Multifunctional Branched Gold–Carbon Nanotube Hybrid for Cell

Article pubs.acs.org/Langmuir

Multifunctional Branched Gold−Carbon Nanotube Hybrid for Cell Imaging and Drug Delivery L. Minati,*,† V. Antonini,‡ M. Dalla Serra,‡ and G. Speranza† †

FBK, Via Sommarive 18, 38123 Trento, Italy Istituto di Biofisica, Consiglio Nazionale delle Ricerche, via alla Cascata 56/C, 38123 Trento, Italy

‡

S Supporting Information *

ABSTRACT: Branched gold nanoparticles were grown on oxidized multiwalled carbon nanotubes by one-step reduction of gold chloride in water. The carbon nanotube/gold hybrids were used for the delivery of the anticancer drug doxorubicin hydrochloride into A549 lung cancer cell line. Doxorubicin (Dox) can be adsorbed in high quantity on both inner and outer surfaces of oxidized carbon nanotubes by π−π stacking interactions between doxorubicin aromatic groups and carbon nanotube (CNT) backbone. Carbon nanotube/gold hybrids display a broad absorption band in the red and near-infrared regions allowing their use for imaging applications. In vitro cellular tests showed that the nanostructures can efficiently transport and deliver doxorubicin inside the cells.

■

INTRODUCTION Recently, functional carbon nanotubes are gaining more and more importance as applications for cellular imaging and drug delivery.1−3 The possibility of CNT surface functionalization via covalent or hydrophobic interactions introduces the possibility to design CNTs for specific applications as fluorescent probes,4 MRI contrast agents,5 and delivery systems in cancer therapy.6 Although the CNT uptake mechanism is still debated due to contradictory results, CNT-based drug delivery cargos are very promising weapons for disease treatments, especially for cancer therapy. A great number of carbon nanotube−drug conjugates were recently developed to deliver specific molecules (chemotherapic agents, si-RNA) inside tumor cells.7 Dai et al. produced carbon nanotubes functionalized with Dox for pHdependent drug delivery applications.8 Aromatic molecules like doxorubicin and fluorescein were found to interact with carbon nanotube backbones trough π−π stacking interaction, allowing a high accumulation of drug molecules on the CNT surface in single-walled carbon nanotubes (with a mass ratio drug/CNT value of about 4). On the other hand, because of contradictory results concerning their cytotoxicity9−11 the use of CNTs for clinical purposes was not approved by Food and Drug Administration. Although CNTs were successfully used for in vivo imaging and therapy, the combinations of their chemical and physical properties with other elements like gold and silver can increase their efficiency as imaging contrast and therapeutic agents. For examples, Zhang et al.12 combined CNT with plasmonic nanoparticles, i.e., gold nanospheres and silver nanoparticles. This hybrid material showed a high contrast in dark field imaging of cancer cells. © 2012 American Chemical Society

Kim et al. produced gold-plated CNT that showed a 2 orders of magnitude enhancement of photoacustic and photothermal signals compared to functionalized CNT in in vivo experiments. By further functionalization with rabbit anti-mouse antibody, these golden CNT were used to map the endothelial receptors of lymph vessels in mice.13,14 Star-shaped and multibranched gold nanoparticles represent a new class of nanoparticles with exceptional optical functionalities, which largely expand the use of such engineered nanostructures in different technological fields.15 The big interest on the development of synthetic strategies for metallic nanostars has been largely driven by their special optical properties. A wide variety of wet chemistry-based synthetic methods is currently in use to prepare anisotropic nanoparticles with narrow size and shape distributions.16 The formation of anisotropic gold nanoparticles on the CNT surface represents an exciting tool for both imaging and therapeutic purposes. For example, Kim et al. recently demonstrated the application of 100 nm gold nanostars as contrast agents in the photoacoustic mapping of rat lymphatic system.17 Van de Broek et al.18 have successfully tested nanobody-conjugated branched gold nanoparticles for photothermal therapy in specific cancer cells. An interesting result was obtained also by Lu et al.19 conjugating preformed gold nanostars on thiol-functionalized CNT for photothermal therapy applications. Another important characteristic of the branched gold nanoparticles is that they are an active substrate Received: August 14, 2012 Revised: October 19, 2012 Published: October 22, 2012 15900

