Introduction

Visualization of viral pathways in an infected subject and understanding viral-induced pathologies is of great interest both scientifically and therapeutically, particularly in the era of the COVID-19 pandemic. In this regard, extracellular vesicles (EVs) offer a unique platform for studying viral infection paths and mechanisms due similar size and biomembrane properties as viruses [1]. In our lab we developed genetically engineered EVs displaying the receptor-binding domain (RBD) of SARS-CoV-2 on their surface creating non-infectious corona-virus-like mimetics (EVs^RBD) binding to the ACE2 receptor. Importantly EVs can be labeled magnetically (e.g. with superparamagnetic iron oxide nanoparticles, SPIONs) [2, 3] and visualized by real-time MRI using T2/T2*-weighted images. Here we show MRI visualization of in vivo targetability of the EVs^RBD in a mouse model, which can be further extended to study the manifestation of the SARS-CoV-2 virus or other viruses in different organs in real time.

Methods

HEK293 cells were genetically modified to express the RBD of SARS-CoV-2 on their surface (Fig. 1a). The secreted EVs – SARS-CoV-2 mimetics referred here as EVsRBD (or EVs^noRBD as controls) were isolated by differential centrifugation. Binding of the EVs^RBD to the ACE2 receptor was examined after incubation of EVs^RBD with the ACE2-expressing cells by fluorescence imaging and flow cytometry. For MRI, the parental cells were incubated with SPIONs (BioPal, 2.5-40 µg/mL) for 24 hours (Fig. 2a) and the secreted SPIONs-labeled EVs were characterized by western blot for protein expression, nanoparticle tracking for their size and by T2-weighted MRI (RARE sequence, TR=2800 ms, TE=42 ms, resolution 0.23×0.23×1 mm³). Two types of subcutaneous tumors were induced in nude mice (HEK293T as control or HEK293T overexpressing ACE2) to study in vivo targeting of EVs^RBD to the ACE2 receptor. Genetically engineered EVs (3x10¹¹), either presenting the RBD (EVs^RBD) or control (EVs^noRBD) labeled with SPIONs for MRI or with DiR for fluorescence were intravenously injected followed by in vivo MRI (15.2T MR scanner) and ex vivo fluorescence imaging (IVIS Lumina XR). T2-/T2*-weighted MR images of mice were acquired before and after intravenous administration of the SPIONs-labeled EVs (FLASH sequence, TR=300 ms, TE=2 ms, resolution 0.23×0.23×0.7 mm³). To confirm the presence of iron, tumor tissues were processed by histology (Prussian Blue staining).

Results

Following incubation of the HEK293 cells with SPIONs, the secreted genetically engineered EVs showed expression of RBD (for EVs^RBD) (Fig. 2c) and typical EVs size (~100 nm) (Fig. 2d). Binding to the ACE2 receptors was confirmed by higher uptake of EVs^RBD in ACE2-expressing cells compared to control EVs^noRBD (Fig.1b, c). MRI of isolated EVs showed concentration-dependent accumulation of SPIONs as depicted from the lower MRI signal on T2-weighted images (Fig. 2e, f) confirming successful labeling of the engineered EVs, which was furthered confirmed by TEM images (Fig. 2b). In vivo targetability of EVs^RBD was demonstrated by both MRI and fluorescence imaging of mice after EVs injection. The higher accumulation of the EVs^RBD in ACE2 tumor as compared to control was observed by the lower MRI signal (T2*-weighted images) of tumor tissue (Fig. 3a,b). Ex vivo fluorescence imaging (Fig. 3c) and histology examination of tumors confirmed the higher accumulation of EVs^RBD in the ACE2 tumors compared to EVs^noRBD (Fig. 3d).

Conclusion

Developing genetically engineered, magnetically labeled EVs, we showed here the ability to use MRI for mapping the binding of coronavirus mimetics to the ACE2 receptor in vivo. The principles described in this study could be further extended to better understand the mechanism and development of SARS-CoV-2 infection and the proposed platform can be implemented to study other viruses by engineering a tailored peptide on the surface of EVs using genetically engineering principles and thus to explore the role of receptors in a wide spectrum of viral-induced pathologies.

Acknowledgements

This study was supported by the Ben B. and Joyce E. Eisenberg Foundation.

References

[1] Hoen, EN, et al. PNAS 2016, 113(33) [2] Han Z, et al. Journal of Extracellular vesicles 2021, 10(3): e12054 [3] Busato, A, et al. International Journal of Nanomedicine 2016, 11:2481-2490