Gold Nanoparticles Capped by a GC-Containing Peptide

Article pubs.acs.org/bc

Gold Nanoparticles Capped by a GC-Containing Peptide Functionalized with an RGD Motif for Integrin Targeting Giorgio Scarì,‡ Francesca Porta,*,† Umberto Fascio,▽ Svetlana Avvakumova,† Vladimiro Dal Santo,# Mariarosaria De Simone,⊥ Michele Saviano,∥ Marilisa Leone,§ Annarita Del Gatto,§ Carlo Pedone,⊥ and Laura Zaccaro§ †

Dipartimento di Chimica Inorganica Metallorganica Analitica “Lamberto Malatesta”, University of Milan and CNR−Istituto di Scienze e Tecnologie Molecolari, Via Venezian 21, Milan 20133, Italy ‡ Dipartimento di Biologia, CIMA Center, University of Milan, Via Golgi 19, Milan 20133, Italy § Istituto di Biostrutture e Bioimmagini-CNR, Via Mezzocannone, 16, Naples 80134, Italy ∥ Istituto di Crystallografia-CNR, Via Amendola 122/O, Bari 70126, Italy ⊥ Dipartimento delle Scienze Biologiche, Università di Napoli “Federico II”, Via Mezzocannone, 16, Naples 80134, Italy # Istituto di Scienze e Tecnologie Molecolari-CNR, Via C. Golgi 19, Milan 20133, Italy ▽ C.I.M.A. Interdepartmental Centre of Advanced Microscopy, University of Milan, Via Golgi 19, Milan 20133, Italy S Supporting Information *

ABSTRACT: Gold nanoparticles were obtained by reduction of a tetrachloroaurate aqueous solution in the presence of a RGD-(GC)2 peptide as stabilizer. As comparison, the behavior of the (GC)2 peptide has been studied. The (GC)2 and RGD(GC)2 peptides were prepared ad hoc by Fmoc synthesis. The colloidal systems have been characterized by UV−visible, TGA, ATR-FTIR, mono and bidimensional NMR techniques, confocal and transmission (TEM) microscopy, ζ-potential, and light scattering measurements. The efficient cellular uptake of Au-RGD-(GC)2 and Au-(GC)2 stabilized gold nanoparticles into U87 cells (human glioblastoma cells) were investigated by confocal microscopy and compared with the behavior of (GC)2 capped gold nanoparticles. A quantitative determination of the nanoparticles taken up has been carried out by measuring the pixel brightness of the images, a measure that highlighted the importance of the RGD termination of the peptide. Insight in the cellular uptake mechanism was investigated by TEM microscopy. Various important evidences indicated the selective uptake of RGD-(GC)2 gold nanoparticles into the nucleus.

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INTRODUCTION In the past years gold nanoparticles (AuNPs) have been extensively investigated for biomedical applications due to their unique optical properties, excellent stability, and biocompatibility, relatively easy preparation, and surface modifications.1−4 One of the main challenges in the nanotechnology field is the development of safe and effective systems for tumor targeting and selective therapy. Currently, the main limit of the anticancer agents is the nonspecific action on the healthy cells, limiting the dosages that can be applied and leading to serious side effects. In this context, AuNPs play a pivotal role in providing new types of delivery systems to permit the entry of one or multiple drugs into the primary tumor, as well as at the site of metastasis and its microenvironment by a passive (Enhanced Permeability and Retention, EPR) or active mechanism.5 AuNPs can be indeed used to deliver an anticancer drug or a radionuclide to tumor sites; also, they can be employed in tumor photothermal therapy because of their plasmon resonance properties.6 In both cases, the targeted © 2012 American Chemical Society

drug approach (active mechanism) can be achieved by the exploitation of specific molecular markers, such as receptors, that are overexpressed in cancerous tissues. The functionalization of nanoparticle surface with targeting ligands, able to recognize these markers, allows receptor-mediated endocytosis and nanoparticle internalization, thus, improving therapeutic efficacy for cancer drugs.7 The range of molecules that can be used as targeting agents is greatly expanded since many ligands (antibody, peptides, carbohydrates, etc.) can be linked to NPs to create higher affinity to tumor biomarkers. Among them, the peptides represent the best candidates8 since they have a number of distinct advantages over proteins and antibodies, such as small size, simple preparation by chemical and biological methods, possibility to be functionalized and Received: March 23, 2011 Revised: February 29, 2012 Published: February 29, 2012 340