dx.doi.org/10.1021/la303298u | Langmuir 2012, 28, 15900−15906

Langmuir

Article

Figure 1. Synthesis scheme of multifunctional branched gold−carbon nanotube hybrid functionalized with PEG and doxorubicin. Mn 5000) by dissolving 1 mg of CNT@Au in a 1 mg/mL PEG−SH water solution under stirring for 12 h. The suspension was purified by centrifugation at 12 000 rpm and the supernatant was replaced by water. The procedure was repeated three times. 2.5. Synthesis of CNT@Au-Dox. One milligram of CNT@AuPEG was redispersed in 3.5 mL of 0.1 mg/mL doxorubicin hydrochloride water solution at pH 8 by NaOH and stirred for 24 h. The suspension was purified by centrifugation at 12 000 rpm for 10 min and the supernatant was collected and analyzed by UV−vis absorption. CNT@Au-Dox suspension was further purified by centrifugation−wash cycles until the supernatant luminescence completely disappeared. 2.6. Dox Release from CNT@Au-Dox Sample. Drug release experiments were performed at room temperature. CNT@Au-Dox (0.2 mg) were dispersed in 2.0 mL PBS (pH 7.4 and 5.0). At definite time intervals, the nanoparticle suspensions were centrifuged (12 000 rpm, 10 min) and the supernatants were separated from the pellets and analyzed. Dox concentration in the supernatant was determined spectrofluorimetrically by exciting at 488 nm and acquiring in the 500−750 nm range. 2.7. Cell Culture. The human lung carcinoma epithelial cell line A549 was cultured in RMPI-1640 medium without phenol red (sigma R7509) supplemented with 10% FBS (fetal bovine serum, Euroclone) and 4 mM glutamine and incubated at 37 °C in an atmosphere of humidified air with 5% CO2. 2.8. Cell Uptake and Drug Release Investigations of CNT@ Au-Dox. Typically, 45 000 A549 cells were seeded onto 16 mm glass coverslips in a 24-well plate and allowed to grow for 1 day at 80% confluence. Then, the medium was replaced by 500 μL of fresh culture medium supplemented with 25 mM Hepes in the presence of CNT@ Au-Dox (0.05 mg/mL). The supernatant was left for 1, 4, and 24 h. Before being analyzed by confocal microscopy (Leica 200D microscope equipped with an argon laser source), cells were washed three times with Dulbecco's phosphate buffered saline (DPBS). For CNT@ Au localization analysis, the cell membranes were stained for 10 min with WGA-AlexaFluor 488 and then analyzed by confocal microscopy (excitation 488 nm, emission 520 nm). For the drug release investigation, Dox fluorescence was acquired by setting the excitation wavelength at 488 nm and a 540−620 nm acquisition range. CNT@ Au localization was obtained by exciting at 633 nm and acquiring in the range 620−650 nm. 2.9. Characterization. UV−vis absorption measurements were performed using an UV−visible−near-infrared spectrophotometer (Cary 5000) in dual beam mode. Photoluminescence spectra were acquired using a Xe lamp as excitation source coupled to a single grating monochromator in a spectral range extending from 400 to 750 nm. For TEM analysis, the colloidal suspensions were deposited and dried onto a carbon-coated copper grid. Samples were observed using a Philips CM12 TEM operated at 120 kV and equipped with an energy-dispersive X-ray spectrometer. Particle size and ζ-potentials were determined by dynamic light scattering (DLS) in back-scattering mode at 25 °C, using a laser particle sizer (Malvern Zetasizer Nano ZS, equipped with a He−Ne laser at 633 nm, 5 mW). In this assay, particle samples were diluted with milli-Q water to a final concentration of 0.01 mg/mL. The same experiment was carried out using cell medium and 10% serum solution at 37 °C. Data were analyzed by the Malvern proprietary software. The obtained distributions are expressed as numeric-weight size distribution and compared with the results obtained by TEM analysis.

for surface enhanced raman scattering (SERS). This effect induces a strong Raman signal increase for the molecules adsorbed on the gold nanoparticle surface allowing their use as SERS tags for imaging applications.20 By combining branched gold nanoparticles optical properties with CNT high storage capacity, it is possible to obtain a multifunctional hybrid material for imaging and drug delivery applications. To the best of our knowledge, there are no reports in the literature reporting the direct synthesis of star-shaped gold nanoparticles on CNT surfaces and about applications of CNT/gold hybrids in drug delivery. In this work, nonspherical gold nanoparticles with gold nanostar-like shapes were grown on oxidized CNT (CNT@ Au). The reduction of gold was obtained at pH 10. This basic environment increases the reaction rate with respect to the neutral pH. TEM analysis shows that CNT surfaces and tips were also available for drug functionalization. CNT@Au were then functionalized with doxorubicin in water solution at basic pH and the nanostructures were incubated with A549 cell lines. The laser scanning confocal microscopy (LSCM) analysis revealed that CNT@Au can effectively penetrate the plasma membrane and deliver doxorubicin inside the cells.

