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Gold Nanorods Coated with Multilayer Polyelectrolyte as Contrast Agents for Multimodal Imaging Hong Ding, Ken-Tye Yong, Indrajit Roy, Haridas E. Pudavar, Wing Cheung Law, Earl J. Bergey, and Paras N. Prasad* Institute for Lasers, Photonics, and Biophotonics, UniVersity at Buffalo, The State UniVersity of New York, Buffalo, New York 14260-4200 ReceiVed: May 1, 2007; In Final Form: June 22, 2007
Gold nanorods coated with multilayer polyelectrolyte is reported as a biocompatible optical probe with capability for dark-field imaging and for electron microscopy of cancer cells. Transferrin (Tf) was conjugated to the polyelectrolyte-coated nanorods for targeted in vitro delivery to cancer cells. Dark-field imaging was used to confirm the receptor-mediated uptake of nanorods into HeLa cells, which is known to overexpress the transferrin receptor (TfR). Minimal uptake was observed with untargeted nanorods. Electron microscopy was used to confirm that the intracellular uptake of the nanorods predominantly occurred via the Tf-TfR interaction and the nanorods localized in vesicular structures such as endosomes.
Introduction Metallic nanoparticles such as gold and silver particles have attracted considerable attention for their unique properties in bioimaging, biosensing, and optical spectroscopy applications.1-7 Such nanoscale metallic structures have also been proposed as contrast agents for electron microscopy,8 magnetic resonance imaging, and optical imaging.9-11 Some of the more interesting applications of metallic nanostructures in optical imaging or sensing applications include surface-enhanced Raman scattering (SERS) based sensors,12,13 SERS imaging,14 surface plasmon scattering imaging,15 surface plasmon sensors,16-18 fluorescence enhancement using radioactive decay engineering, etc.19-21 Recently, scientists have increasingly turned to one-dimensional metal22-27 and semiconductor28-32 nanomaterials as an alternative to spherical nanoparticles, particularly in biophotonics applications for their uniquely tunable optical properties (e.g., plasmonic bands and fluorescence). Especially for metallic nanorods, they retain all the advantages of the more conventional nanoparticles, while providing additional tunability of their plasmonic bands. Typical Au NRs have two distinct plasmon resonance absorption bands, a longitudinal band corresponding to electron motion along the axis of the particle and the transverse band corresponding to motion along the short axis of the particle. The absorption maximum for the longitudinal band shifts itself to longer wavelengths with increasing aspect ratio. It has also been reported that Au NRs can exhibit a much greater electric field enhancement at their tips33,34 compared to the field enhancement around Au nanospheres, which should lead to increased SERS activity. Metallic nanostructures have several advantages over other optical contrast agents such as quantum dots, fluorophores, or fluorophore-labeled nanoparticles. These metallic particles can act as optical contrast agents using their plasmonic scattering properties for MRI or electron microscopy. Also, they do not photobleach and can be easily detected at relatively low concentration range (∼10-13-10-16 M). The tunability of longitudinal plasmonic bands of these Au * To whom correspondence may be addressed. E-mail: pnprasad@ buffalo.edu.