dx.doi.org/10.1021/bc200143d | Bioconjugate Chem. 2012, 23, 340−349

Bioconjugate Chemistry

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conjugated, low toxicity, low immunogenicity, and high affinity and specificity for the receptors. Peptides can be used not only as ligands for targeting biomarkers but also as multidentate agents to stabilize gold nanoparticles. Recently, we have reported new peptide sequences displaying thiol (GC) or amine groups (GK) as capping agents.9,10 A special feature of these peptides is that they do not lead to particle aggregation by cross-linking. It is well-known that RGD containing peptides can specifically bind the integrin αvβ3 receptor, generally recognized to be a tumor and angiogenesis marker,11 and RGD-conjugated nanoparticles have been successfully employed for tumor imaging and photo thermal therapy.12,13 In the present article, the design and the synthesis of a chimeric peptide (thereafter named RGD-(GC)2), displaying motifs for both targeting and capping functions, are described. RGD-(GC)2 encompasses an RGD cyclic motif, chosen as a targeting ligand for αvβ3 integrin and a (GC)2 linear part to stabilize the AuNPs. AuNPs stabilized by RGD-(GC)2 and (GC)2 (for comparison) peptides were prepared and fully characterized by UV−visible, ATR-FTIR, and NMR spectroscopies, confocal and TEM microscopy, and ζ-potential and light scattering measurements. The cellular uptake of Au-RGD(GC)2 and Au-(GC)2 nanoparticles into U87 glioblastoma cells was investigated, and a correlation between uptake and brightness of the images collected by confocal microscopy was attempted. Moreover, TEM experiments concerning the presence of Au-RGD-(GC)2 and Au-(GC)2 nanoparticles in U87 cells were performed in order to understand if the receptor-mediated entrance could be favored. Then, the fate of nanoparticles inside the cell was observed, and surprisingly, it was discovered that the RGD-peptide behaves as a nucleus penetrating molecule.

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Figure 1. Peptide sequences of (a) RGD-(GC)2 and (b) (GC)2.

The Fmoc deprotection step was performed with 30% piperidine in DMF for 10 min. Before the final Fmoc deprotection of the Arg 1 in RGD-(GC) 2 , a selective deprotection of the Glu residue from the allyl group was carried out by treatment of the peptidyl resins with PhSiH3 (24 equiv)/Pd(PPh3)4 (0.25 equiv) in DCM twice for 30 min. The cyclization reaction was carried out on the resin with benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop) (1.5equiv)/HOBt (1.5equiv)/DIPEA (2 equiv) in DMF for 3 h and monitored by the Kaiser test. The peptides were cleaved off the resin by treatment with a mixture of trifluoroacetic acid/water/ethandithiol/triisopropylsilane (94:2.5:2.5:1 v/v/v/v) for 3 h at room temperature. The resins were filtered, and the crude peptides were precipitated with diethyl ether, dissolved in a H2O/CH3CN solution, and lyophilized. The products were purified by preparative RP-HPLC on a Shimadzu system equipped with a UV−visible detector SPD10A using a Phenomenex Jupiter Proteo column (21.2 × 250 mm; 4 μm; 90 Å) and a linear gradient of H2O (0.1% TFA)/CH3CN (0.1% TFA) from 5 to 70% of CH3CN (0.1% TFA) in 30 min at flow rate of 20 mL/min. The collected fractions containing the peptides were lyophilized. The identity and purity of the compounds were assessed by an ESI-LC-MS ThermoFinnigan instrument equipped with a diode array detector combined with an electrospray ion source and ion trap mass analyzer on a Phenomenex C18 column (250 × 2 mm; 4 μm; 90 Å) at a flow rate of 200 μL/min (Figure 1, Supporting Information). Preparation of Au-RGD-(GC)2 Nanoparticles (10.2 nm). To 60 mL of mQ water, a NaAuClB4 aqueous solution (0.06 mmol, 5.07 × 10−2 M) was added. Under vigorous stirring, the aqueous solutions of RGD-(GC)2 peptide (3 × 10−5 mmol, 3.95 × 10−3 M) and NaBH4 (0.12 mmol, 0.112 M) were added. A red sol was immediately formed and left under stirring for further 120 min. Then, the particles were purified by dialysis for 48 h, by using Spectra/Por 6 dialysis tubing, 2K MWCO. The dialyzed sol was analyzed by HPLC to verify the absence of free peptide, by using the conditions reported in Peptide Synthesis. Preparation of Au-(GC)2 Nanoparticles (13.9 nm). To 40 mL of mQ water, the aqueous solutions of NaAuCl4 (0.03 mmol, 3.41 × 10−2 M) and (GC)2 peptide (2.1 × 10−4 mmol, 7.96 × 10−4 M) were added under vigorous stirring. After 5 min, a NaBH4 aqueous solution (0.06 mmol, 0.1 M) was quickly added, obtaining a cherry red sol that was left under

EXPERIMENTAL PROCEDURES

Chemicals. Sodium tetrachloroaurate dihydrate (99% w/ w), sodium borohydride (98.5% w/w), phenylsilane, and tetra(triphenylphosphine)palladium were purchased from Sigma-Aldrich. Polypropylene reaction vessels and sintered polyethylene frits were supplied by Alltech Italia. NovaSyn TGR resin, coupling reagents, and all amino acids were from Novabiochem. N,N-Diisopropylethylamine (DIPEA) was purchased from Romil and piperidine from Biosolve. mQ water was used as solvent in all colloidal experiments. Peptide and gold aqueous stock solutions were filtered through 0.45 μm Millipore syringe filters before the use. Peptide Synthesis. The chimeric cyclopeptide RGD(GC)2 and the linear peptide (GC)2 (Figure 1) were manually synthesized using the Fmoc (fluorenylmethyloxycarbonyl) solid-phase strategy (0.1 mmol). The syntheses were carried out on NovaSyn TGR resin (loading 0.28 mmmol/g resin), using all standard amino acids except for Fmoc-Glu-OAll. The amino acids in 10-fold excess were preactivated with 2-(1Hbenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (9.8 equiv)/1-hydroxybenzotriazole (HOBT) (9.8 equiv)/DIPEA (10 equiv) in DMF for 5 min and then added to the resin in the presence of DIPEA (10 equiv) in DMF. The Cys residues were coupled in 5-fold excess with HBTU (5 equiv)/2,4,6-trimethylpyridine (5 equiv) in DMF/DCM (1:1 v/v) for 90 min without preactivation to prevent racemization.14 341