■

EXPERIMENTAL SECTION

2.1. Materials. Tetrachloroauric acid (HAuCl4), doxorubicin, and all other chemicals were purchased from Sigma (Milwaukee, WI, USA) unless otherwise indicated. Doxorubicin standard solution was kept in the dark condition at 4 °C to avoid drug degradation. All reactions were performed using milli-Q water unless indicated in the text. 2.2. Oxidation of CNT (ox-CNT). Commercial multiwalled carbon nanotubes (Nanoamor Inc., mean diameter 10−20 nm, length 0.5−2 μm) were oxidized by sonication in acid mixture (HNO3/H2SO4, 1:3 v/v) for 12 h (see Minati et al.21 for details). The black suspension was then diluted in water and allowed to stand overnight for precipitation. After supernatant removal, the pellet was resuspended with deionized water and filtered with a 0.05 μm filter membrane under vacuum. The precipitate was successively washed with a 0.1 M NaOH water solution to eliminate carbon fragments or oxidation debris that could have been produced during the acid treatment and then rinsed with 0.1 M HCl water solution until neutrality of the filtrate was reached. The oxidized carbon nanotubes (ox-CNT) were found to form stable colloidal suspensions in different solvents including water, ethanol, and dimethylformamide. 2.3. Synthesis of CNT@Au. 0.01 mg of the functionalized carbon nanotubes was dispersed in 1 mL water solution at pH 10 by NaOH. After the introduction of 80 μL of 10 mM HAuCl4 water solution the mixture was incubated for 5 min under stirring at room temperature. The reducing agent hydroxylamine hydrochloride NH2OH·HCl (100 μL, 200 mM) was then added to the suspension to start the gold reduction. The solution color changed from pale yellow to purple and finally to dark blue in around 100 s (Figure S1). The suspension was purified by 10 min centrifugation at 12 000 rpm and washed three times with milli-Q water to remove the unreacted carbon nanotubes. 2.4. Synthesis of CNT@Au-PEG. CNT@Au were further functionalized with poly(ethylene glycol) methyl ether thiol (PEG, 15901

dx.doi.org/10.1021/la303298u | Langmuir 2012, 28, 15900−15906

Langmuir

Article

Figure 2. Transmission electron microscopy images of CNT@Au (A, B, C). Scanning electron microscopy image of a single CNT@Au deposited on silicon substrate (D). XPS measurements were performed using an ESCA200 instrument (Scienta-Gammadata ESCA 200, Uppsala, Sweden). Wide scans were acquired in the binding energy (BE) range 1200−0 eV using 500 eV pass energy, while the core lines were acquired at 150 eV pass energy to increase the energy resolution. Scanning electron microscopy analysis was carried out in a JSM7001F instrument equipped with a thermal field emission gun. Images were acquired with samples placed at 90° with respect to the analyzer direction.

The images emphasize the peculiar shape of these gold nanostructures, which show a high number of protrusions and edges with irregular shapes. As evidenced by the images, carbon nanotube surfaces are partially exposed and this is an indication of the incomplete gold coating of the CNT. The dimensions of the gold nanostructures are between 70 and 100 nm as revealed by TEM images. The complete reduction of the HAuCl4 precursor on the oxCNT was checked also by XPS measurement. The Au4f core line of gold shows the principal peak Au 4f7/2 placed at 84.0 eV confirming the complete reduction of the Au3+ ions to metallic gold22 (Figure S3A). CNT@Au were further functionalized with thiolated PEG by simple chemical adsorption of the SH terminal groups on the gold surface (CNT@Au-PEG). PEG has emerged as one of the most popular polymers for drug delivery because of its nonfouling properties. The PEG coating has a double function. First, it increases the colloidal stability of the nanoparticles and avoids protein absorption on the surface of the CNT. Second, its steric hindrance avoids the adsorption of the Dox molecules on the gold surface. It is known that Dox has good interaction with nanostructured gold surfaces.23 In order to minimize the presence of Dox on gold and to maximize their stacking on the CNT, neutral charged thiol-functionalized PEG was chosen to stabilize the nanoparticles. The effective functionalization of the gold surfaces by thiolated PEG was checked by XPS as reported in Figure S3B. The C1s core line of the CNT@Au shows a principal component at 284.4 eV, a typical component of the CNT sp2-C atoms of carbon nanotube, and a component at 286 eV

