Bioconjugate Chem. 2005, 16, 1176−1180
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Tat Peptide as an Efficient Molecule To Translocate Gold Nanoparticles into the Cell Nucleus Jesus M. de la Fuente*,§ and Catherine C. Berry Centre for Cell Engineering, Institute of Biomedical and Life Science, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, U.K. Received February 9, 2005; Revised Manuscript Received June 10, 2005
The labeling of targeting molecules with nanoparticles has revolutionized the visualization of cellular or tissue components by electron microscopy. A particularly desirable target is the nucleus, because the genetic information is there. To date, utilizing nanoparticles for nuclear targeting has not proved very successful due to the impermeable nature of the plasma and nuclear membranes; thus nanoparticle design and synthesis is a critical factor. We report in this article the synthesis of water-soluble gold nanoparticles functionalized with a Tat protein-derived peptide sequence by a straightforward and economical methodology. The particles were subsequently tested in vitro with a human fibroblast cell line by optical and transmission electron microscopy to determine the biocompatibility of these nanoparticles and whether the functionalization with the translocation peptide allowed particles to transfer across the cell membrane and locate in the nucleus.
INTRODUCTION
During the last 20 years, there has been a great deal of interest in the self-assembly fabrication of hybrid materials from inorganic nanoparticles and biomolecules (1). Nanoparticles are similar in size range to many common biomolecules and thus appear to be natural companions in hybrid systems. At present, it is straightforward to control and modify the properties of nanostructures to better suit their integration with biological systems, for example, controlling their size and modifying their surface layer for enhanced aqueous solubility, biocompatibility, or biorecognition (2, 3). This proposal of merging biological and nonbiological application systems at the nanoscale level has been used for research in cell and molecular biology for biosensing, bioimaging, masking of immunogenic moieties to targeted drug delivery, and interaction studies (4-6). For example, gold nanoparticles were initially used in biological applications in 1971, when Faulk and Taylor devised the immunogold staining procedure. More recently, the labeling of targeting molecules with nanoparticles has revolutionized the visualization of cellular or tissue components by electron microscopy (7). In fact, metal, semiconductor, polymer, and magnetic particles have been previously used to target cells (8-14). Most of these studies exclusively concerned particle entry into the cell cytoplasm. However, the nucleus is a particularly desirable target, because the genetic information of the cell and transcription machinery resides there. The diagnoses of disease phenotype, the identification of potential drug candidates, and the treatment of disease by novel methods such as antisense therapy could be enhanced greatly by the efficient transport of materials to living cell nuclei (15). However, one of the impediments in the use of nanoparticles for this purpose is their particularly low translocation efficiency into the cell nucleus. * To whom correspondence should be addressed. Tel: +34954489568. Fax: +34-95440565. E-mail:
[email protected]. § Current address: Laboratory of Glyconanotechnology, Grupo de Carbohidratos, Instituto de Investigaciones Quimicas, CSIC, Isla de La Cartuja, Americo Vespucio 49, 41092 Sevilla, Spain.
Our research group is developing biocompatible fluorescence quantum dots and gold nanoparticles functionalized with different peptides to specifically label different receptors on the cell. Recently, we have reported the synthesis of CdS nanoparticles functionalized with a Tat protein-derived peptide sequence (16). This peptide sequence has previously been used as an efficient way of internalizing a number of marker proteins in cells (17, 18). In this study, gold nanoparticles protected with the non-amino-acid tiopronin and functionalized with a Tat protein-derived peptide were synthesized using a straightforward method. These nanoparticles are biocompatible and have been able to cross the cell and the nuclear membranes and accumulate in the cell nucleus. EXPERIMENTAL PROCEDURES
Chemicals. Hydrogen tetrachloroaureate(III) trihydrate (99.9+%), (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, and 2-[N-morpholino]ethanesulfonic acid (99.5%) were purchase from Sigma-Aldrich, N-(2-mercaptopropionyl)glycine (>98%) and N-hydroxysuccinimide (>97%) from Fluka, NaBH4 (98%) from Lancaster, and Tat protein-derived peptide sequence (GRKKRRQRRR) from Southampton Polypeptides Ltd. 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 NMR1 spectra were acquired on Bruker DRX-400 spectrometers, and chemical shifts are given in ppm (δ) relative to the residual signal 1 Abbreviations: UV/vis, ultraviolet-visible; NMR, nuclear magnetic resonance; FT-IR, Fourier transfer infrared; TEM, transmission electron microscopy; EDC, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride; NHS, N-hydroxysuccinimide; MES, 2-[N-morpholino]ethanesulfonic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMSO, dimethylsulphoxide; FCS, fetal calf serum; SP, surface plasmon; HIV, human immunodeficiency virus.
