Article pubs.acs.org/Langmuir
Targeted Raman Imaging of Cells Using Graphene Oxide-Based Hybrids Zhenyu Zhang, Meng Wang, Dongliang Gao, Da Luo, Qinghai Liu, Juan Yang, and Yan Li* Beijing National Laboratory for Molecular Science, Key Laboratory for the Physics and Chemistry of Nanodevices, State Key Laboratory of Rare Earth Materials Chemistry and Applications, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China S Supporting Information *
ABSTRACT: Graphene oxide (GO) is a reliable and multifunctional platform to perform cell imaging. In this work, a controllable Pt-seed-mediated method is used to prepare GO/gold nanoparticle (AuNP) hybrids, and after the covalent binding of folic acid (FA), GO/AuNP/FA hybrids are prepared. Selective labeling and Raman imaging of folate receptor (FR)-positive HeLa cells are realized using such GObased hybrids. In this system, FA is the targeting agent, AuNPs work as surface-enhanced Raman scattering substrates, and GO takes the role of both supporting the AuNPs with FA and acting as a Raman probe. This research further extends the application of GO as a multifunctional platform in bioimaging and other biomedical processes.
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INTRODUCTION Carbon-based nanomaterials (carbon nanotubes, fullerene, graphene, etc.) have attracted great interest in biomedical applications because of their unique structure and properties.1−4 Graphene oxide (GO) is a kind of graphene derivative, and different from the strong hydrophobicity of graphene, GO possesses carboxylic acid groups at the sheet edges and hydroxyl and epoxy groups on the graphitic plane, so it has a very good water solubility without further functionalization.5 Even after being modified or loaded, the hybrids obtained can form stable colloidal dispersion systems in water.6,7 Considering its water solubility, GO might be a more flexible and promising material for biological and biomedical applications in comparison to graphene. It has been found that GO could serve as a unique double-sided easily accessible platform for multiform functionalization and efficiently load and transport various substances for bioapplications, such as drug delivery,8,9 biosensing,10−15 cancer therapeutics,16−19 and cell imaging.6,7,20−23 Cell imaging is a very important technique used to investigate biomechanisms and cellular processes. Cell Raman imaging is a method of excellent spatial resolution and stability. GO has characteristic Raman fingerprints,24 and it has been used as a Raman probe to perform Raman imaging of cells, with the help of Au or Ag nanoparticles loaded as surface-enhanced Raman scattering (SERS) substrates to improve the Raman signal and the imaging ability of GO.6,7,21,22 Previously, we reported that GO could act as a multifunctional platform for fluorescence and Raman imaging of HeLa cells.7 All of these studies have shown that GO has great potential in bioimaging when combined with the SERS technique. In bioimaging, selective labeling and © XXXX American Chemical Society
targeting are very important. Therefore, it is of great interest to develop a targeted imaging technique of cells. Folate receptor (FR) is overexpressed in many epithelial-derived tumors, including ovarian, breast, lung, and colorectal cancers. Hence, integrating the GO-based SERS imaging method with FRtargeted molecules will improve cancer cell imaging and other biomedical studies. Herein, we demonstrate a strategy for using GO as a multifunctional platform in targeted Raman imaging of HeLa cells based on the hybrids comprising GO, Au nanoparticles (AuNPs), thioglycolic acid (TGA), and folic acid (FA). In this hybrid, AuNPs act as SERS substrates and FA is conjugated via TGA to target the FR-overexpressed HeLa cells.