■

RESULTS AND DISCUSSION Figure 1 shows the schematic reaction pathway of Dox conjugated-CNT@Au synthesis. ox-CNT display a high solubility in water and biological media thanks to the presence of a high number of carboxyl groups on their surface. (see Figure S2 and Table S1). Sonication of the CNT in the sulfuric−nitric acid solution leads to a strong oxidation of carbon atoms on the surface of the CNT. In fact, the ζ-potential of the oxidized carbon nanotubes measured at room temperature was −50.4 mV. This low potential value is a consequence of the high concentration of carboxylic groups on the surface of CNT. The fraction of COOH groups in the CNT was estimated by XPS peak fitting of the C1s and O1s core lines, and it resulted in 8% of the total carbon atoms in the CNT. The mean length of the ox-CNT is ∼200 nm. The formation of the branched gold nanoparticles on the CNT was achieved by reduction of Au3+ ions in NH2OH water solution. Scanning and transmission electron microscopy images of the CNT@Au are reported in Figure 2 where the particular shape of the gold particles on the surface of the CNT is highlighted. 15902

dx.doi.org/10.1021/la303298u | Langmuir 2012, 28, 15900−15906

Langmuir

Article

associated with the C2H4−O repetitive units of PEG molecules. Dynamic light scattering analysis (DLS) (Figure S4) shows a mean dimension of about 87 nm, and this is in good agreement with the results obtained by electron microscopy analysis. The wide dispersion of the measured hydrodynamic radii is possibly related to the nonspherical shape of the CNT@Au as evidenced by TEM images. Furthermore, DLS measurements could be efficiently used to monitor the formation of nanoparticle aggregates in liquid suspension. The light scattering analysis of CNT@Au was performed also in cell culture medium at pH 7.5. The hydrodynamic sizes of the CNT@Au in this condition were similar to those measured in water at pH 7.5. After 24 h of incubation, no aggregates were revealed in cell medium by DLS analysis. The visible absorption spectroscopy of the CNT@Au samples (Figure 3) shows a small absorption band at around 530 nm and an intense absorption peak at 684 nm.

shows a reduced doxorubicin absorption peak that confirms the bonding between Dox molecules and the CNT@Au. According to UV−vis spectrum, doxorubicin uptake by CNT@Au was estimated to be around 92%. The amount of Dox adsorbed on the CNT@Au was estimated to be 0.32 mg over 1 mg of CNT@Au. This amount is remarkably higher compared to the one obtained by absorption of doxorubicin on gold nanoparticles of similar dimensions (indicatively lower than 11% w/ w).25 Similar experiments were done by using ox-CNT instead of CNT@Au. The drug/CNT mass ratio value was 1.5. With respect to CNT@Au nanoparticles, oxidized carbon nanotubes possess a higher free surface that can bind more drug molecules. In addition, Liu et al. have shown that the bond strength of the doxorubicin−CNT complex depends on the diameter of the CNT wall.8 This can be explained by a better interaction within the aromatic rings of anthracycline groups present on Dox and the CNT surfaces. The effective binding of the Dox molecules on the CNT@Au is enlightened by the UV−vis absorption spectrum reported in Figure 4B. The spectrum shows the presence of a new feature in the 450−550 nm region associated to the doxorubicin molecules adsorbed on the CNT@Au. This is also confirmed by photoluminescence spectrum reported in Figure 4C, where the fluorescence emission of the CNT@Au-Dox is reported. Weak Dox fluorescence signal was observed after the drug binding to the CNT@Au compared to the Dox solution at the same concentration. This phenomenon was reported in the literature by other groups.26 CNT@Au-Dox nanoparticle behavior was also investigated in physiological and acidic condition. We found that Dox stacked on CNT@Au is stably bound in basic buffer solutions, in physiological buffers, and in bovine serum at pH 7.4. At pH 5.0, we observed a faster release of Dox from CNT@Au (Figure 4D) due to the increased hydrophilicity and solubility of Dox at lower pH. This causes an increased protonation of NH2 groups of Dox, thereby reducing the hydrophobic interaction. The pHdependent drug release from CNT could be exploited for drug delivery applications since the environments of tumor tissues and intracellular organelles are acidic.27,28 These acidic regions can potentially facilitate the active drug release from CNT delivery vehicles. In addition, the optical properties of the gold nanoparticles can be exploited for in vitro imaging application without using fluorescent probes as recently reported by Yuan et al.29 Simulation of the absorption and scattering contributions in the extinction spectrum of branched gold nanoparticles reported in ref 30 showed that for 100 nm star-shaped gold nanoparticles the scattering contribution is predominant on the absorption in the red-NIR region. The light scattering of the branched gold nanoparticles on the surface of the CNT was exploited for CNT@Au localization in cells by laser scanning confocal microscopy. To investigate the intracellular behavior of the carbon nanotube−gold hybrid, we first studied the CNT@Au uptake into cells. A549 cancer cells were incubated with a 0.05 mg/mL CNT@Au suspension for 1 h. Cellular membranes were then stained with WGA-Alexafluor 488 fluorescent probe, and the localization of the CNT@Au was obtained in vivo by using a 633 nm excitation laser source and a 620−650 nm band-pass filter at very low laser power (0.5 mW). Because of the low scattering of biological tissue in the visible range and thanks to the possibility to analyze small slices of

