NANO LETTERS
In Vivo Molecular Probing of Cellular Compartments with Gold Nanoparticles and Nanoaggregates
2006 Vol. 6, No. 10 2225-2231
Janina Kneipp,*,† Harald Kneipp,† Margaret McLaughlin,‡ Dennis Brown,‡ and Katrin Kneipp† Wellman Center for Photomedicine, HarVard UniVersity, Medical School, Boston, Massachusetts 02114, and Renal Unit, Massachusetts General Hospital, HarVard UniVersity, Medical School, Boston, Massachusetts 02114 Received June 30, 2006; Revised Manuscript Received September 8, 2006
ABSTRACT Surface-enhanced Raman (SERS) signatures were measured from single living cells at different times after the uptake of gold nanoparticles. The spectra are indicative of chemical changes in the environment of the nanostructures over time. The increase of the SERS signal strength and parallel TEM studies indicate the formation of nanoaggregates providing optimum SERS enhancement for ultrasensitive probing inside the endosomal compartment. The results have implications for medical and biotechnology applications of SERS nanosensors in cells.
Owing to their favorable physical and chemical properties, gold nanoparticles have been useful tools in biology for several decades. Their high electron density has made them popular labels in electron microscopy, and because their surface properties enable a great variety of functionalization procedures1 they can be used to target selected structures in biological samples.2-4 The latter advantage, along with the universal biocompatibility of their material, has contributed to the increasing interest in utilizing gold nanoparticles as vehicles for drug and gene delivery.5,6 In past years, a number of studies on the behavior of gold nanoparticles in cultured cells have been conducted, investigating, for example, potential cytotoxic effects and uptake efficiency as a function of various particle parameters such as size, shape, and surface functionalization.7-9 The plasmonic properties of gold nanoparticles allow one to detect them10 or to measure distances between them.11,12 It is possible to improve spectroscopic signals from molecules in their immediate vicinity by exploiting the local optical fields of noble metal nanoparticles. Raman spectroscopy performed in the local optical fields of noble metal nanostructures, termed surface-enhanced Raman scattering (SERS),13,14 can give detailed information on molecular structure and chemical composition in the nanometer-scaled environment of the nanoparticles.15 In bioanalytical applica* Corresponding author. Present address: Federal Institute for Materials Research and Testing, FG I.3, Richard-Willsta¨tter-Str. 11, D-12489 Berlin, Germany. Phone: +49-30-8104-5738. Fax: +49-30-8104-1137. E-mail:
[email protected]. † Wellman Center for Photomedicine, Harvard University. ‡ Renal Unit, Massachusetts General Hospital, Harvard University. 10.1021/nl061517x CCC: $33.50 Published on Web 09/23/2006
© 2006 American Chemical Society
tions, SERS on gold nanostructures can serve two different purposes: (i) In the vast majority of experiments, they are used as SERS labels. There, a reporter molecule (e.g., a dye) linked to a gold nanoparticle is detected by its very specific Raman signature.16 The use of SERS labels has also been demonstrated in cells, using gold or silver nanoparticles and reporter molecules.17,18 Moreover, SERS spectra of selected reporter molecules were shown to depend on the pH value in the environment of the metal nanostructures.19 (ii) Gold nanostructures can serve as nanosensors by delivering the enhanced Raman spectroscopic signatures of biological molecules and structures in their environment.20,21 In general, both purposes can also be combined, and the SERS signature of a reporter on a gold nanoparticle can be obtained along with the enhanced Raman signature of its cellular environment.17 The cellular SERS signature contains information on the molecular composition of the cellular substructure and also other chemical properties, such as the local pH. In particular, SERS nanosensors enable the chemical characterization of the nanometer vicinity of the gold nanoparticles and the measurement of vibrational spectra at a sensitivity and lateral resolution unachieved so far in other experiments. There is compelling evidence that high SERS enhancement levels are associated mainly with enhanced local optical fields.22,23 This implies that the SERS enhancement factor depends strongly on the morphology (e.g., the size, shape, or aggregation) of the nanoparticles. So far, the highest SERS enhancement factors have been obtained exploiting extremely high field enhancement on aggregates or clusters formed by individual silver or gold nanoparticles 20-60 nm in size.24