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EXPERIMENTAL SECTION
Materials. Kish graphite, H2PtCl6, HAuCl4, FA, N-hydroxysuccinimide (NHS), sodium citrate, ascorbic acid, and TGA were purchased from Sigma-Aldrich. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) was obtained from TCI. Cell Counting Kit-8 (CCK-8) was purchased from Dojindo. The dialysis bag MD34 (3500) was purchased from Union Carbide. The HeLa human cervical carcinoma cell line and the A549 human lung cancer cell line were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China). The RPMI 1640 culture medium and fetal bovine serum (FBS) were purchased from Invitrogen. Cell culture dishes and plates were purchased from Corning Incorporation. All other reagents were obtained from commercial sources at the highest purity available Received: June 16, 2016 Revised: August 8, 2016
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DOI: 10.1021/acs.langmuir.6b02248 Langmuir XXXX, XXX, XXX−XXX
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Langmuir Scheme 1. Scheme Showing the Preparation Route for GO/AuNP/FA Hybrids
and were used without further purification. Deionized water was used throughout the work. Preparation of GO. Graphite oxide was synthesized using an improved Hummers method,25 and GO was obtained through the sonication of graphite oxide. In a typical process, first, a 9:1 mixture of H2SO4/H3PO4 (135:15 mL) was added to a mixture of graphite flakes (1.0 g) and KMnO4 (6.0 g), then heated to 50 °C, and stirred for 24 h. Afterward, the reaction mixture was cooled to room temperature and poured onto ice (made from 600 mL water) with 30% H2O2 (9 mL). Then, the mixture was filtered, and the filtered dispersion was centrifuged at 10 000 rpm for 20 min followed by repeated washing with deionized water until the pH of the solution reached ∼6. The precipitates were added to 100 mL of deionized water and resuspended to give a solution containing large GO sheets. Subsequently, the solution was vigorously sonicated for 5 min each round and 10 rounds in total using a 600 W ultrasonic cell disruptor (Ningbo Scientz Biotechnology Co., Ltd.). Very small GO sheets were obtained by removing the precipitates via ultracentrifugation (Micro Ultracentrifuge, Hitachi) at 100 000g for 25 min. Loading Pt Seeds on GO. In a typical process, 5 mg of GO sheets were dispersed into 15 mL of ethylene glycol via sonication, and then 0.1 mL of H2PtCl6 solution (50 mM) was added. After sonication for 20 min, the pH of the dispersion was adjusted to 11−12 by using 2.5 M NaOH−ethylene glycol solution. Then, the dispersion was kept in an oil bath at 130 °C for 2 h under continuous stirring. Finally, the raw dispersion of the GO/Pt hybrids was centrifuged at 50 000 rpm for 20 min followed by repeated washing with water to remove the residues. The obtained precipitates were resuspended with 5 mL of deionized water through sonication. The GO/Pt suspension was adjusted to 1 mg/ mL in terms of GO. Preparation of GO/AuNP Hybrids. In a typical process, 1 mL of the GO/Pt suspension (1 mg/mL) was diluted with 50 mL of deionized water and sonicated for 30 min. Then, 600 μL of 40 mM HAuCl4 solution and 3.0 mL of 1.0 wt % sodium citrate solution were added. After sonication for 20 min, 0.45 mL of 0.1 M ascorbic acid solution was added, and the mixture was stirred at room temperature for 30 min. Finally, the raw dispersion of GO/AuNP hybrids was centrifuged at 50 000 rpm for 20 min followed by repeated washing with water to remove