Figure 3. Visible absorption spectrum of CNT@Au sample. Inset: image of the CNT@Au colloidal suspension in water.

The absorption spectrum of the CNT@Au is similar to that reported by Kumar et al.24 for star-shaped gold nanoparticles. In this paper, the simulated absorption spectrum of gold nanostars was composed by two peaks: a lower-intensity features falling in the green part of the spectrum similar to that of spherical gold nanoparticles and a higher-intensity component whose position strictly depends on the tip lengths. In a similar way the UV−vis absorption spectrum of the CNT@Au shows a small tail at 530 nm assigned to the plasmon modes localized near the central sphere, while the main absorption peak at 680 nm is attributed to the plasmon modes localized near the tips. On the basis of the position of the band at maximum absorbance, the CNT@Au extinction spectrum can be associated with that of branched gold nanoparticles with small tips. CNT@Au nanoparticles were further functionalized with doxorubicin for drug delivery applications. Since Dox binding energy to CNT is higher at basic pH where the drug is deprotonated,8 CNT@Au were incubated with doxorubicin (0.1 mg/mL in water solution) at pH 8 for 24 h under magnetic stirring. After incubation, the nanoparticles were centrifuged and the supernatant was analyzed by UV−vis spectroscopy to estimate the Dox loading content in CNT@ Au. The spectrum of the supernatant was compared with that of a doxorubicin solution at the same concentration used for the CNT@Au loading (Figure 4A). The supernatant spectrum 15903

dx.doi.org/10.1021/la303298u | Langmuir 2012, 28, 15900−15906

Langmuir

Article

Figure 4. (A) UV−vis spectra of doxorubicin solution and supernatant obtained by the separation of the nanoparticles after 24 h incubation. (B) UV−vis spectrum of CNT@Au−Dox sample. (C) Doxorubicin fluorescence spectra of CNT@Au−Dox sample and of the Dox solution before incubation with CNT@Au nanoparticles. (D) Doxorubicin cumulative release of CNT@Au-Dox nanoparticles at different pH values.

Figure 5. (A,B) Laser scanning confocal microscopy analysis of A549 cancer cell line incubated with CNT@Au−PEG sample for 1 h. (A) CNT@Au (red spots) and cell membrane (green). Z-series and orthogonal views (xz and yz) of A549 cancer cells incubated for 1 h. Optical images were collected every 0.7 μm beginning at the bottom of the coverslip and moving upward. Orthogonal images in the xz and yz planes indicate the CNT@ Au localization inside the cell. White oval labeled by N indicates the nucleus position in the cell. (B) Detail of one cell showing the scattering of nanoparticles localized inside the cell (white arrows) and on the cell membrane (blue arrows).

