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Nanoparticle Targeting at Cells Jesus M. de la Fuente,*,† Catherine C. Berry, Mathis O. Riehle, and Adam S. G. Curtis Centre for Cell Engineering, Institute of Biomedical and Life Sciences, UniVersity of Glasgow, Glasgow G12 8QQ, UK ReceiVed NoVember 10, 2005. In Final Form: January 19, 2006 Gold nanoparticles have been used for analytical and biomedical purposes for many years. In fact, the labeling of targeting molecules with nanoparticles has revolutionized the visualization of cellular or tissue components by electron microscopy. We report in this study the derivatization of tiopronin-protected nanoparticles with ethylenediamine and poly(ethylene glycol) bis(3-aminopropyl) terminated and their functionalization with the GRGDSP peptide sequence by a straightforward and economical methodology. The particles were subsequently tested in vitro with a human fibroblast cell line to determine the biocompatibility, and the cell-particle interactions, using fluorescence and scanning electron microscopies. The results indicate that tiopronin gold nanoparticles aggregate due to culture medium proteins, whereas the tiopronin gold nanoparticles derivatized with ethylenediamine induce endocytosis, and the same nanoparticles derivatized with poly(ethylene glycol) derivative promote particle-cell adhesion.
Introduction Nanometer-sized metallic and semiconductor particles have been a focus of intensive research over the last 20 years.1 Gold nanoparticles (AuNPs) are the most stable and popular of these nanoparticles.2 These nanocrystals play important roles in different branches of science, such as chemical catalysis, catalysis for the growth of nanowires, nanomedicines, nanoelectronics, etc.2 While numerous routes exist for the production of colloidal gold nanoparticles, the description by Brust et al.3 and its variations4,5 are the most popular synthetic schemes in the field. These alkanethiolate nanoparticles have received considerable attention due to their advantages of stability, suspendability in different solvents, and facile characterization by standard analytical techniques. As nanoparticles and biomolecules are of a similar length scale, it seems logical that the combination of biomacromolecules to nanomaterials can provide interesting tools for mimicking the biomolecules present in cellular systems, probing the mechanisms of biological principles, as well as developing chemical means for the handling and manipulating biological components.6 The use of gold nanoparticles in biological applications was first highlighted in the 1970s with the immunogold staining procedures.7 Since then, the labeling of target molecules with gold nanoparticles has revolutionized the visualization of cellular and/ or tissue components by electron microscopy. During the last 10 years, several groups have prepared gold nanoparticles linked with sugars,8,9 proteins, and DNA.10 These nanoparticles are * Corresponding author. E-mail:
[email protected]. † Current address: Grupo de Carbohidratos, Instituto de Investigaciones Quı´micas, CSIC, Isla de La Cartuja, Ame´rico Vespucio 49, 41092 Sevilla, Spain. (1) Murray, C. B.; Kagan, C. R.; Bawendi, M. G. Annu. ReV. Mater. Sci. 2000, 30, 545-610. (2) Daniel, M.-C.; Astruc, D. Chem. ReV. 2004, 104, 293-346. (3) Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.; Whyman, R. J. Chem. Soc., Chem. Commun. 1994, 801-802. (4) Collier, C. P.; Saykally, R. J.; Shiang, J. J.; Henrichs, S. E.; Heath, J. R. Science 1997, 277, 1978-1981. (5) Cliffel, D. E.; Zamborini, F. P.; Gross, S. M.; Murray, R. W. Langmuir 2000, 16, 9699-9702. (6) Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042-6108. (7) Polak, J. M.; Varndell, I. M. Immunolabeling for electron microscopy; Elsevier Pub.: Amsterdam, 1984. (8) Barrientos, A. G.; de la Fuente, J. M.; Rojas, T. C.; Fernandez, A.; Penades, S. Chem.-Eur. J. 2003, 9, 1909-1921.