the residues. The obtained precipitates were resuspended with 1 mL of deionized water through sonication. The GO/AuNP suspension was adjusted to 1 mg/mL in terms of GO. Preparation of GO/AuNP/TGA Hybrids. In a typical process, 1.0 mL of the GO/AuNP suspension (1 mg/mL) was diluted with 10 mL of deionized water and sonicated for 20 min. Then, 1.0 mL of 48 mM TGA solution was added and sonicated for 30 min. Finally, the raw dispersion of GO/AuNP/TGA hybrids was centrifuged at 50 000 rpm for 20 min followed by repeated washing with water to remove the residues. The obtained precipitates were resuspended with 1 mL of deionized water through sonication. The GO/AuNP/TGA suspension was adjusted to 1 mg/mL in terms of GO. Preparation of GO/AuNP/TGA/FA (GO/AuNP/FA) Hybrids. In a typical process, 1 mL of the GO/AuNP/TGA suspension (1 mg/mL) was diluted with 10 mL of deionized water and sonicated for 20 min. Then, 12 mg of EDC·HCl and 18 mg of NHS were added and sonicated for 2 h. After that, 2 mL of 0.5 wt % FA solution (dissolved in the 0.05 M NaHCO3 solution) was added, and the mixture was stirred at room temperature for 1 day. The raw dispersion of the GO/AuNP/FA hybrids was centrifuged at 50 000 rpm for 20 min followed by dialysis for 2 days to remove the unbonded FA. After being dialyzed, the dispersion was centrifuged, and the obtained precipitates were resuspended with 1 mL of deionized water through sonication. The GO/AuNP/FA suspension was adjusted to 1 mg/mL in terms of GO. Cell Culture. HeLa cells and A549 cells were cultured in the RPMI 1640 media supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin. There were two samples of HeLa cells: one was cultured and maintained in the FA-containing (FA+) RPMI 1640 media, which was named FR-negative (FR−) HeLa; the other one was cultured and maintained in the FA-depleted (FA−) RPMI 1640 media to induce the upregulation of FR expression, which was named FR-positive (FR+) HeLa. FR− A549 cells were also cultured in the FA− RPMI 1640 media. The cells were all maintained at 37 °C and 5% CO2 in a watersaturated incubator. After at least five passages, the cells could be used for other cell experiments. Cytotoxicity of the GO/AuNP/FA Hybrids. The cytotoxicity was tested using the CCK-8 assay in FR− HeLa cells cultured in the FA+ RPMI 1640 media. The HeLa cell suspension (100 μL, 1 × 104 cells per B
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Langmuir well) was dispensed into a 96-well plate and preincubated at 37 °C and 5% CO2 for 12 h. Then, the GO/AuNP/FA hybrids were diluted with a medium to various concentrations (5, 20, 30, 40, and 60 μg/mL in terms of GO), and 100 μL of the above-mentioned solutions were added to the 96-well plate after the removal of the old medium. Subsequently, the 96well plate was incubated for another 12 h in the incubator (37 °C and 5% CO2). And then, 100 μL of CCK-8 solution (CCK-8/medium 1:9) was added to each well after the removal of the old solution, and the 96-well plate was incubated for another 1 h. The absorbance at 450 nm with the reference wavelength of 800 nm was measured on a Tecan infinite M200 Pro NanoQuant. Cell Imaging. Cells were cultured in the corresponding media. The cell suspension (1 mL, 1 × 105 cells per well) was put into the wells of 24-well plates possessing silicon wafers inside to let cells seed for 4 h before incubation. To investigate the imaging ability of the hybrids, FR− HeLa cells cultured in the FA+ RPMI 1640 media were used. After the removal of the old medium, the diluted solutions of the GO, GO/Pt, GO/AuNP, GO/AuNP/TGA, and GO/AuNP/FA hybrids (5 μg/mL in terms of GO) were added to each well. The incubations were carried out at 37 °C in a 5% CO2 atmosphere for 12 h. At last, the cells on the silicon wafers were washed and held in phosphate buffer saline (PBS, 0.01 M) for Raman imaging. To investigate the imaging selectivity of the hybrids, FR+ HeLa cells, FR− HeLa cells, and FR− A549 cells were used. After the removal of the corresponding old media, GO/AuNP/FA hybrids diluted with the FA− RPMI 1640 media were added to the wells containing the FR+ HeLa cells. As control experiments, GO/AuNP/FA hybrids diluted with the FA− RPMI 1640 media were added to the wells containing the FR− HeLa cells and FR− A549 cells; GO/AuNP/FA hybrids diluted with the FA+ RPMI 1640 media were added to the wells containing the FR+ HeLa cells; GO/AuNP/TGA hybrids diluted with