NRs allows multicolor labeling similar to that with quantum dots or other fluorophores. Recently, there have been reports of applications of gold nanorods for bioimaging15 and optical therapeutic applications.23,35,36 Currently, there is an intense focus on employing these metallic nanorods as optical35,37 and MRI contrast agents,9,11 as well as for electron microscopy applications.38 In the realm of optical scattering imaging using techniques such as darkfield imaging, the longitudinal plasmonic bands can be tuned to obtain wavelength-selective plasmon-enhanced scattering from these rods. Though Au NRs have been used for in vitro imaging,23 there have not been many reports on the use of Au NRs for in vitro targeting of cancer cells. Also no studies were reported about the in vitro cytotoxicity of CTAB- or polyelectrolyte-coated Au NRs, though there are many reports of using CTAB and polyelectrolytes for capping gold nanorods.39 Thus, there is a need to address these issues before Au NRs can be further applied in animal studies. In this study, we report the use of Au NRs coated with multilayer polyelectrolyte (AuMLP) as a biocompatible optical probe with wavelength-selective plasmon-enhanced scattering for dark-field imaging as well as for electron microscopy. Experimental Section Materials. Transferrin (Tf), cetyltrimethylammonium bromide (CTAB), hydrogen tetrachloroaurate(III) trihydrate (HAuCl4‚ 3H2O), silver nitrate (AgNO3), L-ascorbic acid, glutaraldehyde (50% aqueous solution), and sodium borohydride were purchased from Aldrich. All chemicals were used as received. HPLC-grade water was used in all the experiments. Stock solutions of sodium borohydride and L-ascorbic acid were freshly prepared for each new set of experiments. Poly(3,4ethylenedioxythiophene)/poly(styrenesulfate) (PEDT/PSS, molecular weight ) 240 000) and poly(diallyldimethyl ammonium chloride) (PDDAC, 20%) were obtained from Polysciences, Inc. Synthesis of Gold Nanorods for Targeted Imaging. The Au NRs were prepared by the seed-mediated growth method in CTAB surfactant solution. CTAB forms rodlike micelles
10.1021/jp0733419 CCC: $37.00 © 2007 American Chemical Society Published on Web 08/09/2007
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Figure 1. TEM images of (a) CTAB-coated and (b) PDDAC/PSS MLP-coated Au NRs. (c) Visible absorption spectra of (i) CTAB-coated, (ii) CTAB/PSS-coated, (iii) CTAB/PSS/PDDAC-coated, and (iv) transferrin-conjugated CTAB/PSS/PDDAC Au NRs. The scale bars are (a) 100 and (b) 70 nm.
Figure 2. ζ-Potential data for Au NRs at different stages of functionalization. 0 ) Cetyltrimethylammonium bromide; 1 ) poly(3,4ethylenedioxythiophene)/poly(styrenesulfonate) polyelectrolyte; 2 ) poly(diallyldimethyl ammonium chloride).
above its critical micelle concentration (cmc) and therefore forms the template for the subsequent synthesis of Au NRs. Following that, the positively charged CTAB-coated Au NRs were further coated with two successive layers of polyelectrolytes, (a) the negatively charged PEDT/PSS and (b) the positively charged PDDAC. This polymeric multilayering was necessary in order to generate positively charged Au NRs, yet “masking” the CTAB layer, which is known for its cytotoxicity. The cationic AuMLP NRs were then electrostatically attached to the targeting ligand transferrin (Tf). The detailed procedures in each step are described below. (a) Synthesis of Gold Nanoparticle Seeds. The gold seeds were synthesized by the method described by Nikoobakht et al. Briefly, 5 mL of 0.2 M CTAB solution was mixed with 5
Figure 3. (a) pH-dependent effective diameter of CTAB-coated Au NRs and AuMLP NRs; UV-visible absorption spectra of (b) CTABcoated Au NRs and (c) AuMLP NRs at various pH values.
mL of 0.96 mM HAuCl4. Then 0.6 mL of ice-cold 0.01 M NaBH4 solution was quickly added, resulting in the formation of a light-brown solution. The seed solution was vigorously stirred for 2 min and then kept at room temperature. This seed solution was used for the synthesis of gold NRs after 30 min. (b) Synthesis of Au Nanorods. The synthesis method of Au NRs is adapted from Nikoobakt et al. Briefly, 10 mL of 25 mM HAuCl4 and 250 mL of HPLC-grade water were added to 12.5 mL of 4.0 mM AgNO3 solution at room temperature. Then, 250 mL of 0.2 M CTAB and 5 mL of 0.08 M ascorbic acid were added to this solution, and it was gently mixed for 1530 s. We refer to this mixture as the growth solution. After the
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Figure 4. TEM images of CTAB-coated Au NRs at various pH values. Scale bar ) 200 nm. It is evident that aggregation of CTAB-coated Au NRs takes place at pH values over 8.
