Nuclear Targeted Nanoprobe for Single Living Cell Detection by

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Bioconjugate Chem. 2009, 20, 768–773

Nuclear Targeted Nanoprobe for Single Living Cell Detection by Surface-Enhanced Raman Scattering Wei Xie,† Li Wang,‡ Yuying Zhang,§ Le Su,† Aiguo Shen,*,† Jinquan Tan,‡ and Jiming Hu*,† College of Chemistry and Molecular Sciences, School of Basic Medical Science, Wuhan University, Wuhan 430072, People’s Republic of China, and Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, People’s Republic of China. Received October 29, 2008; Revised Manuscript Received January 7, 2009

We present a novel nuclear targeting nanoprobe based on peptide functionalized gold nanoparticles and its surfaceenhanced Raman scattering (SERS) in living cells. For the first time, we probe an original SERS signal from the living cell nucleus by using high-selectivity functionalized gold nanoparticles. The gold nanoparticles conjugated with SV-40 large T nuclear localization signal (NLS) peptide successfully enter the cell nucleus after the incubation with Hela cells and deliver the spatially localized chemical information of the nucleus, as well as the signature of chemicals that intruded subsequently. This new targeted nanoprobe is a nontoxic, biocompatible method for biological research, provided with multiple functions comprising subcellular targeting, intracellular imaging, and real-time SERS detection.

INTRODUCTION Raman spectroscopy has long been regarded as a valuable tool for the identification of chemicals and biological samples (1-3). Within the past decade, this novel diagnostic method has attracted more and more interest because of its nondestructivity and low aqueous interference (4). At the same time, also along with the development of biomedical science, chemists and biologists have paid more attention to the components present in very low absolute amounts in living organisms. While the small cross sections required for Raman scattering have limited its use to the analysis of relatively concentrated samples, surface-enhanced Raman scattering (SERS) enables its advantage in the detection of very low concentration analytes, even single molecules (5-7). Generally, the Raman scattering in SERS takes place in the high local optical fields of noble metallic nanostructures, and this is the most important factor of the targeted study within a complex biological environment. As an effectual SERS substrate, gold nanoparticles are easily dispersed and are chemically stabler than silver nanoparticles, which enable them to be widely used for SERS in biomedical research (8-10). Studying single living cells by SERS based on gold nanoparticles was first reported by Kneipp et al. (11). From then on, probing structural information of intracellular components using this biocompatible substrate was always a major purpose of such studies (12-14). In fact, there is an unarguable fact that the distribution of bare gold nanoparticles in cells is not under control. In recent years, purposeful SERS applications have been performed by functionalizing the nanoparticles with antibodiesorotherbiomoleculesthathaveaspecialreceptor(15,16). The functionalized nanoparticles that also can be described as SERS targeting nanoprobes always contain Raman reporter * Corresponding author. E-mail: [email protected] and agshen@ whu.edu.cn, Tel: 0086-27-62258931, Fax: 0086-27-68754067, Address: College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, P. R. China. † College of Chemistry and Molecular Sciences, Wuhan University. ‡ School of Basic Medical Science, Wuhan University. § Department of Polymer Science and Engineering, Zhejiang University.

molecules to highlight the positions of themselves by their high spectral specificity (16-18). More recently, this method has been successfully used in tumor detection on living animals (8). However, the Raman reporter modified targeting nanoprobe provides only the signature of the reporters and tends to be an imaging instrument rather than a detection probe, like the role of a fluorescent reagent or quantum dot; the application of the latter in targeting research is already proving to be excellent (19). In fact, delivering molecular structural information from the target analyte is the most important characteristic or advantage of SERS, which until now has not been possible by any other technique. The cell nucleus is a desirable target because the genetic information of the cell and transcription machinery reside there (20). Although many methods have succeeded in the nuclear targeted studies using gold nanoparticles, SERS has not been involved, because the functionalization might have blocked the metallic surface or imposed influence on the SERS spectra (20-22). Oyelere et al. (23) recently reported their nuclear targeting method by using gold nanorods, which has been shown to provide SERS information from the local biological environment together with significant influences originating from the functional molecules on the nanorods. Moreover, the targeting effect of the nanorods is not comparable to those using gold nanoparticles (20-22). The nanorods are distributed into both nucleus and cytoplasm, which is very similar to the distribution of bare gold nanoparticles in living cells observed in other work (13). Thus, developing a SERS probe for the detection of special targets remains urgent and will open a window for SERS application in the biomedical field. In this paper, we show that the nuclear localization signal (NLS) peptide functionalized gold nanoparticles can locate effectively at the cell nucleus and deliver the molecular structural information from the nucleus without notable influence arising from the functionalization. By staining the cell nucleus with DNA intercalator, we are able to evaluate the ability of the nanoprobe to monitor the chemical changes in the nuclear environment.