the FA− RPMI 1640 media were added to the wells containing the FR+ HeLa cells. The concentrations of the hybrids were all 5 μg/mL in terms of GO. The incubations were carried out at 37 °C in a 5% CO2 atmosphere for 2 or 4 h. At last, the cells on the silicon wafers were washed and held in PBS (0.01 M) for Raman imaging. Characterization. A Veeco DiMultiMode V atomic force microscope and a Tecnai T20 transmission electron microscope were used to characterize the size and morphology of various GO-based hybrids. The UV−vis absorption spectra were recorded on a Lambda35 UV−vis spectrometer (Perkin Elmer). Fourier transform infrared spectra (FTIR) were recorded on a Nicolet iNIO MX spectrometer (Thermo Fisher). Energy-dispersive X-ray (EDAX) spectra were recorded on a Tecnai T20 transmission electron microscope equipped with an EDAX analysis system. An aqueous sample (5 μL) was dropped on a silicon wafer and dried for Raman measurements with the excitation wavelength of 632.8 nm on a Horiba Jobin Yvon LabRAM ARAMIS Micro-Raman spectrometer. The Raman spectra were taken using a 100× objective, with a 10 s acquisition time at each pixel under 0.4 mW laser irradiation. Replicate measurements on different areas of each sample (at least eight spots) were carried out to verify that the spectrum was reproducible. As for the cell Raman imaging, spatial Raman mapping was carried out with a 50× objective, 1 × 1 μm2 spot, and 1 s acquisition time at each pixel under 0.4 mW laser irradiation. No more than three images were recorded on a single silicon wafer to minimize the heat effect. After the Raman imaging, cells were recultured under normal conditions to evaluate the damage to cells.
Figure 1. TEM (a) and HRTEM (b) images of the GO/Pt hybrids; TEM (c) and HRTEM (d) images of the GO/AuNP hybrids; and UV− vis absorption (e) and FTIR (f) spectra of the FA (1), GO/Pt (2), GO/ AuNP (3), GO/AuNP/TGA (4), and GO/AuNP/FA (5) hybrids.
Figure 2. Dispersion stability of the GO/AuNP/FA hybrids with a concentration of 100 μg/mL in water and media.
(AFM), it is found that Pt nanoparticles exhibit a (1 1 1) plane and their diameter is about 1−2 nm (Figures 1b and S1a). The EDAX spectrum of the hybrids proves the existence of Pt (Figure S2a). In the UV−vis absorption spectra of GO and GO/Pt (Figure S3), the peak at ∼225 nm of GO is shifted to ∼260 nm of GO/Pt, which is attributed to the partial reduction of GO. Using the Pt nanoparticles in GO/Pt hybrids as seeds, GO/ AuNP hybrids were obtained. In Figures 1c,d, S1b, and S4, it can be seen that AuNPs exhibiting the (1 1 1) plane are successfully prepared and loaded on the GO sheets with very small interparticle distances, which is desired for stronger SERS.26 EDAX results demonstrate the presence of Au (Figure S2b). Using Pt nanoparticles as seeds can obviously increase the density of AuNPs loaded on GO. The size of the AuNPs can be altered in the seeded growth process. Larger AuNPs are obtained
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RESULTS AND DISCUSSION Scheme 1 shows the procedures for material preparation. GO was prepared via an improved Hummers method,25 and then vigorously sonicated to reduce the size of GO sheets. H2PtCl6 was added to the GO−ethylene glycol solution and heated to obtain GO/Pt hybrids. Transmission electron microscopy (TEM) results (Figure 1a) indicate that Pt nanoparticles are loaded on GO sheets; according to the characterizations using high-resolution TEM (HRTEM) and atomic force microscopy C
DOI: 10.1021/acs.langmuir.6b02248 Langmuir XXXX, XXX, XXX−XXX
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Figure 3. Optical (a−e) and Raman (h−j) images of the HeLa cells. Cells were incubated with the GO/AuNP (c and h), GO/AuNP/TGA (d and i), and GO/AuNP/FA (e and j) hybrids at 37 °C for 12 h before imaging. Cells incubated in a medium with GO (a and f) and with GO/ Pt (b and g) are shown as controls. The scale bar is 10 μm. The intensity scale bar is 0−60.
