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Cite this: Nanoscale, 2014, 6, 12403 Received 14th August 2014, Accepted 5th September 2014 DOI: 10.1039/c4nr04687k

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Identification of intracellular gold nanoparticles using surface-enhanced Raman scattering† Hai-nan Xie,a Yiyang Lin,a Manuel Mazo,a Ciro Chiappini,a Ana Sánchez-Iglesias,b Luis M. Liz-Marzánb,c and Molly M. Stevens*a

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The identification of intracellular distributions of noble metal nanoparticles is of great utility for many biomedical applications. We present an effective method to distinguish intracellular from extracellular nanoparticles by selectively quenching the SERS signals from dye molecules adsorbed onto star-shaped gold nanoparticles that have not been internalized by cells.

Rapid developments in the application of nanotechnology are providing increasing insight into cellular-level biomedical applications. Indeed, various functional nanomaterials have been extensively used as imaging agents,1–3 drug carriers4 and direct therapeutic sources.5 In particular, gold nanoparticles (AuNPs) are one of the most widely employed nanomaterials due to their unique optical properties, biocompatibility and the facile manipulation of their surface chemistry.6 So far, different biomolecules such as drugs,7 siRNA,8 DNA9 and proteins10 have been coupled with AuNPs for biomedical applications ranging from in vitro biosensing to in vivo cancer treatments.6 Significant efforts are directed at the study of the interactions of gold nanoparticles with cells (e.g., toxicity,11 internalization,12 membrane penetration13 and endosomal escape8), to increase fundamental understanding and in turn help to engineer highly effective nanomaterials for diagnostics and therapeutics. During the study of cell–nanomaterial interactions, being able to elucidate the location of the nanomaterials within cells is of high relevance toward the interpretation of the results. Transmission electron microscopy (TEM) is a straightforward method to localize nanoparticles at the cellular interface. However, imaging cell samples by TEM generally involves a complex preparation process (e.g., cell fixation, staining and dehydration)14 and a low throughput. Another com-

a Department of Materials, Department of Bioengineering and Institute for Biomedical Engineering, Imperial College London, London SW7 2AZ, UK. E-mail: [email protected] b BioNanoPlasmonics Laboratory, CIC biomaGUNE, Paseo de Miramón 182, 20009 Donostia - San Sebastián, Spain c Ikerbasque, Basque Foundation for Science, 48011 Bilbao, Spain † Electronic supplementary information (ESI) available: Experimental details and additional figures. See DOI: 10.1039/c4nr04687k

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monly used method is confocal fluorescence microscopy which offers spatially resolved information of fluorophore labelled biomolecules along the cells and provides three dimensional distributions from the top membrane to the bottom membrane. Although confocal microscopy is a powerful tool to track fluorescent biomolecules in real time, its application to metal nanoparticles is greatly restricted since the fluorescence from organic dyes is likely to be quenched through energy transfer unless the distance between dyes and nanoparticle surface is carefully adjusted.15 Recently, darkfield microscopy has also been utilized to follow the internalization of nanoparticles into cells.16 However, it requires complicated modifications from the commercially available setups. Therefore, a new method such as the one proposed here, which can discriminate the locations of noble metal nanoparticles between intracellular and extracellular environments should be of great utility. Raman scattering spectroscopy is able to provide a molecular fingerprint of the molecules under study. However, only one in every 106–108 photons involves a change in energy thus making Raman scattering an intrinsically weak effect.17 In the presence of noble metal nanostructures such as gold or silver nanoparticles, Raman scattering of molecules that are on or in close proximity to the nanostructure surface is significantly enhanced,17 a phenomenon known as the surface-enhanced Raman scattering (SERS). The recent development of SERS has provided a highly sensitive method for biosensing18 with a detection limit approaching the single molecule level.19,20 Although the 3D localization of metal nanomaterials in cells using SERS has been reported, it requires an expensive confocal Raman microscope and the process is extremely time consuming.21 On the basis of the above considerations, this work aims at providing an alternative strategy to identify intracellular metal nanoparticles based on SERS. As shown in Fig. 1a, the principle of this strategy comprises the selective quenching of the SERS signals from extracellular nanoparticles through a reaction with tris(2-carboxyethyl)phosphine (TCEP), a reducing agent commonly used in biochemistry and molecular

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Fig. 1 (a) Schematic representation of the selective identification of intracellular noble metal nanoparticles using SERS. In the presence of TCEP, SERS signals from dyes on nanostars outside cells are selectively quenched. (b) Extinction spectrum and (c) representative TEM image of gold nanostars used in this assay. Scale bar is 50 nm.