sample with a high z-resolution, it was possible to directly visualize the nanoparticle localization inside the cells. Confocal microscopy analysis shows that internalization of CNT@Au is already achieved after 1 h incubation. The cell

membranes stained with WGA-Alexafluor 488 appears green, while the nanoparticles are red (Figure 5). In Figure 5A, a representative image of A549 cells is reported as well as vertical 15904

dx.doi.org/10.1021/la303298u | Langmuir 2012, 28, 15900−15906

Langmuir

Article

Figure 6. Laser scanning confocal microscopy analysis of A549 cancer cell line incubated with the CNT@Au−Dox sample for 4 and 24 h. Panel: Doxorubicin fluorescence (DOX), gold scattering (Au), and merged images of A549 cancer cells incubated for 4 and 24 h. N indicates the nucleus positions. Right: xyλ analysis (extraction of the photoluminescence line) of the two channels (cyan and red) reported in the LSCM images.

presented in Figure 4D that shows that only the 12% of doxorubicin molecules are released from CNT@Au-Dox at pH 5 after 24 h.

(yz) and horizontal (xz) planes of the 3D reconstruction of a single cell obtained by the z-stack analysis. The results revealed that the nanoparticles are mainly localized inside the cell (Figure 5B white arrows) and only a small fraction of the nanoparticles are localized in proximity of the cell membrane (Figure 5B, blue arrows). Functionalized carbon nanotubes were reported to display a high uptake speed in different cell lines. Recently, it was reported that CNT can be internalized into cells either via the classic endocytic pathways or by direct penetration of cell membrane apparently without side effects.31,32 For CNT@Au nanoparticles, the endocytosis pathway seems to be most probable because of the steric hindrance of the gold nanoparticles on the surface of the CNT. To study the CNT@Au-Dox drug release kinetics, A549 cell lines were incubated for 4 and 24 h in the same conditions of the previous experiments. Doxorubicin was detected by confocal microscopy analysis using an excitation wavelength of 488 nm and a 550−620 nm detection region. Images of Dox fluorescence and gold scattering and their merged images (Figure 6) show that doxorubicin is localized near the CNT@ Au-Dox (cyan spots) inside the cell. In Figure 6, on the right panels the xyλ analysis (extraction of the photoluminescence line) of the cyan and red channels is reported showing the doxorubicin photoluminescence spectrum and the gold scattering. After 4 h incubation, the CNT@Au-Dox were internalized into the cell. Dox was also delivered into the cell by the CNT@ Au, and thereby, its fluorescence (cyan channel) appeared localized in close proximity to the CNT@Au (red channel). CNT@Au as well as doxorubicin are placed in the cytosolic area around the nucleus. However, no Dox fluorescence nor gold scattering spots were observed inside the nucleus in our experimental conditions. After 24 h incubation, Dox is localized both in the cytoplasm and in the nucleus. This demonstrates that after 24 h incubation Dox loaded on CNT was partially released and penetrated into the nucleus, while the CNT@Au nanoparticles were localized in the perinuclear region. LSCM analysis revealed that the doxorubicin was slowly released from the CNT@Au-Dox, and the drug required almost 24 h to be partially delivered in the cytosol and into the nucleus of the cell. This is in agreement with the release profile

■

CONCLUSIONS In conclusion, carbon nanotubes decorated with anisotropic gold nanoparticles were produced by simple and low-cost chemical routes. Transmission electron microscopy analysis confirms the formation of gold nanostructures around the CNT. The visible absorption spectroscopic analysis puts in evidence the presence of a broad absorption band in the nearinfrared region attributed to the plasmon resonance of the branched gold nanoparticles on the CNT. Finally, LSCM analysis on cell culture demonstrates that CNT@Au can be exploited for drug delivery vehicles and image contrast agent in in vitro cell experiments.

■

ASSOCIATED CONTENT

S Supporting Information *

Additional figures and tabular data. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +39 0461314656. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This research was performed in the framework of the PAT Nanosmart research project. We thank the Laboratory of Biomolecular Sequence and Structure Analysis for Health (LaBSSAH) for the technical support.

■

REFERENCES

(1) Liu, Z; Tabakman, S. M; Chen, Z.; Dai, H. Preparation of carbon nanotube bioconjugates for biomedical applications. Nat. Protoc. 2009, 4, 1372−1382.