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microscopy studies on the cells were performed by a Leica TCS-NT instrument using reflected-light optics at a magnification of 63× (1.53 NA Plan-Apochromat). The samples were illuminated with a 488 nm Argon/Krypton laser, using an intensity of AOTF filter by 10%. A neutral filter RT 30/70 was used as the beam splitter and placed at a 45° angle in the path of beam. Quantitative analyses were carried out measuring the brightness of the images of the samples, containing Au-RGD(GC)2 and Au-(GC)2 nanoparticles inside U87 cells. By using Leica Confocal Software, similar cellular areas, containing AuRGD-(GC)2 and Au-(GC)2 nanoparticles (about 780 μm2), were examined. The pixels, concerning the brightness of the two cellular systems of similar area, were measured and compared. In TEM studies, the cells were fixed in a 2% glutaraldehyde solution buffered with 0.1 M sodium cacodylate, at pH 7.2 for 15 min, successively washed with 3 portions of a 0.2 M sodium cacodylate buffer solution at pH 7.2, and postfixed for 10 min with a solution containing 1% (v/v) OsO4 solution buffered with 0.1 M sodium cacodylate at pH 7.4. Finally, the cells were washed three times with the 0.2 M sodium cacodylate buffer solution and dehydrated in an ascending ethanol series.20 Propylene oxide was used as solvent. The cells were infiltrated for 2 h in a 1:1 mixture of Epon-Araldite and propylene oxide, and then the mixture was removed and replaced with 100% Epon-Araldite-resin. The specimens were polymerized for 24 h at 60 °C. The resin embedded specimens were thinly sectioned (30 nm) by ultramicrotome (Ultracut Reichert), and then mounted on a 300 mesh copper grid. The specimens were observed using an EFTEM LEO 912AB transmission electron microscope operating at 100 kV. Digital images were obtained by a CCD Camera System and Leo Image software. Dynamic Light Scattering and ζ-Potential Measurements. Dynamic Light Scattering (DLS) measurements were performed at 90° with a 90 Plus Particle Size Analyzer from Brookhaven Instrument Corporation (Holtsville, NY) working at 15 mW of a solid-state laser (λ = 661 nm). Measurements were carried out at 25 °C in aqueous media. The ζ-potential was determined with the same instrument equipped with an AQ-809 electrode, and data were processed by ZetaPlus Software. The ζ-potential was automatically calculated from electrophoretic mobility based on the Smoluchowski theory. For measurements, Au-(GC)2 and Au-RGD-(GC)2 NPs were diluted with mQ water in order to obtain a concentration of Au NPs close to 0.01 mg/mL. The samples were sonicated for several minutes to avoid the formation of large aggregates and filtered through a 0.45 μm cellulose acetate membrane. ICP Analysis. Au contents were determined by ICP-OES (ICAP 6300, Thermo Fisher Scientific) and an external calibration methodology, after digestion of the samples. The Au-RGD-(GC)2 NPs and Au (GC)2 NPs dispersed over TiO2 samples have been microwave digested in a mixture of 2 mL of HCl (36% by vol) and 1 mL of HNO3 (68% by vol), diluted with Milli-Q water. Finally, the support residues have been eliminated by filtration. TGA Analysis. TGA analyses were performed on PerkinElmer 7 HT thermobalance. Au-(GC)2 and Au-RGD-(GC)2 NPs were prepared scaling up the amounts of reagents in order to obtain 10−3 mmol of peptides in the resulting sols. The sols were lyophilized and subsequently washed several times with mQ water (5 mL each) and centrifuged at 2000 rpm for 5 min. The resulting dark brown powders were used for TGA measurements. The analyses were performed as follows: the