color of the solution changed from orange to colorless, 0.6 mL of the seed solution was added to the growth solution. The resulting mixture was left undisturbed and aged for 16-18 h at room temperature. Au NRs were concentrated and separated from the excess surfactant solution by centrifugation. (c) Preparation of Transferrin-Conjugated Au NR. The assynthesized NRs were capped with a bilayer of CTAB, which is positively charged. These NRs were centrifuged at 9000 rpm to remove excess CTAB. Then, the positively charged surface of NRs was changed to a negatively charged one by coating the NRs with PEDT/PSS polyelectrolyte solution. The extra PSS was separated by centrifugation. Following that, using the same strategy, the negative charged surface of NRs were reconverted to positive charge by further coating with a layer of PDDAC polyelectrolyte. The cationic PDDAC-capped NRs (AuMLP NRs) were then mixed with Tf solution (∼3 mg/mL) and was allowed to react for 30-40 min. The Tf probably linked to the PDDAC-coated NRs by electrostatic interaction. The transferrin-conjugated NRs were further purified by centrifugation. Characterization Methods. UV-Visible Absorbance. The absorption spectra were collected using a Shimadzu model 3101PC UV-vis-NIR scanning spectrophotometer over the range from 300 to 800 nm. The samples were measured against water as reference. All samples were loaded into a quartz cell for measurements. Cell-Staining Studies. HeLa cells were cultured to 70-80% confluency in 35 mm plates using Dulbecco minimum essential media (DMEM) with 10% fetal bovine serum (FBS), 1% penicillin, and 1% amphotericin B. For in vitro imaging with NRs, cells were treated with the Tf-conjugated AuMLP or AuMLP (control) NRs at a final concentration of ∼8.1 microg/ mL at 37 °C. After incubation, the cells were washed three times with PBS and imaged using dark-field microscopy. Dark-Field Microscopy. The light-scattering images were recorded using an upright Nikon Eclipse 800 microscope with a high numerical dark-field condenser (N.A. 1.20-1.43, oil immersion) and a 100×/1.4 NA oil Iris objective (Cfi Plan Fluor). In the dark-field configuration, the condenser delivers a narrow beam of white light from a tungsten lamp and the high NA oil immersion objective collects only the scattered light from the samples. The iris of the objective can be adjusted to optimize the collection and to reduce the leakage of transmitted light. The dark-field imaging was captured using a QImaging Micropublisher 3.3 RTV color camera. The Qcapture software from the camera manufacturer was used for image acquisition
Ding et al.
Figure 5. TEM images of AuMLP NRs at various pH values. Scale bar ) 200 nm. No aggregation was observed for AuMLP NRs at pH ranging from 4 to 10.
and has a feature for adjusting the white color balance for accurately capturing the color differences in samples. Transmission Electron Microscopy (TEM). TEM images were obtained using a JEOL model JEM-100CX microscope, operating with an acceleration voltage of 80 kV. The specimens were prepared by drop-coating the sample dispersion onto a holey carbon-coated 200 mesh copper grid, which was placed on filter paper to absorb excess solvent. Results and Discussion The Au NRs were prepared by the seed-mediated growth method in CTAB surfactant solution, as described in the Experimental Section. These CTAB-coated NRs were further coated with multilayers of polyelectrolytes, in order to generate positively charged Au NRs, while masking the CTAB layer, which is known for its cytotoxicity. The cationic AuMLP NRs were then electrostatically attached to the targeting ligand transferrin (Tf), whose corresponding receptors (transferrin receptors or TfRs) are overexpressed in a variety of cancer cells. In vitro experiments have demonstrated the nontoxicity, as well as robust cell-labeling efficiency, of the Tf-conjugated AuMLP NRs. The cell labeling is attributed to Tf-TfR receptor-mediated endocytosis. Figure 1a shows a TEM image of Au NR samples prepared with CTAB surfactant solution. The length and width of the Au NRs are estimated to be 70 and 35 nm, respectively. In general, the aspect ratio of the Au NRs can be tuned easily upon varying the concentration of AgNO3 in the growth solution. However, there is a critical silver ion concentration, above which the aspect ratio of the NRs starts to decrease again. In all cases, along with a large fraction of NRs, 10-15% of different shapes such as spherical and smaller rods are also observed. Coating gold nanocrystals with polyelectrolytes has been previously reported.39,40 The studies have generated a series of guidelines for successful coating of metal nanocrystals. Figure 1b shows the TEM image of the Au NRs coated with two layers of polyelectrolytes. From this image, it can be observed that the Au NRs are uniformly coated with the polymer. The thickness of the polymer coating of Au NRs is estimated to be ∼3-5 nm. Figure 1c shows the UV-vis spectra of Au NRs, wherein two bands were observed. The longitudinal and transverse plasmon resonance bands are located at ∼645 and ∼516 nm, respectively. From Figure 1c, it can be seen that there is a small red shift (∼3 nm) in the transverse plasmon band maxima after coating with two layers of polymer (516 nm for CTAB-coated Au NRs and 521 nm after two layers of polyelectrolyte coating).