EXPERIMENTAL PROCEDURES Preparation of Nuclear Targeted SERS Probe. Gold nanoparticles with a diameter of ∼20 nm for SERS studies were prepared by reduction of gold (III) chloride hydrate using sodium

10.1021/bc800469g CCC: $40.75  2009 American Chemical Society Published on Web 03/06/2009

Nuclear Targeted Nanoprobe

citrate. After centrifugation, the nanoparticles were resuspended in pH 7.3 borate buffer solution by ultrasonication. The concentrated suspension was then diluted in deionized water to a concentration of ∼1012 particles/mL. The nanoparticles were subsequently modified with 11-mercaptoundecanoic acid (11MUA) (Aldrich) by adding a 30 µL aliquot of a 1 mM ethanol solution of 11-MUT to 3 mL of the aqueous nanoparticle suspension. Five microliters of 1 mM 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) (Sigma) and 2 µL of 1 mM N-hydroxysuccinimide (NHS) (Sigma) were added to the modified nanoparticle solution. After that, a 5 µL aliquot of a 0.75 mM SV-40 large T NLS peptide (GL Biochem Ltd.) aqueous solution was added, and the mixture was stirred for 24 h. At last, the product was centrifuged and resuspended in pH 7.4 PBS to a final concentration of 1010-1011 particles/ mL. The supernatant fluid after centrifugation was tested by HPLC (high performance liquid chromatography) to determine the number of NLS molecules on each nanoparticle. A control experiment was performed by using a NLS mutant peptide (KGGGPKKGRKVGG, GL Biochem Ltd.) connected to gold nanoparticles prepared in the same way as the nuclear targeted SERS probe. Cell Culture and Targeting. Human cervix carcinoma cell line (HeLa) was purchased from China Center for Typical Culture Collection (CCTCC), and all reagents for the cell culture were purchased from Gibco. Cells were cultured in RPMI 1640 medium supplemented with 10% newborn bovine serum, 1% (w/v) glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. After incubation at 37 °C in a humidified atmosphere containing 5% CO2, the cells were resuspended for plating by removing the medium, rinsing three times with PBS, and rinsing the cells with fresh 0.25% trypsin, 1 mM EDTA-Na. Then, after incubation for another 3 min, the activity of trypsin was stopped by adding an equal volume of culture medium, and the cells were diluted to a concentration of 2 × 104 cells/mL. The resuspended cells (3 mL) were placed in a 35 mm Petri dish, permitted to adhere for 24 h, and then the functionalized nanoparticles (200 µL) were added in. After the mixture was incubated overnight at 37 °C, the culture medium was removed and 3 mL fresh medium was added to incubate for another 6 h. Additionally, the cells for DNA intercalator detection were incubated for 5 min with 10 µM 4′,6-diamidino-2-phenylindole (DAPI). Immediately before SERS mapping, the medium was removed, and the cells were rinsed three times in PBS. The cells involved in the SERS mapping (without DAPI staining) were fixed with paraformaldehyde (4% in PBS, pH 7.4) after the point by point scan and incubated subsequently for 40 min in the dark with 5 µM Hoechst 33258 (here, the fixed cells were only for fluorescence imaging). Then, the morphology of the cell nucleus was imaged by a fluorescence microscope (Leica). Viability of the cells after incubation with the nanoprobe was tested by flow cytometric (FCM) analysis. In this experiment, the cells were incubated with the nanoprobe for 12 h and cultured subsequently for 12 h in fresh medium. After incubation, the cells were collected and washed three times with PBS and then suspended in 75% ethanol at 20 °C overnight. Fixed cells were centrifuged and washed with PBS twice. For detecting DNA content, cells were stained in the dark with propidium iodide (PI) (50 µg/mL) and 0.1% RNase A in PBS at 25 °C for 30 min. Stained cells were applied to a flow cytometer (Becton Dickinson). For each analysis, 10 000 events were recorded. Raman Measurement. SERS spectra were obtained using a Jobin Yvon Raman microspectrometer (HR 800) with a 632.8 nm helium-neon laser at 13.6 mW with a 1 µm spot size. The objective used was an Olympus 50× long working distance lens (NA ) 0.5) for both focusing the excitation beam on the sample and collecting the back-scattering light onto an air-cooled