biology.22 Zhuang et al. have demonstrated that TCEP can effectively quench the fluorescence of the cyanine dye Cy5 through 1,4-addition of the phosphine to the polymethine bridge of Cy5 forming a covalent adduct.23 It is thus reasonable to anticipate that the perturbation of the conjugation structure of the fluorophore will not only quench its fluorescence, but will also alter its Raman scattering spectrum.24 Importantly, TCEP has been shown to be impermeant to phospholipid bilayers, which are the main components of cell membranes.25 Consequently, this allows us to selectively quench the Raman signals from molecules adsorbed onto metal nanoparticles that have not been internalized by cells, while retaining the signal from nanoparticles inside cells. Optical bioimaging is greatly facilitated by using near infrared (NIR) light. The main reason is that the penetration depth of photons in biological tissues is limited by the inherent absorption and scattering by tissue components such as blood and water. These components have reduced absorption and scattering effects in the NIR region which provides a relatively transparent optical sensing window for bioimaging.26 Gold nanorods,27 nanocages,28 nanostars29 and hollow shells30 are known to have highly desirable plasmonic properties within the NIR window. Gold nanostars were selected as SERS substrates for this work, since they display a strong localized surface plasmon resonance (LSPR) at 795 nm (Fig. 1b,c), which allows SERS excitation with a 785 nm laser. Superior Raman signals can be obtained by a combination of highly efficient gold nanostars with the dye Alexa-750, which has both absorption (Fig. 2a) and emission (Fig. 2b) bands within the NIR

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Fig. 2 Absorption (a) and emission spectra (b) of Alexa-750 upon addition of 10 mM TCEP. The excitation wavelength for emission was 720 nm. (c) SERS spectra of Alexa-750 after the addition of 50 mM TCEP. The excitation wavelength was 785 nm and the integration time was 1 s, with ∼1 mW power at the sample. (d) Intensity of the highlighted peak in (c) versus time. In (a) and (b), measurements were taken every 2 min. In (a)–(c), the black lines correspond to the sample at time 0 min.

region, so that surface enhanced resonance Raman scattering is registered when a NIR (785 nm) laser excitation is used.17 This minimizes the required excitation time and the potential interference from cells and cell medium due to the presence of the dominant Alexa-750 SERS signals. To verify the reaction between Alexa-750 and TCEP (ESI, Scheme S1†), 5 μM Alexa-750 was reacted with 10 mM TCEP and the addition of the phosphine to the polymethine bridge of the fluorophore was monitored by UV-Vis (Fig. 2a) and fluorescence spectroscopy (Fig. 2b). Both the absorption and emission bands of Alexa-750 decreased rapidly upon TCEP addition and completely vanished after 1 hour. The slight blue-shift observed in the emission band is due to the disruption of the molecule’s conjugation. The conjugation of the fluorophores to gold nanostars was achieved by first forming a cysteamineAlexa-750 complex and then incubating it with polyvinylpyrrolidone (PVP) capped Au nanostars overnight (ESI, Experimental†). The free amine group on the unreacted cysteamine was blocked by carboxyl polyethylene glycol, which also provided greater stability to the nanostars. The dye-coupling step was verified by successfully observing the strong SERS signals of Alexa-750 under 785 nm excitation, as shown in Fig. S1, ESI.† In the presence of 50 mM TCEP, the SERS signal of the fluorophore gradually damped over time and was completely quenched after 1 hour (Fig. 2c and d). To confirm that TCEP cannot penetrate the cell membrane and the SERS signals from internalized nanoparticles could be retained, a combination of TEM and SERS imaging was employed. HeLa cells were incubated with Alexa-750 functionalized gold nanostars (1.2 × 10−11 M) for 48 hours, followed by TCEP treatment (50 mM) for 1 hour. The extracellular nanostars

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were removed by washing with phosphate buffered saline (PBS) for three times before the standard TEM cell sample preparation.14 The cellular uptake of gold nanostars was confirmed by TEM (ESI, Fig. S2†). By using an indexed TEM grid, the sample area where gold nanostars located was identified under Raman microscopy as well as TEM and SERS signals of Alexa-750 are found (ESI, Fig. S2†). This confirms that TCEP cannot penetrate the cell membrane and the fluorophores inside cells remained Raman active. It is evidenced from the above results that TCEP could quench SERS signal from the extracellular nanostars but not from the intracellular ones. To demonstrate the in vitro applications of the system, after being incubated with nanostars, cells were fixed with 10% (v/v) formalin on MgF2 slides, which have negligible Raman background at 785 nm excitation as compared to glass or quartz (ESI, Fig. S3†). Raman mapping was performed using a 63× water immersion objective at 785 nm excitation while fixed cells were kept in 1× PBS. Laser power and integration time not only determine the SERS intensity but may also affect the scanned nanostructures and attached fluorophores.31 As shown in Fig. S4,† less than 10% signal reduction was observed after the second scan on the same area when the laser power was ∼0.5 mW with 5 seconds integration time. Therefore, these optimized parameters were used throughout the whole study. Fig. 3a shows the white light image of the mapped area in which nanostar aggregates can be identified as a darker area. However, it is impossible to identify whether the nanostars are inside or outside the cells solely based on this image. Fig. 3b demonstrates the reconstructed SERS map based on the intensity of the 1300 cm−1 peak of the highlighted area in Fig. 3a. The signal intensities of the attached

Fig. 3 (a) White light image where the mapped area is indicated by a box. (b, c) Reconstructed SERS map based on the intensities of the highlighted peak in Fig. 2c at 1300 cm−1 before (b) and after (c) the addition of 50 mM TCEP. (d) Greyscale values of the valid pixel areas before (left) and after (right) the addition of TCEP. 785 nm excitation wavelength, 5 s integration time, ∼0.5 mW power at the sample. Scale bars are 8 µm.