10.1021/bc050033+ CCC: $30.25 © 2005 American Chemical Society Published on Web 08/26/2005
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of the solvent used. UV spectra were carried out with a UV/vis Shimadzu UV-3101PC spectrometer in Milli-Q water. Infrared spectra of solid 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 JEM1200EX electron microscope working at 80 kV. Particle size distribution of the Au clusters was evaluated from several micrographs using an automatic image analyzer. The number of particles selected for consideration was around 400, which resulted in a stable size-distribution statistic. 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, giving a ruby red solution. NaBH4 (0.30 g, 8.0 mmol, 20 equiv) in 7.5 mL of H2O was added with rapid stirring. The black suspension that was formed was stirred for an additional 30 min after cooling, and the solvent was then removed under vacuum at 40 °C. The crude Au@tiopronin was completely insoluble in methanol but quite soluble in water. It was purified by dialysis, in which the pH of 130 mg of crude product dissolved in 20 mL of water was adjusted to 1 by dropwise addition of concentrated HCl. 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 fresh water approximately 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 (NMR, absence of signals due to unreacted thiol or disulfide byproducts). Yield: 96 mg. Average diameter: 2.8 nm. 1 H NMR (400 MHz, D2O): δ ) 4.40-3.75 (m), 3.70 (bs), 2.20-1.30 (m). UV/vis (H2O): υ ) 450, 560 nm (surface plasmon band). FT-IR (KBr): υ ) 3436, 2920, 2855, 1624, 1385, 1297, 1260, 1019, 804 cm-1. Au@Tat. (N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC; 2 mg, 0.01 mmol) and N-hydroxysuccinimide (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 proceeded for 30 min. After that, Tat peptide (112 µg, 0.000 08 mmol) was added, and the mixture was stirred for 24 h more. This solution was loaded into 6 cm segments of seamless cellulose ester dialysis membrane (Sigma, MWCO ) 10.000), placed in 4 L beakers of water, and stirred slowly, recharging with fresh water approximately every 10 h over the course of 24 h. The dark blue Au@Tat solutions were collected from the dialysis tubes and were lyophilized. Yield: 1 mg. 1H NMR (400 MHz, D2O): δ ) 4.65-4.80 (m), 4.25-3.75 (m), 3.66 (bs), 3.30-3.60 (m), 2.00-1.30 (m). UV/vis (H2O): υ ) 450, 560 nm (surface plasmon band). FT-IR (KBr): υ ) 3419, 2917, 2842, 2357, 2330, 1647, 1635, 1452, 1418, 1385, 1310, 1283, 1111, 1024, 901, 799, 772 cm-1. Biocompatibility Assay. HTERT-BJ1 cells (10 000 cells) were cultivated in a 96 well plate at 37 °C in 5% CO2. After 24 h, the medium was replaced with fresh medium containing the gold nanoparticles in varying concentrations. After cultivation again for 24 h, 20 µL of MTT dye solution (5 mg/mL in PBS) was added to each well. After 3 h of incubation at 37 °C and 5% CO2, the
Scheme 1. Preparation of Au@Tat Nanoparticles
medium was removed, the cells were washed with PBS, and formazan crystals were dissolved in 100 µL of DMSO. 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 gold nanoparticles was calculated by [A]test/[A]control × 100. Cell Culture. Infinity telomerase-immortalized primary human fibroblasts (hTERT-BJ1, Clontech Laboratories, Inc., Palo Alto, CA) were seeded onto glass coverslips (13 mm diameter) at a density of 1 × 104 cells per disk in 1 mL of complete medium. The medium used was 71% Dulbecco’s Modified Eagles Medium (DMEM) (Sigma, U.K.), 17.5% Medium 199, 9% fetal calf serum (FCS), 1.6% 200 mM L-glutamine, and 0.9% 100 mM sodium pyruvate. 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 (≈1 µM) gold nanoparticles for 1 h more. TEM. On removal of excess nanoparticles, cell samples were fixed in 1.5% gluteraldehyde (Sigma, Dorset, U.K.) in 0.1 M sodium cacodylate buffer (Agar, Essex, U.K.). Samples were subsequently post-fixed and stained with 1% osmium tetraoxide in buffer and were dehydrated through a series of alcohol concentrations (30%, 40%, 50%, 60%, 70%, 90%, 96%, 100%), prior to being resinembedded. Sections were further enhanced with lead nitrate and viewed under a Zeiss 902 electron microscope at 80 kV. RESULTS
Au@tiopronin nanoparticles were 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 (Scheme 1). The suspension was shaken for about 30 min, and then the solvent was removed. The nanoparticles were purified by dialysis and characterized by 1H NMR, FT-IR, UV/vis and TEM. Figure 1 shows the TEM image and core size distribution histogram for Au@tiopronin. A mean diameter of 2.8 nm was found for the gold core of the nanoparticles. The
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Figure 1. Transmission electron micrographs and core sizedistribution histograms (inset) of Au@tiopronin nanoparticles.