Figure 4. Optical (a−e) and Raman (f−j) images of cells. FR+ HeLa cells incubated with the GO/AuNP/FA hybrids diluted by FA− RPMI 1640 media (a and f), and FA+ RPMI 1640 media (c and h). FR+ HeLa cells incubated with the GO/AuNP/TGA hybrids diluted by FA− RPMI 1640 media (d and i). FR− HeLa cells (b and g) and FR− A549 cells (e and j) incubated with the GO/AuNP/FA hybrids diluted by FA− RPMI 1640 media. The incubation was carried out at 37 °C for 2 h before imaging. The scale bar is 10 μm.
when using more HAuCl4 to prepare GO/AuNP hybrids (Figures 1c and S5). It indicates that this Pt-seed method is a controllable way to synthesize GO/AuNP hybrids. When TGA was bonded, followed by the bonding of FA, GO/ AuNP/TGA and GO/AuNP/FA hybrids were obtained, respectively. Figure S6 shows the TEM images of the two kinds of hybrids. The conjugation of FA to the GO/AuNP/TGA hybrids was confirmed by UV−vis absorption and FTIR measurements. In the UV−vis absorption spectra (Figure 1e), compared with the GO/AuNP/TGA hybrids (curve 4), GO/ AuNP/FA hybrids (curve 5) show the typical absorption peak of FA at 280 nm (curve 1), indicating the presence of FA. In the FTIR spectrum of GO/AuNP/FA hybrids (Figure 1f, curve 5), peaks at ∼806, ∼1157, and ∼1370 cm−1 have all appeared from FA; the presence of a new peak at ∼1650 cm−1 attributed to the vibration of CONH groups indicates that FA is bonded via the reaction between −COOH of TGA and −NH2 of FA.8 Raman spectra of the FA, GO, GO/Pt, GO/AuNP, GO/AuNP/TGA, and GO/AuNP/FA hybrids under a 632.8 nm excitation are shown in Figure S7. FA does not exhibit observable Raman signals in the frequency region measured. D and G bands of GO located approximately at 1330 and 1595 cm−1, respectively, are observed.24 The GO/Pt hybrids exhibit a Raman signal intensity similar to GO. Because of the remarkable SERS effects of AuNPs, the D and G bands of GO are both enhanced ∼3 times in the GO/AuNP, GO/AuNP/TGA, and GO/AuNP/FA hybrids. First, the dispersion stability of the GO/AuNP/FA hybrids was studied. The aqueous dispersion of the GO/AuNP/FA hybrids (0.5 mg/mL in terms of GO) is stable even after more
than two years (Figure S8). The GO/AuNP/FA hybrids of 100 μg/mL could also stay stable in the media after 7 days of standing (Figure 2). Therefore, when using GO-based hybrids at a much lower concentration of 5 μg/mL in cell culture and imaging, aggregation of the hybrids should not be a problem. HeLa cells were cultured with GO and different GO-based hybrids for 12 h. Then cell Raman imaging was performed by spatially mapping the cells with the integrated intensity of the G band of GO (Figure 3). Cells incubated with GO and GO/Pt hybrids could not be imaged well because of the weak Raman signal. However, when cells were treated with the GO/AuNP, GO/AuNP/TGA, and GO/AuNP/FA hybrids, continuous and distinguishable Raman images were obtained. These results suggest that AuNPs successfully trigger the SERS effect and therefore enhance the Raman intensity of GO to improve the Raman imaging effects of cells, which is consistent with our previous reports.6,7 To perform targeted cell imaging, three cell samples were used. The first one was the HeLa cell line cultured and maintained in the FA-depleted (FA−) RPMI 1640 media to induce the upregulation of FR expression, called FR-positive (FR+) HeLa cells; the second one was the HeLa cell line cultured and maintained in the FA-containing (FA+) RPMI 1640 media (the FR is blocked by free FA in the media), called FR-negative (FR−) D