stirring for further 120 min. The AuNPs were purified by dialysis for 48 h by using Spectra/Por CE dialysis tubing, 500− 1000 MWCO. The dialyzed sol was analyzed by HPLC, to verify the absence of free peptide, by using the conditions reported in Peptide Synthesis. UV−visible, ATR-FTIR, and NMR Spectroscopic Studies. UV−visible spectra were recorded on a JASCO V530 spectrometer using 1 cm path length quartz cuvettes and a solution obtained by diluting three times the original sols with mQ water. ATR-FTIR analysis was performed on a Biorad FTS-40 spectrometer equipped with a Specac Golden Gate ATR platform with a diamond crystal. The spectra of free peptides were recorded in the solid state, while the Au capped nanoparticles were analyzed throughout the formation of a colloidal thin film structured on the ATR crystal surface. NMR samples were prepared dissolving 2 mg of RGD-(GC)2 and (GC)2 peptides or 0.5 mg of the lyophilized nanopowders of Au-RGD-(GC)2 and Au-(GC)2 nanoparticles in 650 μL of a H2O/D2O (90:10) mixture. Before NMR analysis, a part of the lyophilized brown material was suspended in mQ H2O and sonicated for 10 min. Only when a red sol (usually darker than the original one) was obtained after sonication and a visible spectrum confirmed a slightly changed plasmon peak, the NMR analysis was carried out. NMR spectra were collected at 25 °C on a Varian UNITY INOVA 600 MHz spectrometer equipped with a cold probe. Proton resonances of RGD-(GC)2 and (GC)2 peptides were assigned using a standard protocol15 based on the comparison of 2D [1H, 1H] TOCSY16 (mixing time: 70 ms) and 2D [1H, 1 H] ROESY17 (mixing times: 150 and 200 ms) experiments. 2D experiments for resonance assignments were generally recorded with 16 scans, 128−256 FIDs in t1, 1024 or 2048 data points in t2. NMR spectra (1D proton and 2D [1H, 1H] TOCSY (mixing time: 70 ms) of a dispersion of Au-RGD-(GC)2 and Au-(GC)2 nanoparticles were recorded with 1024 scans for 1D and 256 scans for 2D experiments, respectively. Water signal was suppressed by means of either the WATERGATE PFG18 or the DPFGSE (Double Pulsed Field Gradient Selective Echo) techniques.19 Proton resonances were referenced to the water signal at 4.75 ppm. Spectra were processed with the Varian software VNMRJ 1.1D and analyzed with NEASY as implemented in CARA (http://www.nmr.ch/). Cell Culture. The U87 cells (human glioblastoma cell line) (see http://www.ihop-net.org/UniPub/iHOP/gs/157442.html, for the adhesion molecule expressed) were cultivated in Dulbecco MEM, supplemented with 10% heat-inactivated fetal calf serum, 1% (v/v) antibiotic mixture (Sigma), and 2 × 10−3 M L-glutamine. At confluence, the cells were split on sterile coverslips in 3 mL of the above-described medium. Transmission Electron Microscopy Studies on AuNPs. TEM micrographs of the colloidal solutions were obtained using an EFTEM Leo 912 AB instrument at an accelerating voltage of 100 kV. Carbon-supported copper grids were used to support the colloidal sols. The samples were prepared by evaporating a drop of gold sol onto carbon-coated copper grids. The histograms of the particle size distribution and the average particle diameter were obtained by measuring about 150 particles by using Measure IT Olympus Software. Confocal and Transmission Electron Microscopy Studies on Cell Culture. Cells were incubated with 100 μL of Au-RGD-(GC)2 and Au-(GC)2 nanoparticles in 3 mL of the medium for 5, 30, and 60 min (for each sol). Confocal 342



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samples were maintained at 120 °C, under isothermic conditions, for 1 h to remove any residual water and then ramped at 5 °C/min to 900 °C.

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RESULTS AND DISCUSSION The interaction of biocompatible AuNPs with cellular targets is of great interest in biology and medicine for developing new drug delivery systems and setting up various biotechnological applications.21−27 In particular, AuNPs have been capped with cell penetrating peptides to improve internalization28−30 or to target the nucleus,29−33 pointing out that a proper functionalization with peptides can produce high selective systems. However, it was reported that some peptide sequences can act as stabilizers of the colloidal solutions.34,35 In this article, we report on the preparation of two systems of AuNPs capped by the peptides named RGD-(GC)2 and (GC)2 (Figure 1). The RGD-(GC)2 peptide is a chimeric molecule of 12 amino acids that contains two parts, encompassing a RGD-sequence and a (GC)2-motif for integrin targeting and nanoparticle capping, respectively. The portion containing RGD sequence was derived from the c(RGDfK) peptide, an αvβ3 antagonist, where the lysine residue was replaced by glutamic acid to allow the conjugation to the (GC)2-motif by its γ-carboxylic group. The (GC)2 portion contains a sequence of glycines and cysteines similar to the previously reported capping agent GC1510 containing cysteine residues needed for the binding to the gold surface. The two cysteines of (GC)2 portion (either in RGD-(CG)2 or in (GC)2 peptides) supply two thiol groups that can establish covalent bonds to the gold surface9,10 and protect the particles by steric stabilization. A disposal of (CG)2 portion of the peptides parallel to the gold particle surface is suggested (presumably multilayers of peptides lay over the particle).9,10 The peptides were synthesized in solid phase and purified by RP-HPLC giving the pure products with high yield (21% and 95% for RGD-(GC)2 and (GC)2, respectively) (Figure 1, Supporting Information). AuNPs were prepared by borohydride reduction of sodium tetrachloroaurate in the presence of the RGD-(GC)2 or (GC)2 peptides. Afterward, the U87 glioblastoma cell line, overexpressing αvβ3 integrin receptor, was chosen for in vitro cellular uptake assay with the aim of demonstrating an enhancement of the Au-RGD-(GC)2 nanoparticle entrance compared to Au(GC)2 one, lacking the integrin recognizing RGD-sequence. Au-RGD-(GC)2 and Au-(GC)2 sols show the characteristic plasmon absorption bands in the UV−visible spectra (Figure 2, Supporting Information) (λmax (Au-RGD-(GC)2) = 529 nm; λmax (Au-(GC)2) = 514 nm) in agreement with the average mean diameters of the particles obtained by TEM (d Au-RGD(GC)2 = 10.20 nm; d Au-(GC)2 = 13.90 nm) (Figure 2A and B). However, the hydrodynamic diameter of nanoparticles obtained by light scattering experiments highlights the presence of nanoparticle aggregates, being close to 46 and 45 nm for Au(GC)2 and Au-RGD-(GC)2, respectively (Figures 3 and 4, Supporting Information). The superficial charges of both NPs, obtained by ζ-potential measurements, resulted in −27.27 mV and −40.59 mV for Au-RGD-(GC) 2 and Au-(GC) 2 , respectively, confirming good colloidal stability. Although Au(GC)2 NPs show a more negative charge value compared to Au-RGD-(GC)2, the former can specifically absorb proteins from the culture medium on their surface assuming a charge close to the Au-RGD-(GC)2 one, as reported previously.36 Consequently, the higher uptake to U87 cells of Au-RGD(GC)2 respect to Au-(GC)2 is mainly mediated by αvβ3

