Dye-loaded ferritin nanocages for multimodal imaging and photothermal therapy

Abstract

Multimodal imaging-guided photothermal therapy (PTT), for the therapy of cancer, based on a ferritin (FRT) nanocage loaded with the near-infrared dye IR820 (designated DFRT) is demonstrated. The dual roles of DFRT (in imaging and PTT) are successfully balanced by using two different excitation wavelengths: 550 nm for high quantum-yield fluorescence imaging on the one hand and 808 nm for photoacoustic imaging and PTT with high photothermal conversion efficiency on the other.

Keywords: ferritin; fluorescence imaging; photoacoustic imaging; photothermal therapy; theranostics.

Figure 1

(a) Topological image of FRT in partial layer coverage on mica surface with 40 nm color-scale for the height. (b) Selected FRT nanocages are presented on a 20 nm grid in 3D to visualize protein coats with color-scale from 8–12 nm in the insert. (c) A representative AFM image of DFRT on mica surface with 80 nm color-scale for the height. (d) Section plot along the blue line in (a) and white line in (c), showing DFRT are larger and more variable in size. (e) Histogram distribution of the equivalent spherical (Eq.) diameter (Eq. Diameter = (6V/π)^1/3) for the FRT and DFRT nanoparticles, showing significant loading of the dye. inset: 3D visualization of DFRT nanoparticles placed on a 100 nm grid.

Figure 2. Characterization of DFRT

(a) UV-Vis absorbance spectra of FRT, IR820 dye, and DFRT. (b) Fluorescence emission spectra of IR820 dye and DFRT. (c) Temperature elevation of aqueous solution samples with same concentration as a function of irradiation time and (d) Photoacoustic (PA) spectrum of DFRT.

Figure 3. In vitro cell experiments

(a) Fluorescence images of Calcein AM/PI co-stained 4T1 cells with free IR820 dye or DFRT (50 μg/mL dye) incubation for 6 h after being exposed to the 808 nm laser at different power densities of 0.5 and 1 W/cm² for 5 min. The cells incubated with same concentration of free IR820 dye or DFRT without laser irradiation were chosen as control. (b) Viability of 4T1 cells after being incubated with various concentrations of DFRT for 24 h. (c) Viabilities of 4T1 cells after DFRT-induced photothermal therapy at different laser power densities. Cell viability was normalized to the control group without any treatment. Error bars were based on the standard deviations of five parallel samples.

Figure 4. In vivo NIR fluorescence (FL) and photoacoustic (PA) imaging

(a) In vivo NIR FL images after intravenous injection of DFRT in mice at different time points; (b) ex vivo NIR FL images of mouse tissues (from left to right: heart, liver, spleen, lung, kidneys, tumor), harvested at a 24 h time point post-injection of free IR820 dye or DFRT. (c) In vivo two dimensional (2D) and 3D cross-section ultrasonic (US) and PA images of tumor tissues after intravenous injection of free IR820 dye or DFRT in mice at different time points. (d) Time-lapse FL signal change of tumors followed by intravenous injection of DFRT. (e) Biodistribution of free IR820 dye or DFRT in 4T1 tumor-bearing nude mice at 24 h post-injection. (f) Time-lapse PA signal change of tumors followed by intravenous injection of free IR820 dye or DFRT. (mean ± standard deviation; n = 3 per group)

Figure 5. In vivo photothermal therapy

(a) Thermal images of 4T1 tumor-bearing mice with injection of free IR820 dye or DFRT and exposure to 808 nm laser. (b) 3D color Doppler images of tumor before and 12 h after PTT treatment. (c) Heating curves of 4T1 tumors upon laser irradiation as a function of irradiation time. (d) Tumor growth curves of different groups of 4T1 tumor-bearing mice after treatment. Tumor volumes were normalized to their initial sizes. Error bars represent the standard deviations of 4–6 mice per group. * P < 0.01. (e) Survival curves of tumor-bearing mice after various treatments. DFRT injected mice after PTT treatment showed 100% survival over 40 days. (f) H&E staining of tumor sections collected from different groups of mice at 2 h after laser irradiation.