L. Minati‡
*a,
V. Antoninib,
L. Dalboscoc,
F. Benettic,
C. Migliaresic,
M. Dalla Serrab and
G. Speranzaa
aFBK, Via Sommarive 18, 38123 Trento, Italy. E-mail: luminati@fbk.eu
bIstituto di Biofisica, Consiglio Nazionale delle Ricerche, via alla Cascata 56/C, 38123 Trento, Italy
cDepartment of Industrial Engineering & Biotech Research Center, University of Trento, via delle Regole 101, 38123 Mattarello, Trento, Italy
First published on 26th November 2015
This work presents novel magnetite–gold hybrid nanoparticles formed by multiple magnetic cores inside gold nanostars (SPIO@Au). The nanostructures were produced by one-step hydroxylamine reduction of a gold precursor on the surface of functionalized superparamagnetic iron oxide nanoparticles in an aqueous alkaline medium. The nanoparticles show excellent chemical stability, magnetic properties and an intense surface plasmon resonance in the visible-NIR region. In vitro cellular tests showed that these nanostructures could be efficiently internalized inside A549 cells. The optical properties of the SPIO@Au nanoparticles were exploited for in vitro localization analysis through scattering based imaging. These nanoparticles could have enormous potential for application as contrast agents in combined imaging techniques as well as in bio-analytical applications such as biomolecule and cell separation.
1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (PEG)-2000] (DSPE–PEG–amine) and 1,2 distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (PEG)-2000] (DSPE–PEG) were purchased from Avanti Polar Lipid, Inc.
A solution of DSPE–PEG (4.5 ml, 5 mg ml−1) and DSPE–PEG–amine (240 μl, 5 mg ml−1) in chloroform were then added until obtaining a homogeneous mixture. After stirring, the mixture was dried with argon and left in vacuum for 48 hours in order to remove the organic solvent residuals.
The dried film was suspended again in Milli-Q water by ultrasonication for 30 minutes.
The excess lipids were removed from the suspension by repeated ultracentrifugation (25000 rpm for 1 h, 3 times).
For SPIO@Au NPs synthesis, a volume of HAuCl4 aqueous solution was added to SPIO–PEG suspension (pH adjusted to 12) and incubated for 5 min. The reducing agent hydroxylamine was then introduced to start gold reduction. During the reaction, the suspension color changed from pale yellow to dark green-blue in less than a minute (Fig. S2†).
The transmission electron microscope images of the SPIO nanoparticles (Fig. 2A) reveals that the magnetite nanoparticles have a spheroidal shape. The average particle size estimated from TEM images is about 10.4 ± 1.7 nm. TEM (Fig. 2B and C) and SEM (Fig. 2D) images of the SPIO@Au NPs indicate the formation of star-shaped nanoparticles with a size (tip to tip) of about 45–50 nm (Fig. S3†). The elemental composition of the SPIO@Au NPs was investigated by Energy Dispersive X-ray Spectroscopy (EDXS) analysis (Fig. 2E). The Au/Fe molar ratio was estimated to be about 13 confirming the presence of Fe3O4 phase in the nanoparticles. Unfortunately, Fe3O4 nanoparticles cannot be observed in TEM images, because gold coating prevents a deep electron penetration. Moreover, the higher diffraction intensity of Au and the strong overlap with the Fe3O4 features makes the magnetite phase identification in the selected area electron diffraction pattern rather complex (Fig. S4†). Microscopy analysis however did not reveals magnetic nanoparticles adsorbed outside of the SPIO@Au. This was also experimentally confirmed by acid etching experiments. To prove the continuity of the gold coating and rule out the presence of SPIO nanoparticles adsorbed on the gold surface, SPIO@Au nanoparticles were suspended in 1 M HCL solution for 24 h.19 HCl dissolve the iron oxide but not the metallic gold which protected the inner magnetic cores from dissolution, retaining the SPIO@Au magnetic and optical properties (Fig. S5†). The chemical composition of the SPIO@Au after the HCL washing treatment was probed by linear scanning EDXS. Line scan measurements show that Fe Kα line presents multiple peaks located in correspondence of the maximum of the Au Mα spectrum (Fig. S6†).
