Magnetic resonance imaging of ultrasmall superparamagnetic iron oxide-labeled exosomes from stem cells: a new method to obtain labeled exosomes

Abstract

Purpose: Recent findings indicate that the beneficial effects of adipose stem cells (ASCs), reported in several neurodegenerative experimental models, could be due to their paracrine activity mediated by the release of exosomes. The aim of this study was the development and validation of an innovative exosome-labeling protocol that allows to visualize them with magnetic resonance imaging (MRI).

Materials and methods: At first, ASCs were labeled using ultrasmall superparamagnetic iron oxide nanoparticles (USPIO, 4-6 nm), and optimal parameters to label ASCs in terms of cell viability, labeling efficiency, iron content, and magnetic resonance (MR) image contrast were investigated. Exosomes were then isolated from labeled ASCs using a standard isolation protocol. The efficiency of exosome labeling was assessed by acquiring MR images in vitro and in vivo as well as by determining their iron content. Transmission electron microscopy images and histological analysis were performed to validate the results obtained.

Results: By using optimized experimental parameters for ASC labeling (200 µg Fe/mL of USPIO and 72 hours of incubation), it was possible to label 100% of the cells, while their viability remained comparable to unlabeled cells; the detection limit of MR images was of 10(2) and 2.5×10(3) ASCs in vitro and in vivo, respectively. Exosomes isolated from previously labeled ASCs retain nanoparticles, as demonstrated by transmission electron microscopy images. The detection limit by MRI was 3 µg and 5 µg of exosomes in vitro and in vivo, respectively.

Conclusion: We report a new approach for labeling of exosomes by USPIO that allows detection by MRI while preserving their morphology and physiological characteristics.

Keywords: MRI; cellular imaging; exosome labeling; stem cells labeling; superparamagnetic iron oxide nanoparticles.

Figure 1

Intracellular uptake of USPIO. Notes: (A) Prussian blue staining of ASCs to detect the presence of iron nanoparticles after USPIO incubation. The image clearly shows a red nucleus and a blue cytoplasmic inclusion with typical perinuclear localization that indicate the presence of USPIO within the cells (magnification ×20, scale bar 50 µm). (B) Percentage of labeled cells after 24 hours and 72 hours of incubation with USPIO. Data expressed as mean ± SD. (C and D) TEM images of ASC incubated for 72 h with 200 µg Fe/ml of USPIO. In C, the scale bar is 5,000 nm; in D note the endocytic invagination containing nanoparticles and the internalized nanoparticles inside the endosome (scale bar 2,000 nm). In C and D arrows indicate the presence of nanoparticles inside the cell. (E) TEM image of unlabeled ASCs as negative control (scale bar 5,000 nm). Abbreviations: ASC, adipose stem cell; TEM, transmission electron microscopy; USPIO, ultrasmall superparamagnetic iron oxide nanoparticles.

Figure 2

MRI of ASC-USPIO. Notes: (A) R2 values (1/s) of ASCs unlabeled and labeled with increasing USPIO concentrations (12.5 µg Fe/mL, 25 µg Fe/mL, 50 µg Fe/mL, 100 µg Fe/mL, and 200 µg Fe/mL) after 24 hours and 72 hours of incubation. Data expressed as mean ± SD. *P<0.05. R2, relaxivity (1/s) of ASC-USPIO nanoparticles. (B) Representative MR images of gel phantom containing labeled ASCs acquired for the evaluation of the detection limit. Different amounts (from 10² to 10⁵ cells) of ASC-USPIO were used. MRI can efficiently detect up to 10² cells in vitro. In vivo MR images of acquired preintramuscular (C) and postintramuscular (D) injections of 5×10³ (dotted arrow) and 2.5×10³ (arrow) ASC-USPIO. After intramuscular injection (D), we detected up to 2.5×10³ ASC-USPIO. Abbreviations: ASC, adipose stem cell; MR, magnetic resonance; MRI, magnetic resonance imaging; USPIO, ultrasmall superparamagnetic iron oxide nanoparticles.

Figure 3

Characterization and visualization of exosomes-USPIO. Notes: (A) Western blot analysis of the expression of HSP70 (70 kDa) and Alix (95 kDa) in exosomes. Supernatant lacking the ASC and exosomes was used as NC. (B) On the left, MR images of exosomes-USPIO (top) and unlabeled exosomes (bottom) immobilized in gel are shown. The square images on the right show a higher magnification of the areas of interest in each MR image. (C) TEM image of exosomes (arrows) previously visualized by MRI in gel (B) and finally processed for electron microscopy; they are still labeled with nanoparticles (scale bar 100 nm). Abbreviations: ASC, adipose stem cell; CL, ASC cell lysate; EXO, exosomes; MR, magnetic resonance; MRI, magnetic resonance imaging; NC, negative control; TEM, transmission electron microscopy; USPIO, ultrasmall superparamagnetic iron oxide nanoparticles.

Figure 4

In vivo MRI of exosomes-USPIO. Notes: In vivo MR images acquired preintramuscular (A) and postintramuscular (B) injections of exosomes-USPIO (arrow). Prussian blue histological examination of extracted muscle tissue: blue spots inside the muscle confirmed the presence of iron nanoparticles (C) (magnification ×20, scale bar 50 μm). D shows a higher magnification (×40, scale bar 50 μm) of the boxed area shown in C. In vivo MR images acquired preintramuscular (E) and postintramuscular (F) injections of plain USPIO (arrow) containing the same amount of iron of labeled exosomes; the signal is comparable with that detected in exosomes-USPIO (B). Abbreviations: MR, magnetic resonance; MRI, magnetic resonance imaging; USPIO, ultrasmall superparamagnetic iron oxide nanoparticles.