UV/vis absorption spectrum showed a nondetectable surface plasmon band (SP band) (data not shown) as consequence of the small size of the clusters. 1H NMR and FT-IR spectra confirmed the presence of tiopronin in the nanoparticles and the absence of other impurities. The functionalization of Au@tiopronin with the Tat protein-derived sequence (GRKKRRQRRR) was carried out using the reactivity of the free carboxyl group of the tiopronin (Scheme 1). The reaction utilizes the watersoluble carbodiimide EDC to catalyze reactions between the nanoparticle acid groups and any of the amine groups of the peptide (20). We included NHS in the reaction mixture to improve the efficiency of the carbodiimidemediated amide-forming reaction by producing hydrolysis-resistant active ester reaction intermediates (21). The resultant nanoparticles were analyzed by UV/vis, FT-IR, 1 H NMR, and TEM; nonagglutination was observed after Tat peptide coupling. Biocompatibility studies of the Au@tiopronin and Au@Tat nanoparticles were undertaken by evaluating the cell viability of hTERT-BJ1 human fibroblasts by the MTT assay (22) (Figure 2). The metabolic activity and proliferation of fibroblasts was thus measured after 24 h culture and revealed no appreciable cytotoxic effects to cells at concentration as high as 5 µM, and the values reached 80% compared to untreated controls at concentrations higher than 10 µM. TEM images taken after 1 h incubation with the particles (Figure 3) show that Au@tiopronin and Au@Tat were both taken up into the cell body. While Au@Tat particles were mainly located in the nucleus, only a few Au@tiopronin particles appeared to be taken into the cell through membrane invaginations and accumulate in the mitochondrial surroundings. Au@ tiopronin nanoparticles were not detected in the nucleus in any case. DISCUSSION
Based on our previous experience of sulfide-protected glyconanoparticles (3, 23), we now report the simple synthesis of water-soluble and stable gold nanoparticles functionalized with a Tat protein-derived peptide sequence as a vehicle for ferrying nanoparticles into the cell nucleus. The Au@tiopronin particles have been previously used in material science studies due their small size and their water-solubility, although their use in cell biology has not been extensively explored. These nanoparticles
Figure 2. Cytotoxicity profiles of Au@tiopronin (a) and Au@Tat (b) nanoparticles when incubated with human fibroblasts as determined by MTT assay. Percentage of viability of fibroblasts was expressed relative to control cells (n ) 5). Results are represented as mean ( standard deviations.
are useful tools for immunolabeling or as drug carriers due the absence of agglutination in physiological conditions and their easy functionalization. As previously stated, the free carboxyl group of the tiopronin is available for covalent coupling to various biomolecules by cross-linking to reactive amino groups. In this study, a peptide sequence derived from the HIV-1 Tat peptide has been coupled to the nanoparticle surface with a view to transfer gold nanoparticles into the cell nucleus. The biocompatibility of Au@tiopronin and Au@Tat particles was studied using the MTT assay (22). This assay relies on the mitochondrial activity of fibroblasts and represents a parameter for their metabolic activity. Furthermore, it is a simple nonradiactive colorimetric assay allowing simple measurements of cell cytotoxicity, proliferation, or viability. Briefly, MTT is a yellow, watersoluble tetrazolium salt that metabolically active cells are able to convert into a water-insoluble dark-blue formazan by reductive cleavage of the tetrazolium ring. Formazan crystals can be dissolved in dimethylsulfoxide and quantified. Only 20% cytotoxicity was detected in both types of nanoparticles at concentrations up to 10 µM. Below this concentration, cellular metabolic activity did not change much in comparison with control cells (Figure 2). This low cytotoxicity allows us to use concentrations of these nanoparticles up to 10 µM for our cell nuclear labeling purposes. The absence of impurities in the nanoparticles has been confirmed by 1H NMR and FT-IR. Tat specific signals were not found in 1H NMR due the low Tat/tiopronin ratio (≈1:50) (19) and the typical broadening of the 1H NMR signals for this type of gold nanoparticles (3). The TEM images (Figure 1) and the almost nondetectable
Tat Peptide To Translocate Gold Nanoparticles
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particle entry into the nucleus. Au@tiopronin particles were only detected in the cytoplasm in vacuoles or surrounding the mitochondria (Figure 3b), thus demonstrating the specificity of Au@Tat particles to be translocated and accumulate in the cell nucleus. In conclusion, we have prepared and designed stable and water-soluble gold nanoparticles functionalized with a Tat protein-derived peptide sequence with an adequate size range to pass through nucleus pores. This functionalization has allowed the nanoparticles to penetrate the cell membrane and target the nucleus. This research has many potential applications in cell imaging, but also extends to wider biomedical applications, such as gene therapy and drug delivery. ACKNOWLEDGMENT
This work was supported by EC, MEC, and University of Glasgow. J.M.F. thanks the MEC for a postdoctoral fellowship. We thank Mr. Jim Gallagher for help with TEM and Dr L. Cronin, Prof. A. S. G. Curtis, and Dr. M. Riehle for fruitful discussions. LITERATURE CITED
Figure 3. Transmission electron micrographs of human fibroblasts incubated with Au@Tat (a) and Au@tiopronin (b) nanoparticles. The black dots indicated with arrows are nanoparticles, and white arrows show nuclear membrane pores. (scale bars ) 50 nm).
surface plasmon band confirm the small size of the clusters and the absence of aggregates. In the present study, Au@Tat nanoparticles were explored to achieve nuclear targeting of human fibroblasts cells. Targeted nuclear delivery is a challenging task, because the Au@Tat nanoparticles must enter the cell, escape endosomal/lysosomal pathways, possess a nuclear localization sequence, and be small enough (