This is a further confirm that the magnetic cores are located inside the gold nanostars. Scans of several SPIO@Au NPs from various batches also prove the successful SPIO incorporation on the SPIO@Au nanoparticles. Fig. 3 shows the UV-visible spectrum of SPIO@Au NPs in water. It presents a broad surface plasmon resonance band centered at about 695 nm and a weak shoulder at 530 nm. Kumar et al.20 simulated the optical response of gold nanostars by modeling the extinction spectrum of an object composed by a sphere and two cones. Simulated extinction spectra of star-shaped gold nanoparticles were composed by two principal components: a lower intensity peak located in the green-yellow part of the spectrum and a higher intensity component whose position strictly depends on the tip dimensions and shape. The first component was attributed to the modes localized on the central sphere, while the latter one was attributed to modes localized on the nanoparticles tips. In a similar way, the UV-vis absorption spectrum of the SPIO@Au NPs shows a small tail at 530 nm that can be assigned to the plasmon modes localized near the central sphere, and a main absorption peak at 700 nm that can be attributed to the plasmon modes localized on the tips. SPIO@Au NPs can be easily separated from the solution using commercial magnets as shown in Fig. 3. After about one hour, the nanoparticles were quantitatively deposited on the vial in proximity of the magnet while the solution results colourless. The magnetic character of the SPIO@Au NPs was clearly confirmed by the fast response of the particles to the external magnetic field. It is interesting to note that a 10 nm SPIO–PEG nanoparticles suspensions required almost two week for a complete magnetic separation in the same conditions. The small dimensions of the magnetic cores (about 10 nm) limit the size of the magnetic domains, which results in a low magnetic moment value. SPIO@Au NPs required much less time to be separated probably due to the presence of multiple magnetic cores inside each star-shaped nanoparticle. The presence of several magnetic cores inside the same nanoparticle increases the magnetic moment of the material owing to the increase in the dimensions of the magnetic domains. SPIO@Au nanoparticles were characterized by NMR spectroscopy to test their relaxivity rate in water (Fig. S7†). A linear relationship was obtained by plotting the reciprocal relaxation time and the concentration of iron in the sample. The value of the relaxivity for SPIO@Au obtained was 119 s−1 mM−1. This value is similar to that reported for iron oxide nanoparticles used in clinical imaging.21 The high relaxivity rate of the SPIO@Au NPs opens the possibility to use these hybrid nanostructures as MRI contrast agents in in vivo applications. XPS analysis of the SPIO@Au NPs is reported in Fig. 4. A very low signal from iron was detected by XPS (Fig. 4A), whereas an intense doublet in the Au4f region corresponding to metallic gold (84 eV and 88 eV) was clearly observed (Fig. 4B). The Au/Fe atomic ratio obtained by XPS was about 21, a value lower to the one obtained by EDXS analysis. Taking into account that XPS is a surface sensitive technique with a sampling depth of about 5 nm, these results are expected to be a consequence of the incorporation of the magnetic nanoparticles inside the gold nanostars. The change in dimension and morphology of the SPIO@Au NPs were investigated as function of the total Au3+ concentration. At low HAuCl4 concentration (1.0 mM), the average size of the resulting core–shell particles was about 50 nm with a broad absorption peak at around 695 nm. When the concentration of HAuCl4 was increased up to 3.0 mM, the absorption peak experienced a redshift to 880 nm (Fig. 4C). This shift can be attributed to the change of the nanoparticles morphology and to the increase of the nanoparticles size. The average dimensions of the SPIO@Au particles increased to about 150 nm as reported in Fig. 4D (Fig. S8†). It was reported that gold may be reduced by hydroxylamine only if metallic gold is already present in the suspension.22,23 In our previous work, we reported that hydroxylamine at strong basic condition can reduce the gold ions also in absence of external metallic gold.24 In this paper the reduction process performed using the hydroxylamine was carried out in very basic conditions (pH 12). At this pH value the direct reduction of the gold chloride precursor is possible even in absence of preformed gold nuclei as in the seed-growth methods. As shown in Fig. 4D controlling the precursor concentration is possible to tune the dimensions of the synthesized nanoparticles.
