Optically stable biocompatible flame-made SiO2-coated Y2O3:Tb3+ nanophosphors for cell imaging

Abstract

Nanophosphors are light-emitting materials with stable optical properties that represent promising tools for bioimaging. The synthesis of nanophosphors, and thus the control of their surface properties, is, however, challenging. Here, flame aerosol technology is exploited to generate Tb-activated Y(2)O(3) nanophosphors (∼25 nm) encapsulated in situ by a nanothin amorphous inert SiO(2) film. The nanocrystalline core exhibits a bright green luminescence following the Tb(3+) ion transitions, while the hermetic SiO(2)-coating prevents any unspecific interference with cellular activities. The SiO(2)-coated nanophosphors display minimal photobleaching upon imaging and can be easily functionalized through surface absorption of biological molecules. Therefore, they can be used as bionanoprobes for cell detection and for long-term monitoring of cellular activities. As an example, we report on the interaction between epidermal growth factor (EGF)-functionalized nanophosphors and mouse melanoma cells. The cellular uptake of the nanophosphors is visualized with confocal microscopy, and the specific activation of EGF receptors is revealed with biochemical techniques. Altogether, our results establish SiO(2)-coated Tb-activated Y(2)O(3) nanophosphors as superior imaging tools for biological applications.

Figure 1

(a) High-resolution transmission electron microscopy image of the SiO₂-coated Y₂O₃:Tb³⁺ (4 at%) nanophosphors. The crystal core is encapsulated by an amorphous hermetic nanothin SiO₂ film indicated by white arrows. (b) X-ray diffraction analysis of the same sample, showing the characteristic pattern of the Y₂O₃ monoclinic phase with its corresponding weight fraction and average crystal size. (c) Elemental composition of a large area of the SiO₂-coated Y₂O₃:Tb³⁺ nanophosphors, which is verified by the presence of the Y, Tb and Si peaks. The Cu peak comes from the copper grid used for the electron microscopy analysis.

Figure 2

Thermal conductivity (TC) signal as a function of temperature (and time on the right ordinate, dashed black curve) during isopropanol desorption and decomposition on pure Y₂O₃:Tb³⁺, SiO₂-coated, and pure SiO₂. For the pure Y₂O₃:Tb³⁺ the TC decrease with temperature indicates the reaction of the Y₂O₃ surface. In contrast, the behavior of SiO₂-coated nanophosphors is similar to the pure SiO₂ without any TC decrease, indicating that the core particles are hermetically encapsulated by a SiO₂ layer.

Figure 3

Optical properties of the SiO₂-coated Y₂O₃:Tb³⁺ nanophosphors. (a) Excitation spectrum monitoring the emission at 547 nm. (b) Emission spectra upon excitation at 276 nm (solid blue line) or 405 nm (dashed blue line). The strongest emission peak is at 547 nm corresponding to bright green color, as seen also in the inset (image obtained under excitation at 254 nm).

Figure 4

Y₂O₃:Tb³⁺ nanophosphors interference with neuronal differentiation. (a) Length of neurites generated after 6 days of stimulation with Nerve Growth Factor by PC12 cells incubated with increasing concentrations of pure or SiO₂-coated Y₂O₃:Tb³⁺ nanophosphors. The length of neurites generated by control cells is shown as a gray-shaded area. The number of measured neurites is reported in the upper left corner of the graph. Significant differences between the population means are reported (* indicates p < 0.05). Error bars represent the measured standard error of the mean. (b) Length and orientation of neurites generated by PC12 cells in the tested experimental conditions. The black lines in the diagrams correspond to individual neurite profiles.

Figure 5

Optical stability of Y₂O₃:Tb³⁺ nanophosphors. (a) Photobleaching of EGF-Alexa 647 functionalized nanophosphors under continuous laser excitation in a confocal microscopy setup. The panels in the upper row display the emission from a large agglomerated structure of nanophosphors (NP) upon excitation with the 405 nm line of a Solid State laser, while the lower row displays the corresponding red emission from Alexa647 (EGF-Alexa 647) upon excitation with the 633 nm line of a HeNe laser. The corresponding time (expressed in seconds) is reported in the upper left corner of each panel. The scale bar is 10 μm. (b) Signal intensity curves obtained from photobleaching experiments. The intensity of the nanophosphor emission, normalized to the value at t=0, is reported as a black line, while the corresponding intensity of Alexa647 emission is reported as a gray line. The number of averaged experiments is reported in the upper left corner of the graph. Error bars represent the standard error of the mean.

Figure 6

Uptake of EGF-functionalized Y₂O₃:Tb³⁺ nanophosphors by VE11 mouse melanoma cells. VE11 cells were incubated for 4 hours with EGF-functionalized nanoparticles and then fixed and stained with FITC-phalloidin to reveal the actin cytoskeleton. (a) Differential interference contrast (DIC) image of VE11 mouse melanoma cells after 4 hours of incubation with EGF-functionalized Y₂O₃:Tb³⁺ nanophosphors. (b) Confocal image of the nanophosphors emission and (c) the actin cytoskeleton. (d) Merged image of panels (b) and (c).

Figure 7

Western blot analysis of EGFR activation in VE11 mouse melanoma cells by EGF-functionalized Y₂O₃:Tb³⁺ nanophosphors. (a) The activation of EGFR is revealed through the quantification of EGFR phosphorylation in Tyr1068 (upper band) relative to the loading control (lower band). (b) Quantification of Western Blot band intensity from three independent experiments. Significant differences between the population means and the control are reported (** indicate p < 0.01). (c) Time evolution of the plasmon peak position of a localized surface plasmon resonance (LSPR) biosensor with SiO₂-coated nanosilver mounted on a flow-cell. Initially, the biosensor signal is stabilized in the presence of the buffer solution (PBS). Then, the EGF-containing solution is injected, shifting the peak position to higher wavelength. This shift is maintained after the rinsing with buffer (PBS rinse) and incubation with cell medium (medium) for a period over 4 hours.