being used for assembling new materials, developing bioassays and as multivalent systems for interaction studies.10,11 However, there are several aspects that should be considered for these hybrid nanomaterials: The stability of gold nanoparticles in physiological conditions is required for applications in cell biology. Although several groups have prepared water-soluble gold nanoparticles,12 it appears that the resultant particles are not particularly amenable to a broad range of applications; their synthetic scope is limited, or they aggregate in physiological conditions. Although inorganic and metal nanoparticles can be prepared from various materials by several methods, the coupling and functionalization with biological components has only been carried out with a limited number of chemical methods.13-16 To apply gold colloids in newly developed biological assay systems, simple and easy means of anchoring different ligand biomolecules onto particle surfaces are required. A key issue in evaluating the utility of these materials is assessing their potential toxicity, either due to their inherent chemical composition or as a consequence of their nanoscale properties.17,18 One can modify these nanoparticles to better suit biological systems via modification of their surface layer for enhanced aqueous solubility, biocompatibility, and biorecognition. In this study, gold nanoparticles were synthesized using different alkanethiolate capping agents. These NPs have been derivatized with two different diamine functionalized linkers, ethylenediamine (EDA) and poly(ethylene glycol) bis(3-aminopropyl) terminated (PEG), plus the GRGDSP peptide sequence. The influence on human fibroblasts hTERT-BJ1 in vitro was (9) de la Fuente, J. M.; Barrientos, A. G.; Rojas, T. C.; Rojo, J.; Can˜ada, A.; Fernandez, A.; Penades, S. Angew. Chem., Int. Ed. 2001, 40, 2257-2261. (10) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128-4158. (11) de la Fuente, J. M.; Penades, S. Glycoconjugate J. 2004, 21, 149-163. (12) Ackerson, C. J.; Jadzinsky, P. D.; Kornberg, R. D. J. Am. Chem. Soc. 2005, 127, 6550-6551. (13) Ghosh, S. S.; Kao, P. M.; McCue, A. W.; Chapelle, H. L. Bioconjugate Chem. 1990, 1, 71-76. (14) Patolsky, F.; Ranjit, K. T.; Lichtenstein, A.; Willner, I. Chem. Commun. 2000, 1025-1026. (15) Shenton, W.; Davies, S. A.; Mann, S. AdV. Mater. 1999, 11, 449-452. (16) Connolly, S.; Fitzmaurice, D. AdV. Mater. 1999, 11, 1202-1205. (17) Rojo, J.; Dı´az, V.; de la Fuente, J. M.; Segura, I.; Barrientos, A. G.; Riese, H. H.; Bernad, A.; Penade´s, S. ChemBioChem 2004, 5, 291-297. (18) Derfus, A. M.; Chan, W. C. W.; Bhatia, S. N. Nano Lett. 2004, 4, 11-18.
10.1021/la053029v CCC: $33.50 © 2006 American Chemical Society Published on Web 03/07/2006
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assessed in terms of stability, cytotoxicity, scanning electron microscopy (SEM), and fluorescent observation of cytoskeletal elements F-actin, tubulin, and vinculin as well as clathrin. The results show that, although some of the prepared nanoparticles precipitate in physiological conditions, only nanoparticles protected with the non-natural amino acid tiopronin are stable. Nanoparticles derivatized with EDA-RGD appeared to be largely internalized by the fibroblasts. On the other hand, the PEG-RGD nanoparticles are dispersed and located on the surface of the fibroblasts and, thus, perhaps may serve as a nontoxic and improved way of drug targeting. Material and Methods Materials. All of the chemicals were of reagent grade and were used without further purification. Hydrogen tetrachloroaureate(III) trihydrate (99.9+%) (product no.: 484385), (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, 2-[N-morpholino] ethanesulfonic acid (99.5%) (product no.: M3671), L-cysteine (>97%) (product no.: W326305), poly(ethylene glycol) bis(3aminopropyl) terminated (product no.: 452572), and 11-mercaptoundecanoic acid (95%) (product no.: 450561) were purchased from Sigma-Aldrich; N-(2-mercaptopropionyl)glycine (>98%) (product no.: 63794), N-hydroxysuccinimide (>97%) (product no.: 56480), and ethylendiamine (75-80%) (product no.: 03783) were from Fluka; NaBH4 (98%) (product no.: 5859) was from Lancaster; and GRGDSP peptide sequence (>99.0%) (product no.: 44-0-24) was from American Peptides Co. House distilled water was further purified using a Milli-Q reagent grade water system (Millipore). Buffers were prepared according to standard laboratory procedure. Other chemicals were reagent grade and used as received. General Procedures. 