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fluorophores are revealed by the brightness level of each pixel. It should be noted that at this stage, nanostars, both inside and outside the cells, contributed to this reconstructed map. One hour after addition of 50 mM TCEP, a second SERS map was recorded from the same area. Based on the previous SERS experiments (Fig. 2d, S2†) and also on literature data,25 we find that all the contributions from nanostars outside the cells were eliminated in Fig. 3c and thus the remaining signals shown in this map originated only from internalized nanostars. Image analysis was performed on the basis of the greyscale value changes by the comparison of the valid pixels between Fig. 3b and c. SERS maps were converted by Photoshop® to greyscale values, varying from black at the weakest intensity (0) to white at the strongest intensity (255)32 by measuring changes in pixel brightness. Any grey values above zero in the converted images were treated as valid pixels. As shown in Fig. 3d, the brightness was reduced to ∼20% of the original image after the addition of TCEP (the calculation detail is described in the ESI†). Since TCEP cannot penetrate through the cell membrane, this means that ∼70% of the nanostars were located outside of the cells considering the 10% reduction induced by the laser thus providing a clear indication that ∼30% of the nanostar distribution was intracellular. More than 10 cells were imaged in the same way from different areas of the cell culture and similar results were obtained. To be noted, aggregation of gold nanostars in the intracellular environment was observed (Fig. 3a). This further enhanced the SERS signals from the intracellular nanostars by generating hot spots17 and facilitated the localization of the particles. It is worth mentioning that this method could distinguish the nanostars physically binding to the cell membrane from those inside the HeLa cells, which cannot be achieved by ICP-MS and ordinary SERS mapping. This method is however not intended to provide the exact locations of individual nanoparticles within cells, but rather a verification of whether they are intracellular or extracellular. In our experiment, the samples were washed three times before Raman imaging and the majority of unbound nanostars were removed. Hence the extracellular nanostars in the samples located mostly on the cell membrane (Fig. 3) and displayed ‘identical distributions’ to the nanostars inside the cells. By selectively quenching through TCEP, the SERS signal from the extracellular nanostars was eliminated while those from inside the cells remained. The technique of selectively quenching the SERS signal by TCEP can potentially be applied to investigate the intactness of the cell membrane. As shown above, TCEP is generally impermeable to cell membranes. However, TCEP can enter the cell when the membrane is damaged. Upon entry, we hypothesized that the SERS signals from internalized Raman probes could be quenched which in turn could be utilized to monitor the perturbation of cell membrane. To this end, HeLa cells were incubated with Alexa-750 functionalized Au nanostars and fixed on MgF2 slides. The SERS signals from outside the cells were quenched upon incubating with TCEP for 1 hour, while the signals from inside the cells were retained (Fig. 4a).

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Fig. 4 Reconstructed SERS maps based on the intensities of the highlighted peak in Fig. 2c at 1300 cm−1 overlapped with white light images before (a) and after (b) the disruption of cell membrane with Triton X-100. 785 nm excitation wavelength, 5 s integration time, ∼0.5 mW power at the sample. Scale bar is 10 µm.

Perturbation of the cell membrane was then triggered by adding 1% v/v Triton X-100, which is known to increase the permeability of the cell membrane.33 After 10 minutes, the HeLa cells were immersed with TCEP for another 1 hour to examine the integrity of cell membrane. As shown by Raman mapping on the same area (Fig. 4b), more than 95% of SERS signals are quenched compared to that in Fig. 4a confirming the expected increase in the permeability of the cell membrane. Importantly, this method can be applied utilizing different Raman reporters for localizing noble metal nanoparticles which was confirmed by using Cy5, which is resonant with 633 nm but not with 785 nm excitation. The SERS responses of Cy5-functionalized gold nanostars were assessed before and after addition of TCEP, showing that also in this case the fluorescence and SERS intensities decreased with time after addition of TCEP (ESI, Fig. S5†). Although gold nanostars were chosen in this study because of their high signal enhancement efficiency, this method could also be applied with other plasmonic nanostructures with efficient SERS capability.17 In summary, a general method is presented to localize gold nanoparticles by selectively quenching the SERS signals originating from dye-conjugated nanoparticles outside cells. This approach is expected to help understanding actual intracellular nanoparticle distributions. We also anticipate this localization strategy will provide a means for assessing the internalization efficiency of various cargos coupled with noble metal nanoparticles, such as DNA/RNA, proteins, or drugs, which are of major relevance when studying endocytosis. As demonstrated here, it could also prove very useful as a way of checking cell membrane integrity.

Acknowledgements This research was supported by the European Commission FP7 “Self-assembled virus-like vectors for stem cell phenotyping (SAVVY) project”, project number: 310445.

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Nanoscale, 2014, 6, 12403–12407 | 12407

Identification of intracellular gold nanoparticles using surface-enhanced Raman scattering.

The identification of intracellular distributions of noble metal nanoparticles is of great utility for many biomedical applications. We present an eff...
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