Figure 2. (A) TEM micrograph and histogram of Au-RGD-(GC)2 nanoparticles. Mean diameter = 10.2 nm. (B) TEM micrograph and histogram of Au-(GC)2 nanoparticles. Mean diameter = 13.9 nm.

receptor-based endocytosis due to the presence of an RGD motif on Au-RGD-(GC)2 NPs. The higher Z-potential value of Au-(GC)2 with respect to the Au-RGD-(GC)2 one can be due to various factors such as the different surface concentrations of the two ligands (vide inf ra), eventual different ionic dissociations or zwitterionic forms of the peptides, different conformations of Au-(GC)2 and Au-RGD-(GC)2 systems, different interactions of the two organic chains with water, and, most importantly, different ionic strengths of the solutions (due to some residual electrolyte (NaCl) still being present after the washings).37 The number of gold nanoparticles was calculated by using UV−vis spectroscopy data. Huo Q. et al. have found that the nanoparticle extinction coefficient is independent of the 343



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Figure 3. (a) Comparison of ATR-FTIR spectra of free RGD-(GC)2 peptide (red line) and Au-RGD-(GC)2 nanoparticles (black line). (b) Comparison of the ATR-FTIR spectra of free (GC)2 peptide (red line) and Au-(GC)2 nanoparticles (black line).

N−H stretching region, (GC)2 has a sharper band located at 3292 cm−1, whereas RGD-(GC)2 shows a broader two component band with one component at 3301 and a shoulder at 3205 cm−1. S−H bond stretching vibration falls around 2550 cm−1 for free cysteine; unfortunately, such a vibration was not clearly revealed in any of the recorded FTIR spectra. In pure (GC)2 peptide, a broad feeble band located at 2547 cm−1 appears, but in the literature, a feeble but sharp band of the S− H bond vibration is usually reported.40 Upon interaction with the gold surface, the whole spectrum of (GC)2 is strongly affected (black line in Figure 3b): amide I and II bands undergo significant splitting, and the band located at 1644 cm−1 in the free peptide now shows a main component at 1652 cm−1 plus a shoulder at 1620 cm−1. In addition, two bands at 1698 and 1687 cm−1 appear; the band at 1540 cm−1 is split into three components at 1555, 1544, and 1513 cm−1. The N−H stretching band, which in the free peptide is located around at 3264 cm−1, is almost absent when (GC)2 interacts with gold, probably forbidden by the surface selection rule. This result agrees with a flat and parallel disposition of (GC)2 over gold surface. The FTIR spectrum of the RGD-(GC)2 peptide interacting with AuNPs also shows some changes (black line, Figure 3a): the amide I band shows two feeble components at 1686 and 1646 cm−1, and amide II shows a broad band centered at 1455 cm−1. The N−H stretching band is also affected: in the free peptide, there is a strong absorption at 3301 cm−1 with 3195 and 3065 cm−1 less intense components, whereas only a strong band at 3221 cm−1 is evident upon interaction. These data suggest that both CO and NH groups of the amide bonds are affected by the interaction of the peptides with the gold surface and that the backbone conformation and the hydrogen bonding pattern of the conjugated peptides undergo significant changes. We implemented FTIR results with NMR spectroscopy to further validate the binding of the (GC)2 and RGD-(CG)2 peptides to the gold surface and to identify key organic residues involved in this interaction. In the first stage of the NMR analysis, we determined proton resonance assignments for the (GC)2 and RGD-(GC)2 peptides in their free forms. Because of the fast tumbling of these small peptides, no NOE contacts could be observed in 2D [1H, 1H] NOESY experiments;41 thus, we recorded 2D [1H, 1H] ROESY spectra.17 By comparing 2D [1H, 1H] TOCSY and 2D [1H, 1H] ROESY, we could identify all of the different spin-systems and also sequentially assign them (Figure 7, Supporting Information). In particular, the