Bioconjugate Chem., Vol. 20, No. 4, 2009 769 Scheme 1. Preparation of the Nuclear Targeted Nanoprobe

charge-coupled device detector. The collection time of each SERS spectrum was 1 s over a spectral range from 600 to 1700 cm-1. Cells for the measurement adhered to a CaF2 slide in the culture medium. When Raman mapping was performed, the scan over a single living cell was carried out on a computer-controlled x,y stage in 1 µm steps at a laser spot size of ∼1 × 10-8 cm2. Excitation intensities in the experiments of living cells were less than 1 × 105 W/cm2, which ensures the safety of the living cells within the measurements. Peak frequencies and rapid checking of instrumental performance were calibrated with the silicon phonon line at 520 cm-1. Transmission Electron Micrograph (TEM). Cells incubated with the targeting probe for the TEM experiment were prepared by staining in 1% OsO4, dehydrating in ethanol gradient, immersing in LR white embedding resin, and curing under UV light. Microtomed sections were then placed on grids and poststained with uranyl acetate and lead acetate. TEM measurements for the cells were performed on a JEOL JEM-100CXII microscope, and high-resolution TEM for the probe was performed on a JEOL JEM-2010 microscope.

RESULTS AND DISCUSSION Scheme 1 shows the illustration of preparation of the nucleus targeted nanoprobe. EDC reacts with the carboxyl group on the modified nanoparticles, forming an amine-reactive O-acylisourea intermediate. This intermediate reacts with an amine on the peptide, yielding a conjugate of the two molecules joined by a stable amide bond (24). In our experiment, HPLC was employed to detect the NLS peptides, which were not conjugated to the gold nanoparticles and remained in the solution after the reaction. The result shown in the Supporting Information indicates that only a very small amount of the peptide molecules were detected and almost all of the peptide were immobilized on the gold surface because of the superfluous carboxyl group. Thus, the number of the peptide molecules per nanoparticle is ∼700. More peptide coverage will lead to an increase of nuclear uptake (25) but may also influence Raman signal collection, as we have mentioned in the Introduction. High-resolution TEM images of the nanoprobe and naked nanoparticles are shown in Figure 1a,b, respectively. The functionalization resulted in a

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Figure 2. Cell viability after the addition of varying volumes of nanoprobe into 3 mL aliquots of culture medium. Error bars represent standard deviation of the measurements.

Figure 1. Transmission electron micrographs of the NLS peptide conjugated nanoparticle (nanoprobe) (a) and naked gold nanoparticles (b). (c) Extinction spectra of the nanoprobe and gold nanoparticles (dashed and solid line, respectively). (d) SERS spectra of the nanoprobe (lower spectrum) and CV (10-6 M) enhanced by the nanoprobe (upper spectrum).

low-contrast layer surrounding the metal and contributed a ∼1.5 nm increment to the radius of the sphere. Figure 1c is the extinction spectra of the nanoparticles before and after the peptide modifying, obtained by using a Varian Cary 100 UV-vis spectrometer. The chemical change on the gold surface resulted in a 5 nm red shift of the surface plasmon because the citrate layer of the nanoparticles was disturbed by the conjugation. Also shown are the SERS spectra of the nanoprobe and its mixture together with 1 × 10-6 mM crystal violet (CV) (Figure 1d) obtained at an acquisition time of 1 s. The concentraton of the nanoprobe here is 1010-1011 particles/mL, which is 15 times more than that of the nanoprobe used in the cell incubation. Although the signature of the nanoprobe is obtained at that condition, the relatively low SERS intensity of NLS peptide conjugated 11-MUA on the surface of the gold nanoparticles does not impose any notable influence on the SERS detection of CV. Theoretically, either chemical enhancement or electromagnetic enhancement emphasizes the adsorption or compact adjacency of the analyte and the metal surface (26, 27). The enhancement of CV by the nanoprobe indicates that the functionalization leads to a partial surface coverage of the gold nanoparticles and the product is capable of vibrational information delivery in aqueous solution. Cultured cells were incubated with the nanoprobe as described in the Experimental Procedures, and the cell viability was tested by using FCM analysis in a broad concentration range of nanoprobe. The result shown in Figure 2 indicates that no evident apoptosis was caused by the nanoprobe even at a rather high concentration. Generally, after entry into the cell the NLS peptide will bring the particle to the nucleus by interaction with the pore complex and then cross the nuclear membrane (20). Raman mapping of living HeLa cells incubated with the targeting and control probe performed by the Jobon-Yvon system using a 50× long working distance objective is shown in Figure 3A. The mapping automatically merged by the