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Figure 6. Cytotoxicity studies of HeLa cells treated with Au NR bioconjugates. MTT assays illustrating cell viability upon exposing the cells to different concentrations of Au NR bioconjugates for 24 and 72 h.
It is known that the nanocrystal plasmon band is dictated by the local dielectric function.27 Therefore, the red shift is probably due to the changes in the local refractive index from that of water to that of polyelectrolytes. These red shifts in plasmon band maxima are consistent with the previous reports.39 The water dispersible AuMLP NRs were colloidally stable for weeks at room temperature. To monitor the sequential deposition of oppositely charged polyelectrolytes onto surface of Au NRs, ζ-potential measurements were performed at each stage of polymer coating. Figure 2 shows the ζ-potential values for Au NRs as a function of polymer coatings. The ζ-potential of as-synthesized Au NRs is ∼+20 mV due to the presence of a bilayer of cationic CTAB on the NR surface. It can be clearly observed that there is charge reversal upon sequential deposition of the oppositely charged polyelectrolytes onto the surface of Au NRs. When transferrin is conjugated to AuMLP NRs, a charge reversal from positive to negative (∼5 mV) is again observed. In this study, a light-scattering technique (dynamic light scattering, DLS) was used to determine the colloidal stability of the AuMLP NRs at different pH values as compared with Au NRs. The pH profile of the effective diameter of the Au and AuMLP NRs is shown in Figure 3. From Figure 3, one can observe that the effective diameter of the as-prepared CTAB-coated Au NRs is 40 nm in solution, with a pH around 4. When the pH value increases to 10, the effective diameter of the Au NRs rapidly increases to ∼450 nm, indicating that aggregation of Au NRs is taking place. On the other hand, the effective diameters of Au MLP remain unchanged from pH value 4 to 10, suggesting that the colloidal stability of Au NRs was further improved by coating them with polyelectrolyte. Also, this implies that the AuMLP NRs can be used in a wide pH range, which is suitable for various bioapplications. It is worth mentioning that the size of the as-prepared Au NRs is ∼70 nm in length and ∼35 nm in width. However, light scattering only shows an effective diameter of ∼38 nm. The light-scattering analysis assumes that particles are spherical. Thus, the actual sizes determined by light scattering in our case cannot be taken literally. Therefore, we want to emphasize that the light-scattering technique is only used to monitor the aggregation of the Au NRs. To further prove the aggregation of CTAB-coated Au NRs occurred at higher pH values, both UV-vis spectroscopy and TEM analysis were conducted to support the DLS results. Figure 3b,c shows the UV-vis spectra of both CTAB-coated Au NRs and AuMLP NRs suspension at pH values from 4 to 10. The data show that the prominent longitudinal peaks from CTAB-coated Au NRs disappear at pH values above 8 (Figure 3b), suggesting that the dimension of
the gold nanorods has changed, unlike AuMLP NRs, where the peaks still appeared at ∼645 nm (Figure 3c). Furthermore, careful TEM analysis on these samples (see Figures 4 and 5) has revealed that the aggregation of CTAB-coated Au NRs is indeed dependent on the pH values as compared to polyelectrolyte-coated Au NRs, which show good colloidal stability even at pH value at 10. The in vitro cytotoxicity of the various Au NR samples was evaluated using a a colorimetric cell-viability (MTT) assay,41 as given in Figure 6. In the MTT assay, the absorbance of formazan (produced by the cleavage of MTT by dehydrogenases in living cells) at 570 nm is directly proportional to the number of live cells. The detailed protocol for MTT assay can be found elsewhere.41,42 It can be clearly seen that the CTAB-coated NRs display marked cytotoxicity following treatment with the cells for 24 h (Figure 6a) and 72 h (Figure 6b), even at low dosages. In stark comparison, for the cells treated with AuMLP and TfAuMLP NRs, the relative viability was always extraordinary high for both 24 and 72 h posttreatment. The cell viability is as high as 80% for the maximum dosage. This data not only confirms the cytotoxicity of the CTAB-coated NRs, thereby justifying our strategy of the polyelectrolyte multilayering, but also demonstrates the nontoxicity of the final Tf-AuMLP NRs, highlighting their potential for in vitro and in vivo imaging. It is also worth noting that the viability of the cells treated with the AuMLPs and Tf-AuMLPs is more or less independent of the treatment time (24 and 72 h). The Tf-conjugated AuMLP NRs were then used for in vitro imaging. HeLa cells are chosen as the target cell line, which is known to overexpress