Tracking of multimodal therapeutic nanocomplexes targeting breast cancer in vivo

Abstract

Nanoparticle-based therapeutics with local delivery and external electromagnetic field modulation holds extraordinary promise for soft-tissue cancers such as breast cancer; however, knowledge of the distribution and fate of nanoparticles in vivo is crucial for clinical translation. Here we demonstrate that multiple diagnostic capabilities can be introduced in photothermal therapeutic nanocomplexes by simultaneously enhancing both near-infrared fluorescence and magnetic resonance imaging (MRI). We track nanocomplexes in vivo, examining the influence of HER2 antibody targeting on nanocomplex distribution over 72 h. This approach provides valuable, detailed information regarding the distribution and fate of complex nanoparticles designed for specific diagnostic and therapeutic functions.

Figure 1

Characterization of multifunctional nanocomplexes. (a) Schematic representation of antibody and PEG conjugation to nanocomplexes. (b) Fluorescence (FL) intensity of nanocomplexes at 830 nm compared with standard unenhanced ICG showing ∼50-fold enhancement by nanocomplexes. Inset: Near-IR FL image of vials with enhanced ICG (nanocomplexes) and standard ICG of equivalent concentration. (c) T₂-weighted MR images of nanocomplexes in aqueous media at various concentrations. The [Fe] concentration in each sample is provided at the bottom of the respective images. (d) Spin-spin relaxation rate (T₂⁻¹) as a function of [Fe] of the nanocomplexes. Optical image of nanocomplexes (e) dispersed in aqueous media and (f) with magnet (shown with arrow).

Figure 2

Nanocomplexes delivery in vivo imaged via NIR-FOI (a) NIR images of mice with HER2 low expressing MDAMB231 xenografts (top) and HER2 overexpressing BT474AZ xenografts (bottom) at 0.3, 2, 4, 24, 48, and 72 h postinjection of nanocomplexes. (b) Fluorescence (FL) intensity of tumor-to-body ratio at different time points of mice with BT474AZ xenografts (n = 6) and MDAMB231 xenografts (n = 3) and showing maximum fluorescence at 4 h. Significant variation across tumor types, p = 0.007, determined by ANOVA is observed. (c) Fluorescence intensity comparison of tumors only between BT474AZ (n = 6) and MDAMB231 (n = 3) showing 71.5% increase in signal at 4 h in BT474AZ tumors compared to MDAMB231 tumors, p = 0.003 across tumor types. The dotted lines have been provided as guide to the eye.

Figure 3

Monitoring uptake of nanocomplexes in vivo via MRI. (a) T₂-weighted MR images of mice with HER2 overexpressing, BT474AZ, xenografts (top) and HER2 low expressing, MDAMB231, xenografts (bottom) preinjection, 0 h, and 4, 24, 48, and 72 h postinjection of nanocomplexes. The tumor is shown in red circle. (b) MR image intensity of tumor-to-body ratio at different time points of mice with BT474AZ xenografts (n = 3) and MDAMB231 xenografts (n = 3) and showing T₂-weighted contrast for BT474AZ even at 72 h. Significant variation across tumor types, p = 0.002, determined by ANOVA is observed. (c) MR image intensity comparison of tumors only between BT474AZ (n = 3) and MDAMB231 (n = 3) showing 50.5% darker contrast at 24 h in BT474AZ tumors compared to MDAMB231 tumors, p = 0.038.

Figure 4

Nanocomplexes biodistribution in vivo. (a) NIR fluorescence images of mice tissues harvested from BT474AZ (left) and MDAMB231 (right) 72 h postinjection of nanocomplexes. (b) Surface averaged fluorescence intensity analysis of mice organs of BT474AZ (red, n = 5) and MDAMB231 (black, n = 3) showing maximum fluorescence in tumors, p = 0.005. (c) Gold distribution per mass of tissue obtained from ICP-MS in various mice organs of BT474AZ (red, n = 5) and MDAMB231 (black, n = 3) showing significant Au content in tumors, p = 0.019.

Figure 5

Immunohistochemistry based analysis of microvessel density via CD31 antibody staining for (a) BT474AZ and (b) MDA-MB-231 xenografts. Images are depicted at 200× magnification. The endothelial cells are stained in red; cell nuclei are stained in purple. (c) Vessel density counts for two tumor sections. Error bars are drawn with standard error of mean over eight random locations per tumor type.

Figure 6

TEM images of BT474AZ tumor sections retrieved 72 h postinjection. (a) Low-resolution image showing nanocomplex bound to the cell surface (red) and nanocomplexes internalized in the cytoplasm (black). (b) High-resolution image of the nanocomplex shown within red box in part a. (c) High-resolution image of the nanocomplexes shown within black box in part a. (d) Low-resolution image of a different area of the tumor section showing nanocomplex in cytoplasm. High-resolution image of the nanocomplex provided as inset.