Figure 3. (A) Merged SERS image made up of a SERS map by the intensity from 600 to 1700 cm-1 and an optical transmission image. The arrows indicate the positions where the spectra in Figure 4 were collected. Scale bar: 10 µm. The insert is the cell incubated with NLS mutant functionalized nanoparticles as the control experiment. Scale bar: 5 µm. (B) Fluorescent image of the cell after nuclear staining by Hoechst 33258. Scale bar: 10 µm. (C) Differential interference contrast micrograph of the cell. Scale bar: 10 µm. (D) Transmission electron micrograph showing the entry of the nanoprobe to the cell nucleus. Scale bar: 400 nm. (E) Transmission electron micrograph of the nanoprobe inside the cell nucleus. Scale bar: 100 nm.

detection system contains an optical transmission image and a SERS image constructed by the total SERS intensity between 600 and 1700 cm-1. These images only give the spatial information of the SERS. Several spectra which are selected as examples from 812 spectra (indicated by white arrows) in the mapping of the targeted cell will be discussed later. As shown

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Bioconjugate Chem., Vol. 20, No. 4, 2009 771 Table 1. Raman Frequencies Observed in Nuclear SERS Spectra of a Living HeLa Cell and Their Tentative Assignments

Figure 4. SERS spectra obtained from the nucleus of a single living cell incubated with the targeting nanoprobe at points 359, 334, 450, 506 and 532, respectively.

in the mapping, the SERS signals provided by the targeted probe tend to be in the center of the cell. In order to determine the distribution of the probe in the cell, or the position of the cell where the SERS signals come from, immediately after the SERS detection the cell was stained by Hoechst 33258, a bisbenzimide DNA intercalator which can highlight the nucleus by producing fluorescent emission at 461 nm while bound with DNA. The fluorescent image together with a differential interference contrast micrograph of the stained cell is presented in Figure 3 (B and C, respectively). It clearly shows in the figure that most of the SERS signals in the targeted mapping are generated in the location of cell nucleus and the signals in the control experiment are mainly from the cytoplasm. Additionally, in the electron micrograph (see Figure 3D) the nanoparticles were found inside the nucleus, which proves again that the nanoprobe is able to cross the nuclear membrane. Figure 3E is a typical electron micrograph of the nanoparticles inside the nucleus, and no evidence of membrane structure was found close by, which indicates that these nanoparticles were not in a special chemical environment inside the nucleus. If this nanoprobe is to be a useful intracellular targeting tool, then one would expect to see the ability of chemical probing in the nucleus. Figure 4 shows the typical SERS spectra obtained from the cell nucleus at points 359, 334, 450, 506, and 532 of the mapping. Each of the spectra has received contributions originating from DNA backbone and bases. Deformations and rocking vibrations of CH2 groups in deoxyribose appear at 1455-1462 cm-1 and 992-996 cm-1, respectively. Spectrum a contains signatures of adenine base at 1305 cm-1 and of DNA backbone phosphate at 832 cm-1, the frequency of both suggesting that the DNA molecule investigated has a right-handed double helix conformation, also known as the B form. Similar bands can also be found in spectra b and e, but no evidence of other DNA conformations (A and Z) was obtained during the experiment, which confirms that DNA of B form dominates

Raman bands (cm-1)

assignmentsa,b

642 653 734 832, 833 868 891 910, 920, 921 963 982 996 1000, 1003 1034 1069, 1090, 1093 1120 1134 1148 1159, 1160, 1164, 1167, 1168 1180, 1189 1207 1215 1232, 1273, 1275, 1288 1242 1297, 1305 1320 1342, 1345, 1355 1410 1454, 1459 1473 1490, 1497, 1500, 1501 1530 1546, 1550, 1554 1580, 1581 1589 1656