1H NMR spectra were acquired on a Bruker DRX-400 spectrometer, and chemical shifts are given in ppm (δ) relative to the residual signal of the solvent used. UV spectra were carried out with a UV/vis Shimadzu UV-3101PC spectrometer in milliQ water. Infrared spectra of solid NPs samples pressed into a KBr plate were recorded from 4000 to 750 cm-1 with a JASCO FT/IR 410 model spectrometer. For TEM examinations, a single drop (10 µL) of the aqueous solution (0.1 mg mL-1) of the gold nanoparticles was placed onto a copper grid coated with a carbon film. The grid was left to dry in air for several hours at room temperature. TEM analysis was carried out in a JEM-1200EX electron microscope working at 80 kV. Synthesis of Gold Nanoparticles. Au@tiopronin. Hydrogen tetrachloroaureate(III) trihydrate (0.15 g; 0.4 mmol; 1 equiv) and N-(2-mercaptopropionyl)glycine (tiopronin) (0.19 g; 1.2 mmol; 3 equiv) were codissolved in 20 mL of 6:1 methanol/acetic acid, resulting in a ruby red solution. Sodium borohydrate (0.30 g; 8.0 mmol; 20 equiv) in 7.5 mL of H2O was subsequently added via rapid stirring. The resultant black suspension was stirred for an additional 30 min after cooling, with the solvent removed under vacuum at 40 °C. The crude sample was completely insoluble in methanol but reasonably soluble in water. It was purified by dialysis, in which the pH of 130 mg of crude product dissolved in 20 mL of water (NANOpure) was adjusted to 1 by dropwise addition of concentrated hydrochloric acid. This solution was loaded into 15 cm segments of seamless cellulose ester dialysis membrane (Sigma, MWCO ) 10.000), placed in 4 L beakers of water, and stirred slowly, recharging with freshwater ca. every 10 h over the course of 72 h. The dark blue Au@tiopronin solutions were collected from the dialysis tubes and were lyophilized. The product materials were found to be spectroscopically clean and produced a yield of 96 mg. 1H NMR (400 MHz, D O): δ ) 4.40-3.75 (m), 3.70 (bs), 2.202 1.30 (m). UV/vis (H2O): υ ) 450, 560 nm (surface plasmon band). IR (KBr): υ ) 3433, 2925, 2852, 1722, 1644, 1531, 1384, 1199, 1014, 794 cm-1. Au@cysteine. A solution of L-cysteine (0.012 M; 9 mg; 0.0715 mmol; 5.5 equiv) in H2O (6 mL) was added to a solution of hydrogen
Langmuir, Vol. 22, No. 7, 2006 3287 tetrachloroaureate(III) trihydrate (0.025 M; 5 mg; 0.013 mmol; 1 equiv) in water (0.5 mL). An aqueous solution of NaBH4 (1 M; 11 mg; 0.286 mmol; 22 equiv) was added in small portions with rapid stirring. The black suspension formed was stirred for an additional hour and purified by precipitation out of MeOH and lyophilized. The product materials were found to be spectroscopically clean and produced a yield of 1.5 mg. UV/vis (H2O): υ ) 540 nm (surface plasmon band). IR (KBr): υ ) 3436, 2923, 2360, 1635, 1384, 1074, 941 cm-1. Au@MUA. A solution of 11-mercaptoundecanoic acid (0.012 M; 16 mg; 0.0715 mmol; 5.5 equiv) in MeOH (6 mL) was added to a solution of hydrogen tetrachloroaureate(III) trihydrate (0.025 M; 5 mg; 0.013 mmol; 1 equiv) in water (0.5 mL). An aqueous solution of NaBH4 (1 M; 11 mg; 0.286 mmol; 22 equiv) was added in small portions with rapid stirring. The black suspension formed was stirred for an additional hour and purified by precipitation out of MeOH and lyophilized. The product materials were found to be spectroscopically clean and produced a yield of 2 mg. UV/vis (H2O): υ ) 380, 410 nm (surface plasmon band). IR (KBr): υ ) 3432, 2917, 2846, 1560, 1411, 719 cm-1. Derivatization of Gold Nanoparticles. Au@tiopronin-EDA. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (10 mg; 0.05 mmol) and N-hydroxysuccinimide (NHS) (15 mg; 0.125 mmol) were added to 4 mL of the above Au@tiopronin solution (10 mg) in 2-[N-morpholino]ethanesulfonic acid (MES) (50 mM, pH 6.5). The reaction was permitted 30 min. Subsequently, ethylenediamine (0.012 mL; 0.17 mmol) was added and the mixture was stirred a further 24 h. This solution was loaded into 6 cm segments of dialysis membrane, placed in 4 L beakers of water, and stirred slowly, recharging with freshwater ca. every 10 h over the course of 24 h. The dark blue Au@tiopronin-EDA solutions were collected from the dialysis tubes and were lyophilized producing a yield of 5.5 mg. UV/vis (H2O): υ ) 450, 560 nm (surface plasmon band). IR (KBr): υ ) 3396, 2923, 1649, 1539, 1439, 1391, 1246, 1192, 1111, 1038, 1016, 643, 593, 537 cm-1. Au@tiopronin-PEG. (N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (20 mg; 0.1 mmol) and Nhydroxysuccinimide (NHS) (30 mg; 0.25 mmol) were added to 4 mL of the above Au@tiopronin solution (20 mg) in 2-[Nmorpholino]ethanesulfonic acid (MES) (50 mM, pH 6.5). The reaction was