Magnetite-Gold nanohybrids as ideal all-in-one platforms for theranostics

Abstract

High-quality, 25 nm octahedral-shaped Fe₃O₄ magnetite nanocrystals are epitaxially grown on 9 nm Au seed nanoparticles using a modified wet-chemical synthesis. These Fe₃O₄-Au Janus nanoparticles exhibit bulk-like magnetic properties. Due to their high magnetization and octahedral shape, the hybrids show superior in vitro and in vivo T₂ relaxivity for magnetic resonance imaging as compared to other types of Fe₃O₄-Au hybrids and commercial contrast agents. The nanoparticles provide two functional surfaces for theranostic applications. For the first time, Fe₃O₄-Au hybrids are conjugated with two fluorescent dyes or the combination of drug and dye allowing the simultaneous tracking of the nanoparticle vehicle and the drug cargo in vitro and in vivo. The delivery to tumors and payload release are demonstrated in real time by intravital microscopy. Replacing the dyes by cell-specific molecules and drugs makes the Fe₃O₄-Au hybrids a unique all-in-one platform for theranostics.

Figure 1

Fe₃O₄-Au hybrid NPs serve as a unique theranostics platform. Optimized fundamental properties, i.e. perfect crystallinity, octahedral shape and bulk-like magnetic properties (I), are combined with efficient stabilization of NPs in water by an amphiphilic polymer; subsequent functionalization of the Au surface with fluorescent dye provides a non-toxic system, internalized by tumor cells in vitro and in vivo, important for biomedical research (II). The described combination of properties also leads to high R₂ relaxivity values for improved contrast in magnetic resonance imaging (III), tested both in vitro and in vivo, which significantly contribute to tumor diagnostics. Double functionalization of Fe₃O₄ and Au surfaces with antitumor drug and fluorescent dye/ two fluorescent dyes allows for the simultaneous tracking of NP vehicle and drug cargo for payload delivery and release (IV), shown in vitro and in vivo in real time.

Figure 2

Structural characterization of Fe₃O₄-Au hybrid NPs. (a) Experimental X-Ray diffraction pattern (black). Black and red Miller indices correspond to Fe₃O₄ and Au phases, respectively. The red diffractogram is a Rietveld refinement based on Fe₃O₄ and Au powder diffraction reference data. The intensity is normalized to the strongest peak, i.e. the Fe₃O₄ (311). (b) TEM micrograph of Fe₃O₄-Au hybrid NPs. (c) High-resolution HAADF-STEM image. The right panels show higher magnifications and the crystallographic planes (lines). Their corresponding directions (arrows) are indicated for Fe₃O₄ (dark image) and Au (bright image). The particle is viewed along its [011] direction.

Figure 3

Magnetic characterization of Fe₃O₄-Au hybrid NPs. (a) Hysteresis loops recorded at 5 K and 300 K. Both loops are measured in the field range of ± 9 T. (b) Coercive field as function of temperature T^2/3. The error bar is smaller than the symbol size and the mean magneto-crystalline anisotropy according Sharrock’s formula is determined from the linear fit. Details are discussed in the text. (c) ZFC/FC curves of Fe₃O₄-Au hybrid NPs in 5 mT. The arrow indicates an abrupt change of the magnetization, i.e. the Verwey transition temperature T_V = 123 K. (d) Mössbauer spectrum at 300 K.

Figure 4

Stability, in vitro toxicity and internalization studies of Fe₃O₄-Au hybrid NPs. (a,b) Stability of NP-PEG in deionized water, 1× PBS, RPMI and RPMI with 10% FBS upon the incubation at 25 °C and 37 °C at a NP concentration of 333 μg·ml⁻¹ Fe₃O₄ (51 μg·ml⁻¹ Au) measured by DLS: (a) NPs diameter and (b) polydispersity index (PDI) as function of time. The error bars represent the standard deviation (SD). (c) 4T1 cells viability assessment after 48 h incubation for various concentrations of NP-PEG by MTS test. Results are shown as means ± SD, *p < 0.05 (one-way ANOVA) comparing to cells incubated with PBS. (d) Dynamics of free Cy5 (6 μg·ml⁻¹ Cy5; top panel) and NP-Cy5 (193 μg·ml⁻¹ Fe₃O₄, 30 μg·ml⁻¹ Au, 6 μg·ml⁻¹ Cy5; bottom panel) accumulation in 4T1 cells studied by fluorescence microscopy after 30 min, 6 h and 24 h of co-incubation. (e) XY-, XZ- and YZ-projections of a tumor cell Z-stack (8 steps, 500 nm each) after 30 min of co-incubation with NP-Cy5 (30 μg·ml⁻¹ Fe₃O₄, 5 μg·ml⁻¹ Au, 1 μg·ml⁻¹ Cy5). White arrows point to an example of NPs inside cytoplasm in three orthogonal projections. White dashed line – optical section through tumor nucleus (N, delineated by yellow dashed line) containing NPs (arrow). (f) NP-PEG uptake by 4T1 cells after 48 h of co-incubation (100 μg·ml⁻¹ Fe₃O₄, 15 μg·ml⁻¹ Au). Cells were dissolved in aqua regia, and Fe₃O₄/Au concentrations were determined by AES. Results are shown as means ± SD.