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Figure 4 shows the selective labeling and imaging of FR+ HeLa cells incubated with GO/AuNP/FA hybrids for 2 h before imaging. FR+ HeLa cells exhibit the upregulation of the FR expression, and the affinity between FR and FA induces the GO/ AuNP/FA hybrids gathering at the surface of the FR+ HeLa cells. Therefore, Raman imaging is realized efficiently (Figure 4a,f). Repeated experiments have verified the results (Figure S9). However, if the FR were blocked by the free FA in the media, the cell Raman imaging could not be realized (Figure 4c,h). FR+ HeLa cells incubated with the GO/AuNP/TGA hybrids in the FA− RPMI 1640 media (Figure 4d,i), FR− HeLa cells (Figure 4b,g) and FR− A549 cells (Figure 4e,j) incubated with the GO/ AuNP/FA hybrids in the FA− RPMI 1640 media, which were also used as control samples, could not be imaged either. These results indicate that the GO/AuNP/FA hybrids can selectively label the surface of the FR+ HeLa cells very quickly. Cells incubated with the hybrids for 4 h were also investigated for the labeling of the GO/AuNP/FA hybrids (Figure 5). The targeted labeling of the FR+ HeLa cells was also observed. Compared with the results of 2 h, more intensive signals of the hybrids were found from the cells when 4 h of incubation was employed. This indicates that more hybrids have gathered at the surface of the cells or entered into the cells with the increase in the incubation time. All of the above-mentioned classifications and experimental conditions are summarized in Table 1 for an easy comparison. Cytotoxicity of GO/AuNP/FA hybrids was tested using the CCK-8 assay in the FR− HeLa cells cultured in the FA-containing (FA+) RPMI 1640 media. The viability of the untreated HeLa cells (without hybrids) was set as 100%, and the viability of the hybrid-treated HeLa cells was calculated in accordance. As shown in Figure 6, the hybrids have no obvious cytotoxicity to HeLa cells when the concentration of the hybrids equals to that used for imaging (5 μg/mL in terms of GO). Even when the concentration is 4−6 times higher, the cytotoxicity of the hybrids is still very low.
Figure 5. Optical (a−e) and Raman (f−j) images of cells. FR+ HeLa cells incubated with the GO/AuNP/FA hybrids diluted by FA− RPMI 1640 media (a and f), and FA+ RPMI 1640 media (c and h). FR+ HeLa cells incubated with the GO/AuNP/TGA hybrids diluted by FA− RPMI 1640 media (d and i). FR− HeLa cells (b and g) and FR− A549 cells (e and j) incubated with the GO/AuNP/FA hybrids diluted by FA− RPMI 1640 media. The incubation was carried out at 37 °C for 4 h before imaging. The scale bar is 10 μm.
Table 1. Classification and Experimental Conditions for the Experiments Outlined in Figures 4 and 5 Figures 4 and 5
a,f
b,g
c,h
d,i
e,j
cell: folate receptor positive (+) or not (−) hybrid: folic acid functionalized (+) or not (−) medium: folic acid containing (+) or not (−)
+ + −
− + −
+ + +
+ − −
− + −
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CONCLUSIONS
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ASSOCIATED CONTENT
In this study, a Pt-seed-mediated method was developed to nucleate AuNPs densely onto GO with a controlled size. After the modification with FA, the hybrids were used to label the FR+ HeLa cells, relying on the ligand−acceptor interaction between FR and FA. Then, targeted cell Raman imaging was realized employing the SERS effect of the AuNPs. Besides imaging, this targeted labeling method could also be used for selective drug delivery or thermal ablation therapy. Therefore, it is a prospective method in various biomedical applications.
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b02248. Figure S1−S9 AFM images of GO/Pt and GO/AuNP, EDAX spectra of GO/Pt and GO/AuNP hybrids, UV−vis absorption spectra of GO and GO/Pt, TEM images of hybrids, Raman spectra of hybrids, dispersion stability of GO/AuNP/FA hybrids, and optical and Raman images of cells (PDF)
Figure 6. Cell viability of the HeLa cells incubated with different concentrations of the GO/AuNP/FA hybrids for 12 h (n = 6).
HeLa cells; and the third one was FR− A549 cells cultured in the FA− RPMI 1640 media. E
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Projects 91333105 and 21321001).
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