permitted 30 min. Subsequently, poly(ethylene glycol) bis(3-aminopropyl) terminated (554 mg) was added and the mixture was stirred a further 24 h. This solution was loaded into 6 cm segments of dialysis membrane, placed in 4 L beakers of water, and stirred slowly, recharging with freshwater ca. every 10 h over the course of 24 h. The dark blue Au@tiopronin-PEG solutions were collected from the dialysis tubes and were lyophilized producing a yield of 99 mg. UV/vis (H2O): υ ) 450, 560 nm (surface plasmon band). IR (KBr): υ ) 3433, 2922, 2881, 1637, 1465, 1379, 1344, 1279, 1240, 1115, 962, 843 cm-1. Functionalization of Gold Nanoparticles with RGD Peptide. Au@tiopronin-RGD. (N-(3-Dimethylaminopropyl)-N′-ethyl-carbodiimide hydrochloride (EDC) (2 mg; 0.01 mmol) and Nhydroxysuccinimide (NHS) (3 mg; 0.025 mmol) were added to 4 mL of Au@tiopronin (2 mg) in 2-[N-morpholino]ethanesulfonic acid (MES) (50 mM, pH 6.5). The reaction was permitted 30 min. Subsequently, GRGDSP peptide (44 µg; 0.08 mmol) was added and the mixture was stirred a further 24 h. This solution was loaded into 6 cm segments of dialysis membrane, and treated as previously. The dark blue Au@tiopronin-RGD solutions were collected from the dialysis tubes and were lyophilized producing a yield of 1.6 mg. UV/vis (H2O): υ ) 450, 560 nm (surface plasmon band). Au@tiopronin-EDA-RGD. (N-(3-Dimethylaminopropyl)-N′ethyl-carbodiimide hydrochloride (EDC) (2 mg; 0.01 mmol) and N-hydroxysuccinimide (NHS) (3 mg; 0.025 mmol) were added to 3 mL of Au@tiopronin-EDA (2 mg) in 2-[N-morpholino]ethanesulfonic acid (MES) (50 mM, pH 6.5). The reaction was permitted 30 min. Subsequently, GRGDSP peptide (44 µg; 0.08 mmol) was added and the mixture was stirred a further 24 h. This solution was
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Scheme 1. Preparation of Au@tiopronin, Au@tiopronin-EDA, Au@tiopronin-PEG, Au@tiopronin-RGD, Au@tiopronin-EDA-RGD, and Au@tiopronin-PEG-RGD Nanoparticles
loaded into 6 cm segments of dialysis membrane, and treated as previously. The dark blue Au@tiopronin-EDA-RGD solutions were collected from the dialysis tubes and were lyophilized producing a yield of 1.8 mg. UV/vis (H2O): υ ) 450, 560 nm (surface plasmon band). Au@tiopronin-PEG-RGD. (N-(3-Dimethylaminopropyl)-N′ethylcarbodiimide hydrochloride (EDC) (2 mg; 0.01 mmol) and N-hydroxysuccinimide (NHS) (3 mg; 0.025 mmol) were added to 3 mL of Au@tiopronin-PEG (10 mg) in 2-[N-morpholino]ethanesulfonic acid (MES) (50 mM, pH 6.5). The reaction was permitted 30 min. Subsequently, GRGDSP peptide (44 µg; 0.08 mmol) was added and the mixture was stirred a further 24 h. This solution was loaded into 6 cm segments of dialysis membrane and treated as previously. The dark blue Au@tiopronin-PEG-RGD solutions were collected from the dialysis tubes and were lyophilized producing a yield of 2.6 mg. UV/vis (H2O): υ ) 450, 560 nm (surface plasmon band). Cell Culture. Infinity Telomerase Immortalised primary human fibroblasts (hTERT-BJ1, Clonetech Laboratories, Inc.) were seeded onto glass coverslips (13 mm diameter) at a density of 1 × 104 cells per well in 1 mL of complete medium. The medium used was 71% Dulbeccos Modified Eagles Medium (DMEM) (Sigma, UK), 17.5% Medium 199 (Sigma, UK), 9% foetal calf serum (FCS) (Life Technologies, UK), 1.6% 200 mM L-glutamine (Life Technologies, UK), and 0.9% 100 mM sodium pyruvate (Life Technologies, UK). The cells were incubated at 37 °C with a 5% CO2 atmosphere for 24 h. At this time point the cells were incubated in complete medium supplemented with 0.05 mg mL-1 gold nanoparticles for a further 24 h. All control cells were cultured in the absence of any particles. Cell Viability. To determine cell cytotoxicity/viability, the cells were plated at a density of 1 × 104 cells/well in a 96-well plate at 37 °C in 5% CO2 atmosphere. After 24 h of culture, the medium
in the wells was replaced with fresh medium containing nanoparticles in varying concentrations. After 24 h, 20 µL of MTT dye solution (5 mg/mL in phosphate buffer pH 7.4, MTT Sigma-Aldrich, UK) was added to each well. After 4 h of incubation at 37 °C and 5% CO2 for exponentially growing cells and 15 min for steady-state confluent cells, the medium was removed and formazan crystals were solubilized with 200 µL of DMSO, and the solution was vigorously mixed to dissolve the reacted dye. The absorbance of each well was read on a microplate reader (Dynatech MR7000 instruments) at 550 nm. The spectrophotometer was calibrated to zero absorbance, using culture medium without cells. The relative cell viability (%) related to control wells containing