capping ligands and the sol medium; it depends only on the nanoparticle core diameter (see Supporting Information).38 Thus, correlating the values of mean diameter of gold core and plasmon resonance absorbance, we calculated the quantity of gold nanoparticles in both colloidal solutions that resulted around 5 × 1012 Au-RGD-(GC)2 and 8 × 1011 Au-(GC)2 NPs. The total gold amount, determined by ICP analysis of 1% (w/ w) Au loaded Au-RGD-(GC)2/TiO2 and Au-(GC)2/TiO2 samples, was respectively, 0.97 and 0.94 mg, in perfect agreement with the theoretical values. The number of peptide molecules bound to each nanoparticle was determined by using TGA analyses (see the Experimental Procedures section and Supporting Information for details). TGA proved to be a useful technique for our hybrid inorganic−organic nanostructures allowing us to quantify accurately the organic coating, surrounding the gold nanoparticle, even if its amount was very low. The thermograms of both systems showed a first weight loss between room temperature and 120 °C due to the evaporation of residual and physisorbed water, which can be neglected for our calculations (Figures 5 and 6 in Supporting Information). Then, weight losses of 1.609% and 1.058% were recorded for lyophilized Au-RGD-(GC)2 and Au-(GC)2, respectively, in the 120−450 °C heating range. Since AuNPs were thoroughly washed from peptide excess, the weight losses can be entirely assigned to the combustion of the peptide ligand capping gold particles. Such weight loss is fully consistent with the one observed in thermograms performed on the pure free peptides (data not shown). Following the method described by De Palma and co-workers,39 we calculated the number of peptide molecules residing on each gold nanoparticle. Each gold nanoparticle is surrounded by about 94 RGD-(GC)2 molecules, while Au-(GC)2 NPs have about 298 (GC)2 molecules residing on their surface. Structural investigations of gold ligand shell were performed by the ATR-FTIR technique in the solid state and NMR spectroscopy. A comparison between the ATR-FTIR spectra of the free RGD-(GC)2 peptide and Au-RGD-(GC)2 nanoparticles is shown in Figure 3a, while the comparison of the spectra of the free (GC)2 peptide and Au-(GC)2 nanoparticles is illustrated in Figure 3b. Pure (GC)2 and RGD-(GC)2 peptides (red lines in Figure 3b and a) show similar FTIR spectra; the main differences are in the amide I and II regions where (GC)2 shows two peaks located at 1644 and 1540 cm−1, whereas RGD-(GC)2 peaks fall at 1655 and 1534 cm−1. In the 344



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presence of strong sequential ROE contacts of the type HNiHαi‑1 allowed us to clearly connect all the residues. Then, we investigated by NMR spectroscopy the binding to the nanoparticles of the (GC)2 peptide lacking the integrin binding motif. The strong interaction between this peptide and AuNPs is evident from inspection of 1D proton spectra (Figure 8, Supporting Information), where a large broadening of all the NMR signals can be observed upon binding to the nanoparticles. Cysteine residues are highly influenced by the interaction, and their β-CH2 proton signal either moves or totally disappears with respect to the corresponding peak in the spectrum of the free (GC)2 peptide (Figure 8, Supporting Information). Similarly, we compared the 1D proton and 2D [1H, 1H] TOCSY spectra of the RGD-(GC)2 peptide with those of the Au-RGD-(GC)2 nanoparticles (Figures 4 and 5).

Figure 5. Comparison of 2D [1H, 1H] TOCSY spectra of the RGD(GC)2 peptide (left) and Au-RGD-(GC)2 nanoparticles (right). The HN-aliphatic protons correlation region is reported and spin system assignments indicated.

HN and side-chain protons disappear for the two Cys residues. Line broadening is accompanied by small chemical shifts changes that are mainly affecting the backbone HN group of the Arg (8.05 ppm in the apo-form and 8.16 ppm in the boundform) as well as the β-CH2 protons of the Asp (2.75−2.61 ppm in the apo-form and 2.82−2.64 ppm in the bound-form), thus indicating that only a small structural rearrangement takes place in the peptide-cyclic portion. From the perusal of spectroscopic ATR-FTIR and NMR data, some common conclusions can be drawn, and a hypothesis about (GC)2 and RGD-(GC)2 interactions with gold nanoparticles can be proposed. Both the techniques indicate a strong interaction of the (GC)2 moiety with the gold surface via the sulfur atom of cysteine residues, which results in a strong perturbation of (i) CO and NH groups (as shown by FTIR data) and (ii) absence of the Cys β-CH2 proton signal (due to the proximity to the Au surface, as shown by NMR data). On the contrary, the rigid RGD ring seems to be almost unaffected upon interaction with gold: from NMR spectra, only a minor structural rearrangement of the peptide-cyclic portion can be detected. In the FTIR spectra, the different band patterns of amide I and amide II between Au-RGD-(GC)2 and Au-(GC)2 (Figure 3a and b) can be assigned to the NH and CO contributes to the RGD moiety (the amide I band can be forbidden by the surface selection rule causing its low intensity). Summing up, the interaction between (GC)2 or RGD-(GC)2 and AuNPs mainly consists of the formation of S−Au bonds between the cysteine thiol residues of (CG)2 and gold surface atoms, leaving the RGD moiety almost unaffected and free for the interaction with receptor. The U87 cells, which express the appropriate receptor for RGD-motif,42 were incubated with Au-RGD-(GC)2 and Au(GC)2 systems for comparison. The cellular uptake of both systems was studied after 5 and 30 min of incubation by confocal microscopy using the reflected-light method. In particular, Figure 6 shows the Au content after 30 min and

Figure 4. Comparison of 1D proton spectra of the RGD-(GC)2 peptide (upper panels) and Au-RGD-(GC)2 nanoparticles (lower panels). An expansion of the HN and aromatic protons region is reported in panel a, while signals from side-chain protons are shown in panel b.