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Fig. 3 Up: Extinction spectrum of the SPIO@Au nanoparticles. Bottom: Digital photographs of star shaped SPIO@Au suspension before (left) and after magnetic separation for 120 minutes (right). |
It may be reasonably assumed that, during initial reduction of the gold precursor on the magnetic core, small gold nuclei are formed. The growth of these gold islands on the surface of the cores decrease the iron oxide nanoparticles water solubility since the PEG coating (which is principally responsible for the stability of the magnetic nanoparticles) is partially covered by gold. As a result, a certain number of magnetic cores aggregate, forming a nanoparticle clusters. At this point, the gold rapidly reduced on the magnetic nanoparticles clusters, covering the core and forming the star-shaped nanoparticle. For their possible use in biological applications, which require the use of a medium containing salts, proteins and other organic molecules, a further stabilization of the nanoparticles may be obtained by conjugation with various polymers containing functional groups. For the in vitro cell test measurement the SPIO@Au nanoparticles were functionalized by polyethylene glycol methyl ether thiol. The nanoparticles functionalized with PEG showed excellent stability both in high ionic strength solution and in biological media. The high scattering of the branched gold nanoparticles particularly in the red region of the spectrum was exploited for SPIO@Au localization in cells by laser scanning confocal microscopy analysis.25 Conventional imaging techniques are normally performed by conjugating fluorophores on the gold nanoparticles. These strategies however suffer from fluorescence quenching and require fluorescent probe immobilization of the nanoparticles surface.
On the contrary, the intense scattering of SPIO@Au NPs can be exploited for high resolution cell imaging, with no quenching issue and without the need of fluorescent probes.26 A549 cancer cells were incubated with a 0.05 mg ml−1 SPIO@Au suspension for 2 h. Cellular membranes were then stained with WGA-Alexafluor 488 fluorescent probe and the localization of SPIO@Au was obtained by a 633 nm excitation laser source and a 620–650 nm band pass filter at very low laser power (0.5 mW). The low power and the very short acquisition times (about 1 minute) were chosen to limit possible heating of the SPIO@Au NP due to plasmonic effect. Negative controls were done by analyzing cells and cells incubated with WGA-Alexafluor 488 (data not show). For both controls no signal were detected at 633 nm indicating that the scattering can be unambiguously assigned to the SPIO@Au scattering.
The possibility to analyze simultaneously the membrane fluorescence and the nanoparticles scattering allow a precise determination of the nanoparticles position inside the cell. This information cannot be obtained by dark-field imaging, where the z-stacking is impossible and the advantage of fluorescent co-localization by LSCM cannot be exploited. Confocal microscopy analysis of A549 cancer cell line (Fig. 5) shows that a partial internalization of SPIO@Au is achieved already after 2 h of incubation. In Fig. 5A the red spots were localized mainly on the external membrane, while in Fig. 5B and C a fraction of the nanoparticles was localized inside the cell in close vicinity to the membrane. In Fig. 5D nanoparticles are equally localized both on the external membrane and inside the cytosol. The dimensions of the red spots in the LSCM images are around 100–300 nm, bigger than that of a single SPIO@Au NP. These spots can be reasonably associated to multiple SPIO@Au NPs aggregates. The Au scattering signal was acquired with a narrow band pass (30 nm) to minimize the contribution from other fluorescent signals, like organelles autofluorescence and WGA Alexafluor. Furthermore, the precise control of the stage in the vertical axis allows a fine XY images acquisition. In Fig. 6, z-stack analysis of a single A549 cells is reported as well as vertical (yz) and horizontal (xz) planes of the 3D reconstruction.
The images revealed that the Au spots were mainly localized in the cytoplasm as small clusters distributed overall the cell (Fig. S9†). The formations of big nanoparticles clusters in the cytosol regions could indicate an endocytosis uptake pathway, as reported in the literature for similar nanoparticles.27,28 Even though SPIO@Au NPs have demonstrated promising magnetic and optical properties much more efforts are required for the application of these nanoparticles in clinical studies. On the other hand, the combination of magnetic and optical properties present in a single nanoparticle can also be exploited to develop all-in-one functional nanoprobes for the isolation and recognition of biomarkers in blood and biological fluids. Gold surface functionalization with biomolecular targeting ligands will also allow simple conjugation to develop compact and efficient nanoprobes for molecular recognition and targeting. The magnetic properties can be exploited for the separation of the target from the biological media, while the plasmonic properties of the nanostars should provide molecular analysis capabilities.
Footnotes |
† Electronic supplementary information (ESI) available: Additional XPS, EDS, TEM and confocal microscopy images are presented. See DOI: 10.1039/c5ra20321j |
‡ Present address: Istituto di Biofisica, Consiglio Nazionale delle Ricerche, via alla Cascata 56/C, 38123 Trento, Italy. |
This journal is © The Royal Society of Chemistry 2015 |