cell culture medium without nanoparticles was calculated by [A]test/[A]control × 100. Scanning Electron Microscopy (SEM) of Cell Morphology. The cells were fixed with 1.5% gluteraldehyde (Sigma-Aldrich, UK) buffered in 0.1 M sodium cacodylate (Agar, UK) (4 °C, 1 h) after 24 h incubation in the particles. The cells were then post-fixed in 1% osmium tetroxide for 1 h (Agar, UK), and 1% tannic acid (Agar, UK) was used as a mordant. Samples were dehydrated through a series of alcohol concentrations (20%, 30%, 40%, 50%, 60%, and 70%), stained in 0.5% uranyl acetate, followed by further dehydration (90%, 96%, and 100% alcohol). The final dehydration was in hexamethyl-disilazane (Sigma-Aldrich, UK), followed by air-drying. Once dry, the samples were sputter coated with gold before examination with a Hitachi S800 field emission SEM at an acceleration voltage of 10 keV. Clathrin Immunofluorescence and Cytoskeletal Observation. After 24 h culture with the nanoparticles, cells were fixed in 4% formaldehyde/PBS, with 1% sucrose at 37 °C for 15 min. The samples were then washed with PBS, and permeabilizing buffer was added at 4 °C for 5 min. The samples were then incubated at 37 °C for
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Results
Figure 1. UV-vis spectra of Au@tiopronin (a), Au@tioproninEDA (b), and Au@tiopronin-PEG (c). 5 min in 1% BSA/PBS. This was followed by the addition of antivinculin, anti-tubulin, or anti-clathrin primary antibody (1:100 in 1% BSA/PBS; monoclonal anti-human raised in mouse (IgG1), Sigma, UK) for 1 h (37 °C). Simultaneously, rhodamine conjugated phalloidin was added for the duration of this incubation (1:100 in 1% BSA/PBS. Molecular Probes, OR). The samples were then washed in 0.5% Tween 20/PBS, and a secondary, biotin conjugated antibody (1:50 in 1% BSA/PBS, monoclonal horse anti-mouse (IgG), Vector Laboratories, UK) was added for 1 h (37 °C) followed by more washing. A FITC conjugated streptavidin third layer was added (1:50 in 1% BSA/PBS, Vector Laboratories, UK) at 4 °C for 30 min, and given a final wash. Samples were then viewed by fluorescence microscopy (Vickers M17).
The nanoparticles were obtained using two different procedures: Au@cysteine and Au@MUA were prepared using a variation of the procedure of Brust et al.3 for the synthesis of monolayerprotected gold nanoclusters. The reaction was executed in an aqueous or methanolic solution rather than in toluene. The nanoparticles were obtained by adding a water solution of L-cysteine or a methanolic solution of 11-mercaptoundecanoic acid to an aqueous solution of tetrachloroauric acid (HAuCl4). By reduction of the resulting mixtures with NaBH4, a yellow to dark suspension was immediately formed. The suspensions were shaken for about an hour, and then the solvent was removed. Au@tiopronin was prepared using the procedure of Murray et al.19 The reaction was executed in a methanolic/acetic acid mixture. Codissolution of HAuCl4 and tiopronin gave a stable ruby-red solution. The addition of NaBH4 reductant provided a dark brown solution. The suspension was shaken for about 30 min, and then the solvent was removed (Scheme 1). Although the three different types of nanoparticles were watersoluble, only Au@tiopronin was stable in physiological conditions. The molecules were purified by dialysis and characterized by 1H NMR, FTIR, UV-visible, and transmission electron microscopy (TEM). TEM images showed a mean diameter of 2.8 nm for the gold core of the Au@tiopronin nanoparticles. The UV-vis absorption spectra showed an almost nondetectable surface plasmon band (SPB) as a consequence of the small size of the clusters (Figure 1a). 1H NMR and FTIR spectra confirmed the presence of tiopronin in the nanoparticles and the absence of other impurities.20 The derivatization of Au@tiopronin with ethylendiamine (EDA) and poly(ethylene glycol) bis(3-aminopropyl) terminated (PEG) was carried out using the reactivity of the free carboxyl group of the tiopronin. The reactions utilized the water-soluble carbodiimide N-[3-(dimethylamino)propyl]-N′-ethylcarbodiimide hydro-chloride (EDC) to catalyze reactions between the nanoparticle acid groups and EDA and PEG amine groups.21 We included N-hydroxysuccinimide in the reaction mixture to improve the efficiency of the carbodiimide-mediated amideforming reaction by producing hydrolysis-resistant active ester reaction intermediates.22 Excess in EDA and PEG was used to minimize their double-functionalization with the nanoparticles. The functionalizations of Au@tiopronin, Au@tiopronin-EDA, and Au@tiopronin-PEG with the GRGDSP peptide sequence