As can be seen in Figure 4, the interaction of the peptide with the large hydrophobic gold surface causes a severe enlargement of all the NMR lines. Line broadening is, in fact, widespread in the 1D proton spectrum in the HN and aromatic region (Figure 4a) as well as in the side-chain region (Figure 4b). It is worth noting that the peak around 2.9 ppm, corresponding to the β-CH2 groups of the cysteines in the free peptide, (Figure 4b upper panel) vanishes when the peptide binds to the nanoparticles (Figure 4b, lower panel). This feature has been previously observed for resonances belonging to the protons that are located rather close to the gold surface.9 Changes can be better seen in 2D [1H, 1H] TOCSY experiments (Figure 5). The large broadening of the resonances causes the loss of many peaks and highly reduces the quality of the 2D spectrum. The use of a cold-probe allowed us to partially overcome this problem. In fact, spin systems for Ala, Arg, Asp, and Glu can be easily recognized in the spectrum of the Au-RGD-(GC)2 nanoparticles (Figure 5, right panel). Correlations between 345



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Table 1. Comparison of Au-RGD-(GC)2 and Au-(GC)2 Nanoparticles Taken Up by U87 Glioblastoma Cellsa peptide

pixel brightness (5 min)

pixel brightness (30 min)

RGD-(GC)2 (GC)2

0.87 0.07

13.3 0

a The brightness (pxl) of similar areas (780 μm2) measured by the images collected by confocal microscopy of Au-RGD-(GC)2 and Au(GC)2 nanoparticles was calculated by the Leica Confocal Software.

entrance mechanism by the recognition between the integrin receptor expressed on the U87 cells and the RGD sequence in the peptide. The cellular uptake mechanism was further investigated by TEM microscopy. Both Au-RGD-(GC) 2 and Au-(GC)2 systems were investigated. The behavior of Au-(GC)2 nanoparticles is shown in Figure 8, in which we can observe that even after 1 h, the AuNPs are outside the cells and located near the cellular membrane.

Figure 6. Confocal microscopy reflected-light image of Au-RGD(GC)2 nanoparticles taken up by U87 cancer cells, after 30 min. The AuNPs are colored yellow for clarity. A large amount of AuNPs are located inside the cells.

highlights a high concentration of Au-RGD-(GC)2 nanoparticles taken up in comparison with a very poor concentration obtained when the (GC)2 peptide was used as the stabilizer after 30 min (Figure 7). Indeed, further details of

Figure 8. TEM micrograph of Au-(GC)2 nanoparticles after 1 h of incubation into U87 cancer cells. The AuNPs are located outside the cellular membrane (black arrows). Bar = 1 μm. Figure 7. Confocal microscopy reflected-light image of Au-(GC)2 nanoparticles taken up by U87 cancer cells, after 30 min. The AuNPs are colored yellow for clarity. Most of the AuNPs are located outside the cells.

On the contrary, the Au-RGD-(GC)2 nanoparticles were easily taken up by U87 cells, and after 1 h (Figure 9A), we found some of them enclosed in the endosome (Figure 9B); thus, we relate these results with the mechanism of internalization by αvβ3 receptors recognition.43 However, after 1 h, we observed also a lot of free Au-RGD-(GC)2 nanoparticles in the cell cytoplasm, some others close to the nucleus (Figure 9C and D), and a relevant number of them in the nuclear membrane complex (Figure 9F and G). These findings let us conclude that there is a fast cellular traffic not yet well understood. In recent times, some articles have appeared in the literature highlighting the cellular uptake mechanisms and the different pathways of nanoparticles inside the cells.44−46 Moreover, in our experiment, the observations of gold particles in the nucleolus (Figure 9D) and the presence of AuNPs located across nuclear cisternae (Figure 9F) let us believe that the endosomial escape mechanism could be operative also in this case, as observed with TAT oligopeptides.45 The presence of many Au-RGD-(GC)2 nanoparticles in the nucleus was confirmed by confocal microscopy as well (Figure 10). In the overlaid image, corresponding to the treatment time

the systems can be derived by the images of the gold incubated cells (collected in reflection, transmission, and overlaid modalities, reported in Figures 9 and 10 in Supporting Information). In order to determine the amounts of Au-RGD-(GC)2 and Au-(GC)2 nanoparticles taken up by the cells, quantitative determinations were carried out, measuring the pixel brightness of the images obtained by confocal microscopy containing capped AuNPs. By using Leica Confocal Software, similar cellular areas (780 μm2), containing Au-RGD-(GC)2 and Au(GC)2 nanoparticles, respectively, both incubated for 5 and 30 min, were examined. The pixels related to the brightness of the two cellular areas were compared and reported in Table 1. The results indicate that the system based on AuNPs functionalized with theRGD motif promotes cellular uptake (0.87 pixels after 5 min and 133 pixels after 30 min of incubation). In contrast, Au-(GC)2 nanoparticles did not enter into the cells after the same period of time. This behavior is reasonably due to an 346



