bundles thus increased antibody adsorption more than 2-fold. This increased antibody adsorption was qualitatively verified using immunogold-labeled antibodies on the functionalized and untreated SWNT surfaces using high-resolution SEM imaging (Figure 4C). Nanogold labeled antibodies can be seen as a white dot of about 10 nm at higher magnification. Consistent with the adsorption isotherms in Figure 4B, we observed a higher density of proteins in f-bSWNT compared to bSWNT. Interestingly, we noted a nonuniform distribution of proteins on the SWNT surface (Supporting Information Figure S7). In addition, analysis of SEM images at high magnification reveals significant differences between bSWNT and f-bSWNT in terms of surface variation at the length scale of proteins (Supporting Information Figure S6 and Table S3). Ultimately, these surface roughness changes could play an important role in increasing protein adsorption for antigen presentation by triggering the steric stabilization of a high density of T cell stimulus on the surface of f-bSWNT. To examine how this affects the morphology of antigen-presentation, we quantitatively assessed the geometry of bound proteins using fluorescence resonance energy transfer (FRET).53-56 Measuring Clustering of Antibodies on Treated bSWNT. To probe the possibility that f-bSWNT may give rise to regions of enhanced protein adsorption and hence aggregate formation, we chose to investigate the plausibility of cluster formation during the immobilization of antibodies on the f-bSWNT surface. FRET microscopy allows for examination of features at the nanometer length scale,55 important for determination of protein arrangements and ascertaining the formation of high density protein aggregates. Here, pairs of antibodies with acceptor and donor fluorescence probes were adsorbed in a similar manner as previously described.16 Next, we performed acceptor photobleaching (AP)57 as a mode of evaluation for FRET efficiency across the FRET pairs (Materials and Methods). Representative FRET images of acceptor photobleaching are shown in Figure 5A. The acceptor channel showed significant bleaching of selected regions of interest (ROIs) as indicated by the decrease in fluorescence intensity before and after bleaching (Supporting Information Figure S8B,C). The donor fluorescence intensity in f-bSWNT, however, increased considerably because of acceptor bleaching as compared to other platforms (not shown). A FRET efficiency map was derived on the basis of the changes in donor fluorescence intensity and the selection of the FRET pair and is shown in Figure 5B. The f-bSWNT panel (top) shows the presence of multiple clusters of high FRET efficiency (>0.6) (51) Niyogi, S.; et al. Acc. Chem. Res. 2002, 35, 1105–1113. (52) Wigginton, J. M.; et al. J. Immunol. 2002, 169, 4467–4474. (53) F€orster, T. Discuss. Faraday Soc. 1959. (54) Kruisbeek, A. Curr. Protocols Immunol. 2003, 18.10.1–18.10.18. (55) Kenworthy, A. K. Methods 2001, 24, 289–296. (56) Lippincott-Schwartz, J.; Snapp, E.; Kenworthy, A. Nat. Rev. Mol. Cell Biol. 2001, 2, 444–456. (57) Gastard, M. Confocal Appl. Notes 2006, 5, 1–10.

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Figure 4. Chemical treatment of a bundled SWNT enhances protein adsorption. (A) Raman spectra of untreated SWNT (top) and treated SWNT (bottom) measured at an excitation wavelength of 785 nm. Numbers enclosed in squares are reference intensities as indicated by the red dashed line. Numbers above peaks are normalized as described in the Materials and Methods section. G is the reference graphitic peak, D is the defect peak, and (1, 2, 3) are families of tubes at a specific diameter (Materials and Methods). (B) Comparison of IgG adsorption isotherms for bSWNT and f-bSWNT (y=x line represents an ideal 100% adsorption case). Data was fit using a one-phase exponential association model. (C) High resolution SEM images of bSWNT and f-bSWNT after adsorption of an equal amount of nanogold labeled antibodies. Right panels represent a higher magnification of the selected region of interest. The yellow arrow points toward a single nanogold-labeled antibody molecule.

ranging from 5 to 6 μm in diameter. The average distance between FRET pairs was deduced from the F€orster equation based on the given F€orster value for the FRET pair (Supporting Information 5652 DOI: 10.1021/la902068z

Figure S8A). This distance was estimated to be approximately 4.5 nm. This feature was unique to f-bSWNT and was found to be absent in the activated carbon and untreated bSWNT groups Langmuir 2010, 26(8), 5645–5654

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Figure 5. FRET acceptor photobleaching reveals increased clustering of antibodies on functionalized bundled SWNT (f-bSWNT). (A) Confocal images of both acceptor and donor labeled antibodies during the process of acceptor photobleaching. The first two columns show an image of antibody-Alexa Fluor 647 (acceptor) on f-bSWNT before and after bleaching at 633 nm, showing regions of interest (ROI)4 selected on the surface of the SWNT platform. At least 10 regions were selected for bleaching. Right column: imaging of antibody-Alexa Fluor 555 (donor) on f-bSWNT before and after bleaching at 633 nm. (B) Representative confocal and FRET efficiency images for f-bSWNT as compared to AC and bSWNT. The legend represents the degree of FRET efficiency. (C) Schematic depicting antibody stimulus enhancement in T cell activation mediated by f-bSWNT. (1) f-bSWNT is rich in defects and nanotube entanglements, facilitating preferential stimulus adsorption into local aggregated (clustered) regions. This step proceeds with a high kAds and a very low kDes. (2) Longer T cell dwell times on the support lead to enhancements in the kinetics and magnitude of T cell activation. Cells flock to the surface at a rate of k(Cell On) (a weak function of antibody cluster size) and diffuse away at a rate of k(Cell Off) (a strong function of cluster size). The diameter of the cluster(s) was measured to be 5 μm on average.