Figure 5

Delivery of doxorubicin to cancer cells by Fe₃O₄-Au hybrid NPs. (a) pH-dependent kinetics of doxorubicin release from DOX-NP-Cy5 NPs in RPMI (pH = 7.2) and acetate buffer (pH = 4.7) at 37 °C for 48 h; NPs concentration 1000 μg·ml⁻¹ Fe₃O₄, 154 μg·ml⁻¹ Au, 33 μg·ml⁻¹ Cy5, 285 μg·ml⁻¹ doxorubicin. At given time points the absorbance at 480 nm, corresponding to the doxorubicin in supernatant, was measured and plotted as the portion of loaded drug total absorbance. (b,c) The dynamics of DOX-NP-Cy5 (63 μg·ml⁻¹ Fe₃O₄; 2 μg·ml⁻¹ Cy5; 18 μg·ml⁻¹ (31 μM) doxorubicin; top panel) or free doxorubicin (31 μM; bottom panel) accumulation in 4T1 cells: (b). Representative fluorescent images of DOX-NP-Cy5 (top panel) and free doxorubicin (bottom panel) accumulation in 4T1 cells after 30 min and 6 h of co-cultivation (see also Supplementary Fig. S10); green – doxorubicin; red – NP-Cy5. (c) Quantification of doxorubicin fluorescence intensity in 4T1 cells nuclei co-incubated with DOX-NP-Cy5 (blue columns) and free doxorubicin (red columns). Results are shown as means ± SD, *p < 0.05 (one-way ANOVA). (d) 4T1 cells viability assessment after 48 h of incubation with serial dilutions of DOX-NP-Cy5 (blue columns) and free doxorubicin (red columns) by MTS test. Results are shown as means ± SD, *p < 0.05 (one-way ANOVA) comparing to cells incubated with PBS.

Figure 6

Accumulation of Fe₃O₄-Au hybrid NPs in 4T1-tumors. (a) Visualization of subcutaneous 4T1-GFP tumor microenvironment by intravital microscopy upon the i.v. injection of NP-Cy5 (6.6 mg·kg⁻¹ Fe₃O₄); blue – CD49b, green – 4T1-GFP, red – NP-Cy5, cyan – Ly6G; yellow circle – intravascular region of interest (ROI), grey circle – interstitial ROI (see also Supplementary Fig. S11a and Supplementary Video S2). (b,c) IVIS imaging of the mouse with two grafted 4T1 tumors upon i.v. injection of NP-Cy5 (6.6 mg·kg⁻¹ Fe₃O₄). (b) Quantification of tumor/liver fluorescence intensity ratio 1 h, 6 h and 24 h after NP-Cy5 i.v. injection. Results are shown as means ± SD; *p < 0.05 (one-way ANOVA) (c). Set of mice photographs with superimposed IVIS images, demonstrating NP-Cy5 accumulation in 4T1 tumors 1 h, 6 h and 24 h after i.v. injection (color code fluorescence intensity, counts). (d) Biodistribution of NP-Cy5 in 4T1 tumor-bearing mice (tumor, kidneys, lungs, heart, spleen, liver) 24 h after i.v. injection (6.6 mg·kg⁻¹ Fe₃O₄, 1.0 mg·kg⁻¹ Au, blue columns) in comparison with control mice (red columns). Corresponding organs were dissolved in aqua regia, and Au concentrations were measured by AES (Fe in Supplementary Fig. S11b). Results are shown as means ± SD; *p < 0.05, ***p < 0.001 (one-way ANOVA).

Figure 7

Study of payload delivery by Fe₃O₄-Au hybrid NPs. (a,b) Confocal intravital microscopy (IVM) of NRed-NP-Cy5 in superficial 4T1 tumor vessels upon i.v. injection to a mouse (6.6 mg·kg⁻¹ Fe₃O₄). (a) NRed-NP-Cy5 and particles aggregates (yellow) circulating in tumor vasculature (dashed lines). Cyan – neutrophils (Ly6G), green – Nile Red, red – Cy5. See also Supplementary Video S3. (b) Nile Red release into tumor tissues (arrow). Time lapsed imaging has been collected for 440 s. Blue – neutrophils (Ly6G), green – Nile Red, red – Cy5. See also Supplementary Video S4. (c) IVM of 4T1-GFP tumor (cyan) accumulating NRed-NP-Cy5 6 h after i.v. injection. Green – Nile Red, red – Cy5. (d) High magnification of the selected area from (c). (e) 4T1-GFP tumor from untreated animal, IVM.

Figure 8

Fe₃O₄-Au Janus NPs as MRI contrast agents. (a) Proton T₂ relaxation time as a function of iron concentration for NP-PEG in water and in 4T1 cells. The R₂ value is determined by the slope of the linear fitting. (b) T₂-weighted images of NPs serial dilutions acquired at TE = 24 ms in water (top panel) and 4T1 cells (bottom panel). (c) Representative T₂-weighted images of BALB/c mouse with both flanks grafted 4T1 tumors captured before and within 24 h after NPs (6.6 mg·kg⁻¹ Fe₃O₄) i.v. injection (see also Supplementary Figure S12). Areas with enhanced tumor contrasting are indicated by arrows.