Tyr: ν(CS) Tyr: γ(CC) A phosphate: ν(OPO) deoxyribose: ν(CC) saccharides Pro: ring ν(CC) proteins: ν(CC) Phe: ω(CH) deoxyribose: F(CH2) Phe: ring breath deoxyribose: ν(CO) phosphate: ν(PO2-), ν(COC) proteins: ν(CN) Pro deoxyribose-phosphate proteins: ν(CN), ν(CC); F(CH3) nucleotides: base ν(CN) Tyr, Phe T, A proteins: amide III A, T, ring ν A, γ(CH2) G proteins: δ(CH2) νs(COO-) deoxyribose: δ(CH2) lipids, proteins: δ(CH2, CH3) G, A G, C proteins: amide II; Trp G, A G, A, Phe, Tyr proteins: amide I

a

Based on refs 12-14, 28, 29. b Abbreviations: ν, stretching; δ, deformation; F, rocking; γ, twisting; ω, wagging; s, symmetrical; Tyr, tyrosine; Phe, phenylalanine; Pro, proline; A, adenine; T, thymine; C, cytosine; G, guanine.

the cell nucleus. On the other hand, the SERS spectra also give conformational information on proteins, another main component of the nucleus. Spectrum c shows an amide I band at 1656 cm-1, which indicates a protein R-helix. In spectra d and e, both characteristics of R-helix and β-sheet conformations are found according to amide III contributions at 1275 (R-helix) and 1232 cm-1 (β-sheet). The detailed SERS band assignments for these spectra are given in Table 1. In addition to the detection of the original components in the nucleus, the nanoprobe is also propitious to deliver vibrational information from molecules intruded. The living cells were stained with DAPI, a DNA intercalator with great SERS activity, immediately after incubation with the nanoprobe, and SERS mapping was performed subsequently. Figure 5 shows the image recorded based on the band intensity at 1615 cm-1, which is assigned to CdN stretching of DAPI in the nucleus. Characteristic DAPI signatures acquired outside using the nanoprobe are presented as trace A together with the SERS spectrum of DAPI obtained inside the stained nucleus at mapping point 510 for comparison. Both spectra show very similar SERS features of DAPI except for some band shifting due to the minor-groove binding at adenine-thymine sites of DNA. Typically, the CdN stretching vibration at 1607 cm-1 (spectrum A) shifted to 1615 cm-1 in spectrum B, which is very likely associated with the formation of conjugated systems between DAPI and DNA bases. Besides DAPI signatures, spectrum A also shows various contributions from cellular components. For example, the band at 1405 cm-1 can be assigned to symmetrical stretching vibration of COO- in protein, and band 735 cm-1

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Figure 5. SERS spectra of DAPI obtained in solution (10 µg/mL) mixed with the NLS functionalized gold nanoparticles (A) and in a living cell nucleus (stained by DAPI) (B). The inset shows the SERS image which was collected according to the Raman intensity at 1615 cm-1. The arrow indicates point 510 where spectrum B was obtained. Acquisition time: 1 s.

is a SERS characteristic of adenine. The targeting nanoprobe entered the cell before DAPI did, and the gold surface was still available for DAPI vibration to be enhanced. This result indicates that the gold surface has not been blocked even inside nucleus and the nanoprobe is capable of delivering real-time chemical changes in its vicinity.

CONCLUSIONS We have demonstrated a targeting probing method for nucleus detection in living cells. The SV-40 large T NLS peptide functionalized gold nanoparticles can locate effectively at the cell nucleus after incubation with the cultured cells, allowing molecular probing of the nuclear components in vivo. Our SERS data provide abundant structural information for both nucleic acids and proteins without notable disturbance attributed to the functionalization. Moreover, by using DAPI as a test sample, we show that the targeting probe can also deliver the vibrations from molecules intruded subsequently, which enables this method to be a real-time targeting detection technique. The nanoprobe described in this study has the potential to be used in the research of drug delivery and cancer therapy.

ACKNOWLEDGMENT W. Xie thanks the National Science Foundation of China (grants no. 30772058 and no. 20705025) for ongoing financial support of our work. Supporting Information Available: HPLC result of the supernatant fluid after centrifugation in the preparation process of the nuclear targeted nanoprobe. This material is available free of charge via the Internet at http://pubs.acs.org.

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