based on several runs. FRET-AP measurements clearly point to the presence of large antibody clusters on the cellular scale. This feature of enhanced clustering is a possible mechanism for the observed enhancement of the magnitude and kinetics of T cell activation observed with treated SWNTs. Proposed Mechanism for Enhanced T Cell Activation by Antibody-Adsorbed f-bSWNT. A possible mechanism Langmuir 2010, 26(8), 5645–5654

describing the observed enhancement in T cell response as induced by f-bSWNT is depicted in Figure 5C. First, the chemical treatment of SWNT increases the overall surface area, facilitating the immobilization of a high density of antibodies for T cell activation. The surface properties of f-bSWNT play a role in increasing the adsorption of proteins (kAds). We determined that protein desorption (kDes) after adsorption is very low as corroborated by T cell DOI: 10.1021/la902068z

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activation results with washed f-bSWNT þ Ab (Supporting Information Figure S4). Additionally, the amount of protein leached after 4 days was less than 20% (Supporting Information Figure S3). Second, antibodies are not uniformly distributed on f-bSWNT but are localized in high-density regions. This localized cluster formation (of average diameter s) on the surface would significantly decrease the off rate of T cells from the surface and facilitate enhanced activation because T cell activation is dependent on the dwell time in the vicinity of the antigen.58,59 In addition, physical confinement of the stimulus on the f-bSWNT surface could also play a role in increasing the effective half-life of this receptor ligand complex.60 Consequently, large antibody clusters on the surface of f-bSWNT impact the rate at which cells leave the surface more than the rate at which they flock to the surface. This longer dwell time k(Cell Off) is needed for efficient T cell antigen receptor engagement and interaction, and leads to a greater magnitude of T cell activation.

tend to aggregate.61,62 Clustering of protein stimuli in those regions would therefore increase the dwell time of T cell substrate interactions, leading to quicker and greater activation. f-bSWNT as such offers a versatile new platform for artificial antigen presentation that recapitulates biological clustering of stimuli on natural antigen-presenting cells. f-bSWNTs are thus useful for applications requiring the development of robust artificial antigen-presenting platforms for ex vivo stimulation and expansion of T cells. Acknowledgment. This work was supported by the Yale Institute of Nanoscience and Quantum Engineering (YINQE) and partially by an NSF Career Award (0747577) to T.M.F. We acknowledge Jonathan Boyd and Brad Calloway from Leica and Tom Ardito, Michael Rooks, Nan Li, Codruta Zoican, Salim Derrouiche, Fang Fang, Erin Steenblock, Cicely Williams, Jason Criscione, Ragy Ragheb, Eric Stern, Udo Schwartz, and Paul Van Tassel.

Conclusions Bundled SWNT is an effective means of T cell antigen presentation, enhancing both the magnitude and kinetics of T cell stimulation. We demonstrated previously that whereas a high surface area is an attractive feature of this substrate, allowing for greater stimulus adsorption, it was not a sufficient factor for the observed enhancement in T cell activation. Here, we show that f-bSWNT with adsorbed stimuli also impacts the kinetics of the activation process. Faster responses are observed with f-bSWNT compared to similar high surface area substrates or substrates with similar chemical microenvironments. These observations hint at the idea that stimulus presentation and configuration on f-bSWNT, in addition to density, could be responsible for the observed enhancement in the kinetics and magnitude of T cell activation. Using FRET microscopy, we investigated the morphology of stimuli on the surface of bSWNT and f-bSWNT and found evidence for the formation of stimulus clusters characterized by a large diameter (5 to 6 μm) and a close interantibody distance (4.5 nm) exclusively on the surface of f-bSWNT. In contrast to untreated bSWNT, which is relatively free of defects, f-bSWNTs are rich in defects with entangled tubes of larger diameter facilitating local regions where protein stimuli would (58) Kalergis, A. M.; et al. Nat. Immunology 2001, 2, 229–234. (59) Van den Berg, H. A.; Rand, D. A. Immunol. Rev. 2007, 216, 81–92. (60) Qi, S.; Krogsgaard, M.; Davis, M. M.; Chakraborty, A. K. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 4416–4421.

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Supporting Information Available: Brunauer-EmmettTeller plot of the carbon groups derived from nitrogen physisorption. FT-IR spectra of bSWNT before and after chemical treatment. Viability studies on splenocytes in the presence of f-bSWNT and bSWNT. Leaching results of IgG adsorbed to f-bSWNT, bSWNT, AC, and PS-OH. Low desorption of protein from the f-bSWNT surface. Brightfield images of splenocytes during 4 days of incubation stained with trypan blue and confocal images of splenocytes and f-bSWNT þ Ab. Semiquantitative analysis of the surface roughness of SWNT bundles using MATLAB. High-resolution SEM of SWNT bundles adsorbing nanogold-labeled antibodies. FRET-AP with the Alexa Fluor 555/Alexa Fluor 647 pair. Parametric values from the sigmoidal dose-response (variable slope) fitting of the results of T cell activation. Parametric values derived from the one-phase exponential fit of IgG adsorption onto f-bSWNT and bSWNT as compared to previously derived values from BSA adsorption. Results from the statistical analysis performed on SEM images of bSWNT and f-bSWNT using MATLAB. Protein adsorption isotherm estimation for bSWNT and f-bSWNT. This material is available free of charge via the Internet at http://pubs.acs.org. (61) Huang, W.; et al. Nano Lett. 2002, 2, 311–314. (62) Burch, H.; et al. Nanotechnology 2008, 19, 384001.

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