were developed using the above-mentioned methodology (Scheme 1). The resultant nanoparticles were analyzed via UV/vis (Figure 1b,c) and TEM. Nonagglutination was observed23 after GRGDSP-peptide coupling. Figure 2 displays the FTIR spectra of solid samples of Au@tiopronin, Au@tiopronin-EDA, and Au@tiopronin-PEG nanoparticles. The main characteristic vibrations of the tiopronin as coating material, the NH bending and CdO stretching modes, appear around 1400-1600 cm-1. Comparing the FTIR spectra of Au@tiopronin and Au@tiopronin-EDA nanoparticles, few differences are observed due to the similarity of both coatings. (19) Templeton, A. C.; Chen, S.; Gross, S. M.; Murray, R. W. Langmuir 1999, 15, 66-76. (20) de la Fuente, J. M.; Berry, C. C. Bioconjugate Chem. 2005, 16, 11761180. (21) Detar, D. F.; Silverstein, R. J. Am. Chem. Soc. 1996, 88, 1013-1019. (22) Templeton, A. C.; Cliffel, D. E.; Murray, R. W. J. Am. Chem. Soc. 1999, 121, 7081-7089. (23) Xiao, Y.; Pavlov, V.; Levine, S.; Niazov, T.; Markovitch, G.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 4519-4522.
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Figure 2. FTIR spectra of Au@tiopronin (a), Au@tiopronin-EDA (b), and Au@tiopronin-PEG (c).
However, new bands from 1200 to 1400 cm-1 are observed in the spectra of Au@tiopronin-EDA due to NHCdO stretching modes. More evident differences can be found from the FTIR spectra of Au@tiopronin and Au@tiopronin-PEG nanoparticles. One clearly sees bands near 2881 and 1114 cm-1. These bands can correspond to the OCH2 and CH2OCH2 stretching modes, respectively, indicating that PEG was covered at the nanoparticle surface. Biocompatibility studies of the Au@tiopronin-RGD, Au@ tiopronin-EDA-RGD, and Au@tiopronin-PEG-RGD were undertaken by evaluating the cell viability of hTERT-BJ1 human fibroblasts by the MTT assay.24 This assay relies on the mitochondrial activity of fibroblasts and represents a parameter for their metabolic activity. The MTT (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetra-zolium bromide) assay is a simple nonradiactive colorimetric assay to measure cell cytotoxicity, proliferation, or viability. MTT is a yellow, water-soluble tetrazolium salt. Metabolically active cells are able to convert this dye into a water-insoluble dark-blue formazan by reductive cleavage of the tetrazolium ring. Formazan crystals, then, can be dissolved in an organic solvent such as dimethylsulfoxide (DMSO) and quantified by measuring the absorbance of the solution at 550 nm, and the resultant value is related to the number of living cells. The metabolic activity and proliferation of (24) Mosmann, T. J. J. Immunol. Methods 1993, 95, 55-63.
fibroblasts was thus measured after a 24 h culture and showed that cell proliferation was more favorable in case of EDA- and PEG-coated particles than with uncoated ones. Au@tioproninEDA-RGD revealed noncytotoxic effects to cell at concentrations as high as ∼2.5 µM, as shown in Figure 3. Au@tiopronin-RGD and Au@tiopronin-PEG-RGD affected the metabolic activity with concentrations higher than ∼2.5 µM, and the values reached 80% as compared to untreated controls. Below this concentration, cellular metabolic activity did not change much in comparison with control cells. SEM images taken at 24 h provided further information on cell morphology in response to particle incubation. It was observed from the SEM results (Figure 4) that the control cells were flat and well spread with small lamellapodia, suggesting cell motility. Au@tiopronin-RGD mainly appeared to form large aggregates (Figure 4c,d), some of which adhered to the cell surface. Only few isolated nanoparticles were found without any visible effect on the cell surface. Conversely, Au@tiopronin-EDA-RGD nanoparticles appeared to be endocytosed by the cells (Figure 4e,f), with evident bumps over the cell surface and the formation of many lamellipodia and filopodia. The Au@tiopronin-PEGRGD appeared to localize and adhere to the cell surface and lamellapodia as demonstrated in Figure 4g,h. Nonaberrations were observed on the cell surface, with the cells being flat and well spread, as control cells.
Nanoparticle Targeting at Cells
Figure 3. Cytotoxicity profiles of Au@tiopronin-RGD (a), Au@tiopronin-EDA-RGD (b), and Au@tiopronin-PEG-RGD (c) when incubated with human fibroblasts as determined by MTT assay. Relative cell viability (%) related to control wells containing cells without nanoparticles was calculated by [Abs]test/[Abs]control × 100 (n ) 3). Results are represented as mean ( standard deviation. Noncytotoxicity has been observed in many cases.