15905

dx.doi.org/10.1021/la303298u | Langmuir 2012, 28, 15900−15906

Langmuir

Article

(2) Liu, Z.; Tabakman, S.; Welsher, K.; Dai, H. Carbon nanotubes in biology and medicine: in vitro and in vivo detection, imaging and drug delivery. Nano Res. 2009, 2, 85−120. (3) Kostarelos, K.; Bianco, A.; Prato, M. Promises, facts and challenges for carbon nanotubes in imaging and therapeutics. Nat. Nanotechnol. 2009, 4, 627−633. (4) Welsher, K.; Liu, Z.; Sherlock, S. P.; Robinson, J. T.; Chen, Z.; Daranciang, D.; Dai, H. A route to brightly fluorescent carbon nanotubes for near-infrared imaging in mice. Nat. Nanotechnol. 2009, 4, 773−780. (5) Chen, B.; Zhang, H.; Zhai, C.; Du, N.; Sun, C.; Xue, J.; Yang., D.; Huang, H.; Zhang, B.; Xie, Q.; Wu, Y. Carbon nanotube-based magnetic-fluorescent nanohybrids as highly efficient contrast agents for multimodal cellular imaging. J. Mater. Chem. 2010, 20, 9895−9902. (6) Liu, Z.; Chen, K.; Davis, C.; Sherlock, S.; Cao, Q.; Chen, X.; Dai, H. Drug Delivery with Carbon Nanotubes for In vivo Cancer Treatment. Cancer. Res. 2008, 68, 6652−6660. (7) Zhang, W.; Zhang, Z.; Zhang, Y. The application of carbon nanotubes in target drug delivery systems for cancer therapies. Nanoscale Res. Lett. 2011, 6, 555−577. (8) Liu, Z.; Sun, X.; Nakayama-Ratchford, N.; Dai, H. Supramolecular chemistry on water-soluble carbon nanotubes for drug loading and delivery. ACS Nano 2007, 1, 50−56. (9) Rothen-Rutishauser, B.; Brown, D. M.; Piallier-Boyles, M.; Kinloch, I. A.; Windle, A. H.; Gehr, P.; Stone, V. Relating the physicochemical characteristics and dispersion of multiwalled carbon nanotubes in different suspension media to their oxidative reactivity in vitro and inflammation in vivo. Nanotoxicology 2010, 4, 331−342. (10) Thurnherr, T.; Brandenberger, C.; Fischer, K.; Diener, L.; Manser, P.; Maeder-Althaus, X.; Kaiser, J. P.; Krug, H. F.; RothenRutishauser, B.; Wick, P. A comparison of acute and long-term effects of industrial multiwalled carbon nanotubes on human lung and immune cells in vitro. Toxicol. Lett. 2011, 200, 176−186. (11) Liu, X.; Hurt, R. H.; Kane, A. B. Biodurability of single-walled carbon nanotubes depends on surface functionalization. Carbon 2010, 48, 1961−1969. (12) Zhang, L.; Zhen, S. J.; Sang, Y.; Li, J. Y.; Wang, Y.; Zhan, L.; Peng, L.; Wang, J.; Li, Y. F.; Huang, C. Z. Controllable preparation of metal nanoparticle/carbon nanotube hybrids as efficient dark field light scattering agents for cell imaging. Chem. Commun. 2010, 46, 4303−4305. (13) Kim, J. W.; Galanzha, E. I.; Shashkov, E. V.; Moon, H. M.; Zharov, V. P. Golden carbon nanotubes as multimodal photoacoustic and photothermal high-contrast molecular agent. Nat. Nanotechnol. 2009, 1, 688−694. (14) Galanzha, E. I.; Shashkov, E. V.; Kelly, T.; Kim, J. W.; Yang, L.; Zharov, V. P. In vivo magnetic enrichment and multiplex photoacoustic detection of circulating tumour cells. Nat. Nanotechnol. 2009, 4, 855−860. (15) Hao, F.; Nehl, C. L.; Hafner, J. H.; Nordlander, P. Plasmon Resonances of a Gold Nanostar. Nano Lett. 2007, 7, 729−732. (16) Guerrero-Martínez, A.; Barbosa, S.; Pastoriza-Santos, I.; LizMarzán, L. M. Nanostars shine bright for you: Colloidal synthesis, properties and applications of branched metallic nanoparticles. Curr. Opin. Colloid Interface Sci. 2011, 16, 118−127. (17) Kim, C.; Song, H. M.; Cai, X.; Yao, J.; Wei, V; Wang, L. V. In vivo photoacoustic mapping of lymphatic systems with plasmonresonant nanostars. J. Mater. Chem. 2011, 21, 2841−2844. (18) Van de Broek, B.; Devoogdt, N.; D’Hollander, A.; Gijs, H. L.; Jans, K.; Lagae, L.; Muyldermans, S.; Maes, G.; Borghs, G. Specific cell targeting with nanobody conjugated branched gold nanoparticles for photothermal therapy. ACS Nano 2011, 54, 319−28. (19) Lu, W.; Kumar Singh, A.; Khan, S.; A Senapati, D.; Yu, H.; Ray, P. C. Gold Nano-Popcorn-Based Targeted Diagnosis, Nanotherapy Treatment, and In Situ Monitoring of Photothermal Therapy Response of Prostate Cancer Cells Using Surface-Enhanced Raman Spectroscopy. J. Am. Chem. Soc. 2010, 9, 132−239.