As experiments and theory show, the clusters can vary in size, ranging from dimers25,26 to fractal structures.27-29 Because of the important role of nanoaggregates and clusters for electromagnetic enhancement, controlling the formation of gold nanoparticle aggregates is of extreme interest for further development of SERS probing in cell biological experiments. In this work, we investigated the behavior of gold nanoparticles in living cells and the potential of the particles to serve as SERS spectroscopic nanoprobes in a varying subcellular environment. The SERS spectra provide us with two observables: (i) the spectral signature of the SERS, which contains information on the nanometer-scaled chemical environment of the gold nanoparticles, and (ii) the signal strength of the SERS, which can give information on the SERS enhancement factor and thereby allows for the monitoring of changes in the morphology of the gold nanostructures, such as the formation of aggregates. The position of the nanoparticles with respect to cellular ultrastructure, as well as the presence and morphology of nanoaggregates in the cells, were also monitored by transmission electron microscopy (TEM). The optimum physicochemical properties of gold nanoparticles, such as size, shape, and surface functionalization, determine which cellular barriers can be crossed by the nanoparticles, and which cellular compartment can be reached. In addition, the sub-environments found in the various cellular compartments influence the properties of the gold nanoparticles, for example, by inducing the formation of aggregates. All of these aspects need to be considered when gold nanoparticle-based SERS probes are designed for applications in living systems. The transfer of particles into eukaryotic cells is known to work in various ways, among them fluid-phase uptake from the culture medium or mechanical methods (e.g., microinjection). As the subject of our study, we chose the endosomal system of cultured eukaryotic cells. This cellular compartment is accessible via endocytotic pathways without additional mechanical or other triggers for particle delivery. The cells take up gold nanoparticles and enclose them in endosomes. The endosomes undergo a maturation process, during which their molecular makeup changes over time, and the pH decreases and drops dramatically in the latest stage, the lysosome.30 Figure 1 gives a schematic description of the applied experimental procedure. The details of the experiment are described in the Supporting Information. The nanoparticles used in our study were generated by a reduction technique, as reported previously.31 Their size ranged from 30 to 50 nm. Recently, it was shown that the endocytosis efficiency of gold nanoparticles is highest for particles of this size.9 The electron micrograph in Figure 1B demonstrates that the gold nanoparticles were stable in cell culture medium (DMEM with 10% FCS). This medium was used for the fluid phase uptake with a nanoparticle concentration of 10-12 M. The observation that the particles are stable in the cell culture medium is in agreement with other recent reports, and with the well-known stabilization effects of serum proteins, the 2226
major constituents of FCS (e.g., bovine serum albumin) in experiments with cells and gold nanoparticles.9,32,33 The particles were delivered into cells grown on cover slips in the form of a gold pulse, lasting 30 min in immortalized rat renal proximal tubule cells (IRPT) and 60 min in the mouse macrophage cell line J774, respectively. The pulse was followed by a chase period in standard culture medium without nanoparticles (Figure 1A). In this way, with the termination of the pulse phase, no new particle uptake takes place, and the sampling of cells is based on the same amount of gold nanoparticles in the cells at all time points after the beginning of the chase period. The pulsed incubation also ensures that all endosomes containing gold nanoparticles at a specific time point exhibit a similar stage of maturation. Figure 1C verifies that the individual particles are taken up by the cells by endocytosis. During the process of endocytosis, a part of the cellular membrane undergoes invagination, thereby enclosing a gold nanoparticle that is contained in the cell culture medium. In the cellular interior, the particle is therefore surrounded by a membrane vesicle. The transmission electron micrograph (Figure 1C) diplays a snapshot of this enclosure process as we observed it in a cell of the epithelial cell line IRPT. Endocytotic uptake of gold nanoparticles was also reported in other cell types, such as macrophages9 and HeLa cells.8 After washing the cells in buffer (PBS), they were examined by Raman microspectroscopy directly in the buffer solution. During these measurements, Raman spectra were collected from individual cells in a raster scan point-by-point in a predefined grid. Excitation intensities