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Figure 10. Confocal microscopy reflected-light image of Au-RGD(GC)2 nanoparticles taken up by U87 cancer cells, after 45 min. The AuNPs are colored red for clarity. Most of the AuNPs are located inside the nucleus as evidenced by the A image collected in the reflection mode.

the coordination of the studied peptides to the gold surface throughout the thiol groups. In particular, the obtained NMR data clearly indicate that the AGCGGCG tail of RGDfunctionalized peptide is responsible for binding to the AuNPs. The changes that can be observed in the NMR spectra are indicative of a strong interaction in which cysteine residues play a crucial role. The uptake of the peptide capped AuNPs by U87 glioblastoma cells gave interesting results and allowed us to discriminate the entrance of Au-RGD-(GC)2 nanoparticles with respect to the Au-(GC)2 ones, owing to RGD targeting. In particular, measuring the pixel brightness of the images obtained by confocal microscopy, we can affirm that only AuRGD-(GC)2 nanoparticles are accumulated in the cells and that their entrance pathway most likely drives them into the endosome. However, further phenomena must occur inside the cell as we observed free AuNPs in the cytosol, in the nucleus, and nucleolus (Figure 9). Thus, we must conclude that further studies are needed to optimize particle delivery, combining the cellular uptake and intracellular targeting.

Figure 9. TEM micrographs of Au-RGD-(GC)2 nanoparticles taken up by U87 cancer cells: (A) AuNPs adhere to plasma membrane (arrow); (B) AuNPs internalized in one endosome; AuNPs close (C) and inside (E,G) the nucleus and nucleolus (D) (arrows). Moreover, we detected a relevant amount of Au-RGD-(GC)2 nanoparticles crossing the nuclear pore complex (F).

of 45 min (Figure 10, panel C), we can observe a large amount of Au-RGD-(GC)2 nanoparticles inside the cells, confirming TEM results and the nucleus penetrating ability of the RGD(GC)2 peptide. However, further studies are needed to explain the appearance of Au-RGD-(GC)2 nanoparticles in the cytosol and the intracellular fate of nanoparticles, as the ability to direct nanoparticles toward the nucleus is a topic of great interest in this field.

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CONCLUSIONS Biocompatible and stable AuNPs can be prepared by using as stabilizer an RGD-(GC)2 molecule, a peptide designed and synthesized ad hoc. It contains an RGD-sequence and a (GC)2motif, important both for coordination to the gold surface and integrin targeting. We compared their behavior with Au-(GC)2 nanoparticles lacking the RGD moiety. Both AuNP systems showed the characteristics (plasmon resonance peaks and size distribution) foreseen for their composition in agreement with previous results.9,10 The Au-RGD-(GC)2 and Au-(GC)2 sols contain 5 × 1012 and 8 × 1011 gold nanoparticles, respectively (ICP measurements), and a ligand/particle ratio equal to 92 and 298, respectively (TGA analyses). The superficial charges, obtained by ζ-potential measurements, confirmed a good colloidal stability of both NPs. Accurate ATR-FTIR and monoand bidimensional NMR studies allowed us to be confident of

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ASSOCIATED CONTENT

S Supporting Information *

RP-HPLC chromatograms and corresponding mass spectra of (GC)2 and RGD-(GC)2; electronic spectra of the sols; 2D [1H, 1 H] TOCSY and 2D [1H, 1H] ROESY spectra of the free peptides and peptide-capped AuNPs; 1D proton spectra of the peptides and capped AuNPs; confocal microscopy images of particles taken up in U87 cells; calculation of AuNP number and Au quantification by ICP analyses; and HPLC analyses of peptides in the mother liquors of both sols. This material is available free of charge via the Internet at http://pubs.acs.org. 347



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AUTHOR INFORMATION

Corresponding Author

*Department of Chimica Inorganica Metallorganica Analitica L. Malatesta, University of Milano, Milano, Italy. Phone: 0039 02 50314361. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Fondazione CARIPLO, PON n. 1078 MUIR and PRIN 2008 n. F5A3AF_001 MIUR are gratefully acknowledged. We thank Dr. Maura Francolini of Pharmacology, Chemotherapy and Medical Toxicology, Department of Milan, Italy for providing U87 cells, Dr. G. Perretta and Mr. L. De Luca from IBB-CNR of Naples for technical assistance, and Dr. Paolo Verderio for Z-potential measurements.

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NOTE ADDED IN PROOF After the submission of the present article, a paper related to the argument herein discussed, titled Cyclic RGD Functionalized Gold Nanoparticles for Tumor Targeting, by D. Arosio, L. Manzoni, E. M. V. Araldi, and C. Scolastico, was published in Bioconjugate Chemistry (2011) 22 (4), 664−672, doi 10.1021/ bc100448r.

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Gold Nanoparticles Capped by a GC-Containing Peptide

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