(20) Rodriguez-Lorenzo, L.; Krpetic, Z.; Barbosa, S.; Alvarez-Puebla, R. A.; Liz-Marzan, L. M.; Prior, I. A.; Brust, M. Intracellular Mapping with SERS-Encoded Gold Nanostars. Integr. Biol. 2011, 3, 922−926. (21) Minati, L.; Speranza, G.; Bernagozzi, I.; Torrengo, S.; Toniutti, L.; Rossi, B.; Ferrari, M.; Chiasera, A. Investigation on the Electronic and Optical Properties of Short Oxidized Multiwalled Carbon Nanotubes. J. Phys. Chem. C 2010, 114, 11068−11073. (22) Information on http://srdata.nist.gov/xps/. (23) You, J; Zhang, G; Li, C. Exceptionally high payload of doxorubicin in hollow gold nanospheres for near-infrared lighttriggered drug release. ACS Nano 2010, 4, 1033−41. (24) Kumar, P. S.; Pastoriza-Santos, I.; Rodrıguez-Gonzalez, B.; Garcıa de Abajo, F. J.; Liz-Marzan, L. M. High-yield synthesis and optical response of gold nanostars. Nanotechnology 2008, 19, 015606− 015612. (25) Chao, X; Guo, L. L.; Zhao, Y. Y.; Hua, K.; Peng, M. L.; Chen, C.; Cui, Y. L. PEG-modifed GoldMag nanoparticles (PGMNs) combined with the magnetic field for local drug delivery. J. Drug Target 2011, 19, 161−170. (26) Ali-Boucetta, H.; Al-Jamal, K. T.; McCarthy, D.; Prato, M.; Bianco, A.; Kostarelos, K. Multiwalled carbon nanotube−doxorubicin supramolecular complexes for cancer therapeutics. Chem. Commun. 2008, 4, 459−61. (27) Pillay, C. S.; Elliott, E.; Dennison, C. Endolysosomal proteolysis and its regulation. Biochem. J. 2002, 363, 417−429. (28) Griffiths, J. R Are cancer cells acidic? Br. J. Cancer 1991, 64, 425−7. (29) Yuan, L.; Wei, W.; Li, J.; Sun, Z.; Wang, H.; Zhang, X.; Chen, Y. Facile method for CLSM imaging unfunctionalized Au nanoparticles through fluorescent channels. J. Nanopart. Res. 2009, 11, 1219−1225. (30) Trigari, S.; Rindi, A.; Margheri, G.; Sottini, S.; Dellepiane, G.; Giorgetti, E. Synthesis and Modelling of Gold Nanostars with Tunable Morphology and Extinction Spectrum. J. Mater. Chem. 2011, 21, 6531−6540. (31) Kostarelos, K.; Lacerda, L.; Pastorin, G.; Wu, W.; Wieckowski, S.; Luangsivilay, J.; Godefroy, S.; Pantarotto, D.; Briand, J. P.; Muller, S.; Prato, M.; Bianco, A. Cellular uptake of functionalized carbon nanotubes is independent of functional group and cell type. Nat. Nanotechnol. 2007, 2, 108−113. (32) Lacerda, L; Russier, J; Pastorin, G; Herrero, M. A; Venturelli, E; Dumortier, H; Al-Jamal, K. T; Prato, M; Kostarelos, K; Bianco, A. Translocation mechanisms of chemically functionalised carbon nanotubes across plasma membranes. Biomaterials 2012, 33, 3334− 3343.

15906

dx.doi.org/10.1021/la303298u | Langmuir 2012, 28, 15900−15906