The observed F-actin and tubulin cytoskeleton was well defined and organized for control cells, and also cells treated with Au@tiopronin-RGD, Au@tiopronin-EDA-RGD, and Au@ tiopronin-PEG-RGD (Figure 5). Furthermore, vinculin was evident throughout the cytoplasm and localized to focal adhesions (Figure 6). The clathrin immunostaining appeared uniform throughout the cell body for control cells and those incubated with Au@tiopronin-RGD and Au@tiopronin-PEG-RGD nanoparticles (Figure 7); however, cells incubated with Au@tiopronin-EDA-RGD nanoparticles showed a higher concentration of staining throughout the cell body (Figure 7f).
Discussion The use of gold nanoparticles in cell biology is well documented with a plethora of different applications from immunolabeling
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to drug delivery. The development of particle stability in physiological conditions, easy functionalization with biologically relevant molecules, and biocompatibility are important issues for these applications. In this study, different alkanethiolate gold nanoparticles have been synthesized; however, only the nanoparticles protected by the non-natural aminoacid tiopronin were stable in physiological conditions. Tiopronin has previously been used to prepare CdS nanocrystals and gold nanoparticles derivatized with Tat peptide by our group.20,25 As previously stated, the free carboxyl group of the tiopronin is available for covalent coupling to various linkers or biomolecules by cross-linking to reactive amine groups. Two different diamine functionalized linkers, EDA and PEG, have been used to derivatize the nanoparticles to study the influence of the organic surrounding layer on the cell response. To target integrins on the cell surface, GRGDSP peptide sequence was coupled to the nanoparticles surfaces.26 The chemistry on the surface of the material is vital in influencing nanoparticle-cell interactions.27,28 Cell culture studies of biocompatibility showed that none of these nanoparticles had any deep effect on the cell viability or cell morphology. Only 20% of cytotoxicity was detected in the case of Au@tioproninRGD and Au@tiopronin-PEG-RGD. The cell cytoskeleton (Factin and β-tubulin) (Figure 5) was well organized in control cells as well as in those exposed to the three different nanoparticles. F-actin is shown structured around the cell periphery, with bundles in the cytoplasm and stress fibers across the cell body, while β-tubulin can be observed as individual fibers radiating out from the organizing center beside the nucleus to the cell periphery. Furthermore, clear focal adhesions were present (Figure 6). Thus, nanoparticle incubation has not interfered with cell cytoskeletal structure or adhesion. The SEM studies showed that the each nanoparticle type with different surface characteristics caused a distinct cell response (Figure 4). The Au@tiopronin-RGD indicated large aggregates, probably due to the interactions between the carboxyl groups on the surface of the nanoparticles with culture medium proteins. This aggregation will therefore disable these nanoparticles for further studies in cell biology. Cells reacted strongly to the presence of Au@tiopronin-EDARGD, with the formation of many lamellipodia and filopodia observed projecting from cell membranes. This is most likely due to particle internalization.29 Clathrin staining provided further evidence to substantiate this finding (Figure 7). The clathrin concentration is elevated throughout the cell, with particular high concentrations at the cell periphery. This suggests to us that the internalization is occurring via an endocytic mechanism.30,31 Thus, while these nanoparticles cannot be considered as systems for cell labeling, they may be useful as internalization vehicles, particularly32 considering their low cytotoxicity even at high concentrations. The last type of gold nanoparticles tested was Au@tioproninPEG-RGD. As was predictable due to the poly(ethylene glycol) (25) de la Fuente, J. M.; Fandel, M.; Berry, C. C.; Riehle, M.; Cronin, L.; Aitchison, G.; Curtis, A. S. G. ChemBioChem 2005, 6, 989-991. (26) Ruoslahti, E. Annu. ReV. Cell DeV. Biol. 1996, 12, 697-715. (27) Berry, C. C.; Wells, S.; Charles, S.; Curtis, A. S. G. Biomaterials 2003, 24, 4551-4557. (28) Gupta, A. K.; Berry, C. C.; Gupta, M.; Curtis, A. S. G. IEEE Trans. Nanobiosci. 2003, 2, 255-261. (29) McCarthy, D. A.; Holburn, C. M.; Pell, B. K.; Moore, S. R.; Kirk, A. P.; Perry, J. D. Ann. Rheum. Dis. 1986, 45, 899-910. (30) Gagescu, R.; Gruenberg, J.; Smythe, E. Traffic 2000, 1, 84-88. (31) McNiven, M. A.; Cao, H.; Piitts, K. R.; Yoon, Y. Trends Biochem. Sci. 2000, 3, 115-120. (32) Nicolazzi, C.; Garinot, M.; Mignet, N.; Scherman, D.; Bessodes, M. Curr. Med. Chem. 2003, 10, 1263-1277.
