Photothermally enhanced drug delivery by ultrasmall multifunctional FeCo/graphitic shell nanocrystals

Abstract

FeCo/graphitic carbon shell (FeCo/GC) nanocrystals (∼4-5 nm in diameter) with ultrahigh magnetization are synthesized, functionalized, and developed into multifunctional biocompatible materials. We demonstrate the ability of this material to serve as an integrated system for combined drug delivery, near-infrared (NIR) photothermal therapy, and magnetic resonance imaging (MRI) in vitro. We show highly efficient loading of doxorubicin (DOX) by π-stacking on the graphitic shell to afford FeCo/GC-DOX complexes and pH sensitive DOX release from the particles. We observe enhanced intracellular drug delivery by FeCo/GC-DOX under 20 min of NIR laser (808 nm) induced hyperthermia to 43 °C, resulting in a significant increase of FeCo/GC-DOX toxicity toward breast cancer cells. The synergistic cancer cell killing by FeCo/GC-DOX drug delivery under photothermal heating is due to a ∼two-fold enhancement of cancer cell uptake of FeCo/GC-DOX complex and the increased DOX toxicity under the 43 °C hyperthermic condition. The combination of synergistic NIR photothermally enhanced drug delivery and MRI with the FeCo/GC nanocrystals could lead to a powerful multimodal system for biomedical detection and therapy.

Figure 1. Structure of FeCo/GC-DOX, drug loading and release

(a) Schematic of DOX π-stacking on FeCo/GC. The FeCo core (shown in green) is surrounded by a single layer of graphite. The nanocrystal is made water soluble by a non-covalent interaction with phospholipid-branched-PEG. Doxorubicin, shown in white, loads non-covalently on the graphitic surface of FeCo/GC. (b) TEM images of FeCo/GC-DOX conjugates. The average diameter is ~4 nm for the FeCo/GC-DOX complex. A high-resolution TEM image of a single FeCo/GC-DOX nanocrystal is shown as the inset. (c) UV-visible absorbance spectra of free DOX, FeCo/GC-DOX (FeCo-DOX) or FeCo/GC (FeCo). The DOX concentration is 450 μM for free DOX and FeCo/GC-DOX samples. The FeCo/GC concentration is ~270 nM for both the FeCo/GC and FeCo/GC-DOX samples. Both free DOX and FeCo/GC-DOX show a characteristic peak around 490 nm. Suspensions of DOX, FeCo/GC-DOX and FeCo/GC are shown in the inset. (d) Loading of DOX on FeCo/GC shows a strong dependence on pH and the DOX concentration during the incubation. The z-axis is the number of DOX molecules loaded on a single FeCo/GC nanocrystal (see methods). (e) The release of DOX from the surface of FeCo/GC is accelerated at lower pH. (f) Concentration-dependent cell survival curves of MCF-7 cells incubated with free DOX or FeCo/GC-DOX for two days. IC₅₀ values of 1.2 μM and 2.9 μM were calculated for free DOX and FeCo/GC-DOX respectively.

Figure 2. Photothermal heating of DOX and FeCo/GC Suspensions

(a) Image of solutions containing 450 μM DOX either as free DOX or FeCo/GC-DOX (FeCo-DOX). The FeCo/GC (FeCo) solution shown has the same FeCo/GC content as the FeCo/GC-DOX solution. (b) Thermal image after 10 minutes of 808 nm laser irradiation at 0.3 W/cm². Both the FeCo/GC-DOX and FeCo/GC samples show significant photothermal heating, while the free DOX solution is not heated by the laser. (c) Temperature measurements from thermal images acquired during the 10 minute laser irradiation show a temperature increase of ~35°C for FeCo/GC containing samples. The free DOX sample showed almost no heating effect from laser irradiation.

Figure 3. Cellular toxicity enhancement of DOX conjugates when combined with photothermal heating to 43°C

(a) Cell viability of MCF-7 cells after incubation with free DOX, FeCo/GC-DOX (FeCo-DOX), FeCo/GC (FeCo). DOX concentrations were either 30 or 60 μg DOX/mL. Samples were incubated for 20 minutes and combined with heating to 43°C through photothermal laser heating (red bars) or thermal heating in a water bath (blue bars). Non-heated samples are shown in black. FeCo/GC, free DOX or untreated control cells showed no viability dependence on the incubation conditions (heated vs. non-heated). FeCo/GC-DOX efficacy was significantly enhanced when incubations were performed at 43°C. The viability of control cells or FeCo/GC (NP) incubated cells were not affected by laser irradiation or thermal heating to 43°C for 20 minutes. (b) MCF-7 viability plots demonstrate the large increase in toxicity resulting from FeCo/GC-DOX (FeCo-DOX) at 43°C over 37°C, while cells incubated with FeCo/GC (FeCo) are not affected by thermal heating to 43°C for 20 minutes. (c) Cell toxicity of DOX at 37°C and 43°C reveals thermal enhancement of DOX toxicity at low doses of DOX (2 μg/mL). DOX efficacy of highly toxic doses (30 μg/mL) is not enhanced at 43°C due to the sufficiently high efficacy of high-dose DOX at 37°C.

Figure 4. Enhanced Cellular Uptake of FeCo/GC-DOX at 43°C

(a) Magnetic resonance image of pelletted, untreated MCF-7 cells or pelletted MCF-7 cells treated with FeCo/GC-DOX (FeCo-DOX) for 20 minutes at 43°C and 37°C. Brightening of samples in the T₁-weighted images indicates FeCo/GC-DOX uptake. Cells incubated at 43°C with FeCo/GC-DOX appeared the brightest due to higher cellular uptake of FeCo/GC-DOX. (b) Region-of-interest (ROI) signal measurements of the cell pellets in part (a). MCF-7 cells treated with FeCo/GC-DOX had the highest signal intensity due to the higher uptake during the 20 minute incubation. (c) DOX fluorescence signal after being extracted from cells incubated with FeCo/GC-DOX (FeCo-DOX) for 20 minutes at 37°C or 43°C. (d) Flow cytometry measurements confirming higher DOX fluorescence in cells incubated with FeCo/GC-DOX at 43°C than at 37°C. Untreated MCF-7 cell signal is shown for comparison. The mean signal of cells treated with FeCo/GC-DOX for 20 minutes at 37°C versus 43°C is plotted in the inset. The ratio of the 43°C treatment versus the 37°C treatment is similar to the signal ratio in part (c).