transferrin receptors (TfRs). Robust receptor-mediated cellular uptake is demonstrated from the darkfield images of HeLa cells treated with the Tf-conjugated NRs (Figure 7c). The orange/red scattering associated with the NRs, as seen in Figure 7c, originates from their strong longitudinal surface plasmon oscillation, which has a resonant frequency within the red region of the optical spectrum. This plasmonenhanced scattering from gold nanorods is much brighter than fluorescence from most fluorophores. In the case of polyelectrolyte-capped gold nanorods (without any Tf conjugation), there were minimal cellular uptake (Figure 7b), and control cells without any treatment also showed no wavelength-selective scattering. This is an elegant demonstration of receptor-mediated targeting of nanorods to tumor cells, which is an improvement over previous attempts for targeted plasmonic scattering imaging using Tf-conjugated gold nanoparticles, where no preferential cellular uptake over nontargeted gold nanoparticles was reported.43
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Figure 7. Light-scattering images of HeLa cells following (a) no treatment, (b) treatment with non-Tf-conjugated AuMLP NRs, and (c) treatment with Tf-conjugated AuMLP NRs (treatment time 2 h). The wavelength-selective scattering (orange/red) associated with the NRs can be clearly distinguished from the background and corresponds to the surface plasmonic enhancement of the longitudinal oscillation in the red region of the optical spectra.
Figure 8. (a) TEM image of HeLa cells with Tf-AuMLPs NRs (the circle points out the Au NRs). (b) The circled area in part a is shown at higher magnification, showing the individual gold nanorods inside the cell cytoplasm.
The main advantages of developing such plasmon-enhanced scattering probes for biological applications are several-fold: (i) the tunability of plasmon-enhanced scattering peak by controlling the aspect ratio of nanorods; (ii) no photobleaching, unlike in case of fluorophores; (iii) the biocompatibility of metallic nanoparticles in comparison with toxic materials such as quantum dots, especially for in vivo applications; and (iv) the capability of using the same targeted particles for multimodal imaging. We have also demonstrated the capability of AuMLP NRs as a nonoptical contrast agent, using transmission electron microscopy. For this, we recorded electron microscopy images of HeLa cells after treatment with the Tf-conjugated and nonconjugated NRs. From Figure 8, it is evident that Tfconjugated NRs are avidly uptaken by HeLa cells, as opposed to non-Tf-conjugated NRs, which show no uptake (data not shown). The endosomal localization of the nanorods within the cells also indicates the receptor-mediated endocytotic uptake (Figure 8b). These observations demonstrate that receptor-mediated targeting of gold nanorods to cancer cells can be validated over multiple imaging platforms, using optical as well as non-opticalbased imaging techniques. This combination of multiple imaging techniques also allows the identification of the precise nature and intracellular localization of the targeted delivery mechanism under study. The possibility of using electron microscopy along with optical imaging allows more precise quantitative estimation of the uptake of targeted nanoprobes.
Conclusions In this study, we have demonstrated the use of biocompatible gold nanorods as targeted contrast agents for imaging of cancer cells, using optical and electron microscopy techniques. We have shown the targeted in vitro uptake of transferrin-conjugated gold nanorods in the Tf receptor positive cell line HeLa, using darkfield imaging. In combination with electron microscopy imaging, which can provide more quantitative information as well as highresolution imaging, this study clearly demonstrates the potential of these metallic nanostructures for use as contrast agents in multimodal bioimaging. The wavelength tunability of the plasmon-enhanced scattering from these nanorods can be utilized for multicolor labeling of different biotargets. Future work will involve expanding the use of these targeted nanorods in multimodal imaging and therapeutic applications, including magnetic resonance imaging and photothermal therapy, along with optical and electron microscopic imaging. Acknowledgment. This study was supported by grants from the NCI RO1CA119397, NIH Grant CA104492, the John R. Oishei Foundation, the Chemistry and Life Sciences Division of the Air Force Office of Scientific Research, and the University at Buffalo Interdisciplinary Research and Creative Activities Fund. Support from the Center of Excellence in Bioinformatics and Life Sciences at the University at Buffalo is also acknowledged.
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