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Figure 4. SEM images of control cells (a,b), cells incubated with Au@tiopronin-RGD (c,d), Au@tiopronin-EDA-RGD (e,f), and Au@tiopronin-PEG-RGD (g,h); white arrows show nanoparticle location. Large aggregates with few isolated nanoparticles are observed in (c) and (d). Formation of many lamellipodia and filopodia projecting from the cellular membrane was observed in cells treated with Au@tiopronin-EDA-RGD, while Au@tiopronin-PEG-RGD was found to attach at the cellular membranes.
Figure 5. F-actin and β-tubulin fluorescent staining for control cells (a,b), and cells incubated with Au@tiopronin-RGD (c,d), Au@tioproninEDA-RGD (e,f), and Au@tiopronin-PEG-RGD (g,h) (scale bar: 30 µm). Well-defined F-actin and tubulin fibers were found in all of the samples.
Figure 6. F-actin and vinculin fluorescent staining for control cells (a,b), and cells incubated with Au@tiopronin-RGD (c,d), Au@tioproninEDA-RGD (e,f), and Au@tiopronin-PEG-RGD (g,h) (scale bar: 30 µm). Well-localized focal adhesions were detected in all of the samples.
surface,33 nonassociation with culture medium proteins was observed. The nanoparticles were found isolated and highly adhered to the cell surface, probably via RGD-integrin interactions, as indicated by the similarity of the staining to those obtained by Groth et al. using anti-β3 integrin antibodies.34 Cells were flat and well spread, and nonmembrane aberrations or increased clathrin staining was observed as compared to the control cells. These results suggest that this type of gold nanoparticle may be used to label different receptors on the cell membrane to be visualized using SEM. It must be highlighted
that the final concentration of the nanoparticles in the culture medium is ∼90 nM, showing the sensitivity of this targeting method. However, more studies to estimate the sensitivity of the labeling using new types of nanoparticles are currently under study. Endocytosis is the conventional mechanism used to remove “foreign” material from the cell surface. Although these materials are endocytosed, the cell will proceed to digest them in vacuoles. Endocytosis can be prevented by appropriately derivatizing the gold nanoparticles and reinforcing the adhesion of the nano-
(33) Mrksich, M.; Whitesides, G. M. Annu. ReV. Biophys. Biomol. Struct. 1996, 25, 55-78.
(34) Groth, T.; Altankov, G.; Kostadinova, A.; Krasteva, N.; Albrecht, W.; Paul, D. J. Biomed. Mater. Res. 1999, 44, 341-351.
Nanoparticle Targeting at Cells
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Figure 7. F-actin and clathrin fluorescent staining for control cells (a,b), and cells incubated with Au@tiopronin-RGD (c,d), Au@tioproninEDA-RGD (e,f), and Au@tiopronin-PEG-RGD (g,h) (scale bar: 30 µm). Uniform clathrin immunostaining was observed in cells incubated with Au@tiopronin-RGD and Au@tiopronin-PEG-RGD, while a higher concentration of staining was observed for cells incubated with Au@tiopronin-EDA-RGD.
particles with specific receptors on the cell membrane. In the present work, the influence of the different chemical surfaces of the nanoparticles on endocytosis has been reported. We have also demonstrated that the nanoparticle uptake can be avoided using an adequate functionalization, thereby increasing their adhesion to the cell surface.
Conclusion In this study, we have presented a straightforward and economical methodology to prepare gold nanoparticles functionalized with different linkers and the GRGDSP peptide sequence. These nanoparticles are stable, soluble in physiological conditions, and exhibit low toxicity. Gold nanoparticles protected with tiopronin induce either endocytosis or adhesion to the cell membrane depending on the chemical composition of the nanoparticle surface. While Au@tiopronin-EDA-RGD nanoparticles were endocytosed without any observed cell-death, Au@tiopronin-PEG-RGD showed high affinity for cell surface
receptor mainly due to ligand-receptor interactions. The internalization without toxicity offers the opportunity to introduce drugs or DNA into the cell cytoplasm using the gold nanoparticles as vehicles, whereas the specific attachment to the cell surface allows the labeling of cell receptors with metallic nanoparticles. This type of directed action using a common skeleton nanoparticle could prove useful in drug delivery and membrane receptor mapping of specific cell types. Further types of coating are currently being investigated that may confer biological activity and also target the particles to the cell membrane without immediate internalization. Acknowledgment. This work was supported by MEC and the University of Glasgow. J.M.F. thanks the MEC for a postdoctoral fellowship. We thank Dr. L. Csaderova for fruitful discussions, M. Mullin for preparing SEM samples, and Dr. L. Cronin for the use of his laboratory facilities. LA053029V
